BIO-OPTICA PAG. Path Settembre 2011_BIO

Transcription

Cited in Index Medicus/MEDLINE, BIOSIS Previews, SCOPUS
Journal of the Italian Society of Anatomic Pathology
and Diagnostic Cytopathology,
Italian Division of the International Academy of Pathology
Editor-in-Chief
Marco Chilosi, Verona
Associate Editor
Roberto Fiocca, Genova
Managing Editor
Roberto Bandelloni, Genova
Scientific Board
R. Alaggio, Padova
G. Angeli, Vercelli
M. Barbareschi, Trento
G. Barresi, Messina
C.A. Beltrami, Udine
G. Bevilacqua, Pisa
M. Bisceglia, S. Giovanni R.
A. Bondi, Bologna
F. Bonetti, Verona
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P. Cossu-Rocca, Sassari
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A.P. Dei Tos, Treviso
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C. Doglioni, Milano
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Società Italiana di Anatomia Patologica e Citopatologia Diagnostica,
Divisione Italiana della International Academy of Pathology
Governing Board
SIAPEC-IAP
President:
C. Clemente, Milano
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G. De Rosa, Napoli
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Past President:
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L. Resta, Bari
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M. Santucci, Firenze
G. Zamboni, Verona
Associate Members
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Vol. 103 August 2011
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Sommario
Relazioni.................................................................................................................................... pag. 79
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relazioni
Pathologica 2011;103:79-134
Giovedì, 27 ottobre 2011
Aula Nova – ore 9.00-13.00
Diagnostica citopatologica
Moderatori: Ambrogio Fassina (Padova), Domenico Messina (Trapani)
Breast citology fine needle aspiration
L. Di Bonito, F. Martellani, A. Romano, A. Zacchi, E. Ober,
A. De Pellegrin, D. Bonifacio, F. Giudici, L. Torelli*, F. Zanconati
UCO Anatomy and Histopathology, Department of Medical Sciences
of Trieste University Italy; *Department of Matematics and Physics,
Trieste University Italy
Breast Fine Needle Aspiration Cytology (FNAC) represents
first choice diagnostic tool to determine mammographic lesions and clinically palpable breast nodules.
This method is considered to bring more advantages, as far as
invasiveness and as costs if compared with other techniques
like core biopsy or surgical biopsy, expecially when it is performed under ultrasound guidance which guarantees perfect
targeting of lesions.
Most literature’s critics towards FNAC are due to the different realities where the exam is performed and to the variety of
breast lesions examined. The most reliable results with FNAC
are obtained in those clinical groups where pathologist, who
is also a cytopathology expert, actively joins the aspiration
sessions taking care of collecting patients’ clinical data, radiological and ecographical features of lesions to be examined.
This way, the pathologist can evaluate macroscopic characteristics of aspirated material (fluid, dense, whitish, jelly like,
smelly etc..) that will be spread on smears and evaluated right
away through a fast stain. Pathologist will be able to evaluate
specimen’s adequacy and to get an idea of clinical issues the
case may arise.
FNAC is affected anyway by a certain degree of subjectiveness, that is more evident with the so called “grey zone “ cases
(the ones not clearly benign or not clearly malignant) and
when there are some doubts about material adequacy. These
situations may create some misunderstandings between clinicians and pathologists with possible over or undertreatment
for patients. The need for clear communication is extremely
important, but it becomes a priority in senology because of the
multidisciplinary aspects of the field. For this reasons a breast
cytology reporting system has been proposed since 1993
within a U.K. mammographic breast screening programme.
The reporting system’s aim is to communicate to clinicians
in an extremely precise way pathologist’s evaluation about
cytological cases:
C1 = inadequate: includes all those cases which do not provide the possibility to solve a specific diagnostic problem
(poor cellularity, bad technical preparation, excessive inflammatory or blood’s elements,...);
C2 = benign: there’s no evidence of malignancy. It includes
all cases characterized by absence of nuclear or morphological
alterations.
C3 = probably benign: includes all cases in which the smear’s
cells are not certainly interpretable as benign. Management of
such cases requires correlation of cytology with clinical and /
or radiological aspect.
C4 = suspicious for malignancy: the cellular appearance,
although highly suggestive for malignancy, is not conclusive.
This category includes the cases with few highly atypical cells
and some very well-differentiated tumors. These lesions must
undergo biopsy to obtain a conclusive diagnosis or, in cases
with low cellularity, FNAC can be repeated.
C5 = malignant: cytological features are diagnostic for malignancy. Sometimes, through FNAC the histotype of malignancy can be determinated.
The five diagnostic categories proposed in the U.K in 1993
were later adopted at a European level; using this system
does not limit pathologists’ possibility to give a description
of morphological aspects observed on smears, but it requires
a conclusive short diagnostic assessment that all other breast
screening unit members can undoubtedly understand to allow
precise and exact indications regarding further exams or treatment to be given. Diagnostic categories have also revealed
being an extremely useful tool for all the audit activities
each anatomic pathology laboratory has to respect within an
organized screening programme, providing periodically data
that show quality control’s respect. As a matter of fact, all
the activity’s reports and correlations regarding cytology’s
diagnostic efficacy are based on working out data obtained by
diagnostic categories and by comparison of cytologic diagnosis with following histology or clinical follow up. Making use
of the five diagnostic categories also permits easy information
exchange and comparison among labs in different regions or
states and any observed abnormality can be easily discussed
and corrected.
We’ve introduced the five diagnostic categories system to report breast cytology in our lab since 1995 in an experimental
way; and they’ve become essential part of cytological reports
since 2000. Mammographic breast screening programme,
which has started in our region, Friuli Venezia Giulia, since
2006, has made them mandatory for all cytological reports
of pathologists involved in the screening programme. This
allows the Public Health Regional Agency (encharged to control and to coordinate the programme) to monitor cytology’s
reports diagnostic quality.
Each diagnostic category is strictly associated with a diagnostic pathway which needs well founded reasons to be modified.
C1, for example requires exam’s repetition or histology which
becomes mandatory for those cases with suspicious radiology;
in case of doubtful radiology the patient can be also early recalled for instrumental follow up. Thanks to the pathologist’s
presence during FNAC sessions, our inadequate rate is very
low; and besides the screening detected lesions, cytology has
represented the first morphological exam for about 90% of
women with breast abnormalities.
In the first table we have represented the number of FNAC in
the period 2004-2010 (Fig. 1). This distribution is representative of breast cytology’s performance in the Trieste’s area and
we can observe the activity’s continuous increase.
For each nodule the correlation with histological examination
was performed or, for benign lesions not undergoing surgery,
correlation with instrumental follow-up was done. No case
80
CONGRESSO NAZIONALE SIAPEC – IAP • PALERMO, 27-29 OTTOBRE 2011
Tab. II.
Fig.1.
Indicators
was lost at follow-up and the correlation was possible even
for those lesions with histology performed at other regional
facilities due to the possibility offered by the Health Information System of Regione Friuli Venezia Giulia (INSIEL) which
allows to share on the network all histological reports made
by the Anatomical Pathology labs operating on the regional
territory. The outcome of cyto histological correlation is summarized in Table I.
Thanks to the cyto-histological correlation, it was possible
to calculate the quality indicators (sensitivity, specificity,
predictive values, false positives and inadequate and compare
them with the corresponding reference standards proposed
in theguidelines for screening mammography summarized in
Table II.
Meeting the imposed reference standards has been made
possible by the direct and continuous participation of the
pathologist in the cytologic sample’s collection. This has
helped to keep the inadequates number very low and to minimize false positives and negatives. Using the rapid cytology
to immediately discuss with the radiologist about patient
management in the same session provides great results for
the patients.
From our experience we think only a cytological exam
with well demonstrated and and well documented quality
which satisfies those criteria suggested by guidelines will
be able to play a key role in patients’ clinical management. It will allow to reduce as much as possible diagnostic biopsies on benign lesions and frozen sections to
confirm malignant lesions.We believe that a good quality
cytologycan provide an important and relevant contribution to define breast lesions’ naturewithout using other
invasive procedures.
Selected references
European guidelines for quality assurance in breast cancer screening and
diagnosis. Fourth edition, 2006.
Guidelines for Cytology Procedures and Reporting in Breast Cancer
Screening. Cytology Sub-Group of the National Coordinating Commitee for Breast Screening Pathology NHS-BSP No. 22 Sept 1993.
Data 2004-2010
Standard
Absolute sensitivity
86,3%
>60%
Complete sensitivity
97,5%
>80%
Specificity (biopsy cases only)
26,8%
Specificity (full)
76,8%
>60%
VPP C5
99,9%
>95%
VPP C4
78,9%
70-80%
VPP C3
9,4%
<20%
False-negative rate
0,2%
<5%
False-positive rate
0,1%
<1%
Inadequate rate
5,0%
<25%
Inadequate rate from cancers
2,2%
<10%
Suspicious rate
14,3%
<20%
Total FNA 2004-2010
5596
Citologia urinaria
M. Bonzanini, P. Dalla Palma
Anatomia Patologica Ospedale S. Chiara, Trento
L’esame citologico delle urine spontanee è caratterizzato da bassi
costi, nessuna invasività e da elevata sensibilità e specificità nella
diagnosi dei carcinomi uroteliali specialmente di alto grado.
Rimane pertanto a tutt’oggi, nella pratica urologica, un esame
di grande valore diagnostico sia come primo approccio nei pazienti con sintomi urologici che come follow-up nei pazienti
con neoplasia uroteliale.
La principale finalità dell’esame citologico delle urine risiede
nella diagnosi delle neoplasie uroteliali, in particolare di quelle di alto grado.
Lo scopo di questa presentazione è quello di affrontare l’iter
diagnostico differenziale morfologico sulla base di alcune
caratteristiche delle cellule e degli aggregati che si possono
incontrare in citologia urinaria nella pratica quotidiana.
1) Aggregati papillari di cellule uroteliali di grandi dimensioni
che pongono il sospetto di carcinoma uroteliale. In questi
casi entrano in diagnosi differenziale alcune condizioni
non neoplastiche come infiammazione, litiasi, terapia,
manovre strumentali, ipertrofia prostatica, patologia renale
che possono provocare atipie reattive e sfaldamento in
papille delle cellule uroteliale o determinare la presenza di
cellule non uroteliali, atipiche nelle urine.
2) Aggregati di cellule piccole: in questo caso la diagnosi differenziale si pone con il carcinoma uroteliale di alto grado
a piccole cellule e con il carcinoma indifferenziato a pic-
Tab. I. Data records 2004-2010.
2004-2010 Data
Malignant histology
C5
C4
C3
C2
C1
Total
1935
217
48
5
53
2258
Benign histology
2
58
228
128
62
478
Malignant at follow-up
93
15
0
0
0
108
Benign at follow-up
0
0
235
2353
164
2752
2030
290
511
2486
279
5596
Total FNA
relazioni
cole cellule. Condizioni come la cistite follicolare, responsabili dell’esfoliazione di linfociti che possono, soprattutto
nei preparati su strato sottile, dare origine ad aggregati
tridimensionali e la presenza di aggregati di cellule basali
possono entrano in diagnosi differenziale.
3) Cellule atipiche singole: la diagnosi differenziale deve tenere in considerazione il carcinoma uroteliale in situ, le alterazioni cellulari indotte da polioma virus o più raramente
da altri virus, e cellule di origine seminale.
4) Cellule con morfologia cilindrica, singole o in aggregati,
possono avere una provenienza esterna all’apparato urinario, possono essere espressione di metaplasia ma possono
essere anche espressione di adenocarcinoma primitivo o
metastatico vescicale.
5) Cellule squamose con aspetti atipici possono essere
anch’esse espressione di metaplasia, di infezione da papilloma virus ma anche essere la spia di carcinoma uroteliale
di alto grado in cui si osservano non di rado aspetti di differenziazione squamosa o del più raro carcinoma squamoso
primitivo della vescica.
Viene infine sottolineata l’importanza di una classificazione
citologica che consenta di standardizzare le risposte e le renda
più immediatamente comprensibili al clinico.
Fine-needle cytology of thyroid lesions
G. Fadda, E.D. Rossi
Istituto di Anatomia e Istologia Patologica, Università Cattolica del
Sacro Cuore, Roma
The fine-needle aspiration biopsy (FNAB) was widely appreciated as a diagnostic tool during the 1950s in Sweden: since then
it has spread worldwide because of its simplicity, safety and the
possibility of repetition.FNAB is regarded as the most accurate
method for the selection of patients with thyroid nodules for
surgery and a very cost-effective diagnostic test. The aspiration
of a thyroid nodule is preferably carried out under sonographic
guidance and only seldom, even if the lesion is palpable, the
maneuver can be performed under manual guidance. All the
nodules in a multinodular goiter should be submitted to the
aspiration maneuver because the risk of malignancy is the same
in each nodule but usually they are selected based on their ultrasound (US) appearance. A hypoechoic solid pattern with irregular margins and the presence of intralesional calcium deposits
are the most important clues for suspecting a malignant lesion.
Another useful method of nodule selection is the evaluation of
its Echo-Power Doppler pattern: if a nodule is vascularized the
likelihood of malignancy is higher compared to poorly vascularized lesions. The aspiration is performed with thin needles
(gauge from 27G to 20G) and it is important to note that the
amount of cells does not depend on the caliber of the needle
but on the sampling time. Therefore, thyroid lesions which are
usually richly vascularized are better sampled using very thin
needles (either 27 or 25G) rather than larger ones (23 to 20G).
After a superficial anesthesia, which may be carried out only by
spraying the skin with ethyl chloride or by injecting lidocaine
in the subcutis, the operator holds the sonographic probe with
one hand and performs the aspiration with the other by means
of a syringe-holding pistol. A FNAB may also be carried out by
simply moving the needle, without any connection to a suction
device (cytopuncture): in this case the material is extruded from
the lesion by capillarity. The danger of complication is low (local pain, limited hemorrhage) even when the number of passes
is up to 5 for each nodule. The procedure can be repeated safely
when the smear shows low cellularity at the on-site assessment
81
and a reliable diagnosis cannot be rendered. The number of
passes is related to the possibility of the on-site assessment of
the material adequacy (2 passes are usually sufficient). When
the on-site check is not possible or when the liquid-based cytology technique is chosen 3 passes might be required depending
on the skill of the operator and on the characteristics of the lesion. Once the needle is withdrawn from the lesion, the material
is extruded onto glass slides and the smear is fixed with 95%
ethyl alcohol for the Papanicolaou stain.
This is the mostly adopted staining technique as it can be
easily compared with the histological specimens stained with
hematoxylin and eosin Alternately, the smear may be air-dried
and then stained with May-Grunwald Giemsa, which also
gives excellent results in thyroid cytology. The thin-layer or
liquid-based cytology (LBC) technique, originally developed
for application to gynecologic cervical smears, has progressively gained consensus after being applied to both nongynecologic and fine-needle aspiration cytology.The LBC
procedure includes two-steps which are the fixation of the
totality of the material in an alcohol-based solution (methanol
or ethanol depending on the technique); and the automated
processing of the material to obtain a thin layer of representative cells. A computer-assisted device allows the transfer of
the fixed and partially disaggregated cells onto a single slide
The two most common methods for processing the cytologic
samples use an alcohol-based fixative solution. In the first
(ThinPrep2000TM, Hologic Co., Marlborough, USA), the cells
are aspirated from a methanol-based solution (CytolitTM) then
filtered and transferred onto a positively charged slide with a
gentle positive pressure. In the second, the cells are collected
in an ethanol-based solution (CytoRichTM), centrifuged twice
then slowly sedimentated onto a poly-L-lysinated slide and
eventually stained with a specific hematoxylin-eosin stain.
(SurePathTM, TriPath Imaging, Burlington, USA). The final
result for both methods is one slide for each lesion where all
cells are concentrated in a thin layer on the central area of the
slide measuring 20 square mms for ThinPrep and 13 square
mms for PrepStain LBC. The LBC method enables the storage
of a variable amount of cells in a preservative solution for up
to 6 months after the biopsy. The remaining material can be
used for the application of ancillary techniques such as immunocytochemistry and molecular biology. The FNA cytology
plays a key role in the preoperative diagnosis of thyroid neoplasms, because of the possibility: a) to select whose patients
must be addressed to surgery or can be simply followed-up; b)
to define the surgical approach and/or c) to give to the patient
correct information regarding the management of their own
lesion. The diagnostic accuracy of the cytology cannot equal
that of histology since the aspiration cytology may yield a diagnosis of “follicular nodule” (or follicular neoplasm) which
defines those lesions composed of follicular aggregates of
thyrocytes which may correspond to a follicular adenoma or
a follicular carcinoma. These differential diagnoses rely on
histologic details (invasion of the capsule and of the vessels,
different patterns between inner and outer portions of the nodule) rather than on the atypical features of the thyrocytes.
Although a diagnosis of follicular nodule (or neoplasm) warrants the surgical removal of the lesion, the possibility that a
benign lesion can be removed affects the figures of specificity
and sensitivity of the procedure cited in the literature. Nonetheless, FNA cytology is a fundamental tool in the management of
thyroid nodules. The cytology of a follicular adenoma yields
a picture of “Follicular Neoplasm” (FN), which is characterized by medium-sized thyrocytes, at times in microfollicular
aggregates, with a background of fibrovascular tissue and
82
Tab. I. SIAPEC-IAP ITALIAN CYTOLOGIC CLASSIFICATION OF THYROID
LESIONS (Fadda G. et al., Pathologica, 2010).
TIR 1. Non diagnostic
TIR 2. Negative for malignant cells
TIR 3. Inconclusive/indeterminate (Follicular proliferation)
TIR 4. Probably malignant
TIR 5. Positive for malignant cells
blood. The colloid amount is scant and features of old hemorrhage (hemosiderin-laden histiocytes) may coexist. Variations
are represented by fire-flare cells (hyperfunctioning FN)
and oxyphilic cells (oxyphilic FN). A diagnosis of follicular
neoplasm means either a possible follicular carcinoma or a
follicular variant of a papillary carcinoma which nonetheless
accounts for about 20% of the corresponding lesions at histology. To avoid unnecessary thyroidectomies, the category
of FN has been divided into subgroups with different risks
of carcinomatous occurrence. Despite these problems, FNA
cytology keeps an important diagnostic role allowing the correct management of patients with thyroid nodules in more than
80% of cases. Papillary carcinoma (PC) is the most frequent
thyroid malignancy, accounting for more than 80% of all epithelial tumors, and this rate has progressively increased due to
the relative decrease of the follicular carcinoma. The cytologic
picture of PC is actually well defined and a high diagnostic accuracy can be reached. It is based on the nuclear details of the
neoplastic thyrocytes rather than on their aggregation. The detection of clear nuclear pseudoinclusions (Orphan Annie’s eye)
is a pivotal clue for the diagnosis. The presence of well formed
papillae and psammoma bodies may strengthen the cytological
diagnosis of PC but this finding is unfortunately rather infrequent. Accordingly, a correct diagnosis of PC can be made
with the presence of such nuclear features even in those cases
showing a diffuse follicular pattern. Follicular carcinoma
(FC) accounts, at least in Western countries, for about 5% of
all thyroid malignancies. The FC yields a cytological picture of
“follicular nodule” either classical or oxyphilic, and its clinical
significance has already been discussed under the paragraph on
follicular adenoma (see above). Nevertheless, two more details
should be discussed: i) the definition of “follicular neoplasm”
(FN – see above) is too often used, becoming a wastebasket
for all those lesions which look suspicious to the pathologist
or which have been poorly sampled by the operator. The category of FN is only defined by the presence of medium-sized
follicular cells along with the absence of colloid, but the correct diagnosis should be “inadequate amount of diagnostic
material”; ii) whenever possible, the presence of nuclear pleomorphism in non-oxyphilic thyrocytes should be emphasized
in the cytological report because such a morphological feature
may be associated with a high frequency of malignancy,
according to some authors. On the other hand, the nuclear
pleomorphism of the oxyphilic cells carries no diagnostic relevance as it is present in most hyperplastic oxyphilic nodules
of a thyroiditis and shows no association with the malignant
potential of the lesion. Medullary thyroid carcinoma (MTC)
is an uncommon malignancy accounting for about 5% of all
thyroid neoplasms which originates from the parafollicular
cells (C-cells). The smear obtained from a MTC is usually cellular, with scanty, if any, colloid. The cells are usually isolated,
medium-sized (though isolated large cells may be detected)
and show a characteristic “plasmacytoid” appearance: a round
peripheral nucleus with “cartwheel” chromatin (a distinctive
feature of endocrine cells) and abundant granular cytoplasm.
Such cells are very suggestive of a MTC and, when detected,
their cytoplasmic immunoreaction for calcitonin and CEA
should be sought to confirm the diagnosis and to guide the
surgical approach. A MTC should be suspected in those cases
showing a moderate cellularity without follicular aggregates
and with fragments of fibrovascular or hyalinized tissue (amyloid).The Insular thyroid carcinoma (ITC) may be defined
as a malignant aggressive tumor showing a solid trabecular
pattern (insular) composed of poorly differentiated thyrocytes
which still exhibits differentiated features.(immunodetectable
cytoplasmic thyroglobulin). Despite their deceptively harmless
appearance, these small thyrocytes with monomorphic dark
nuclei exhibit a brisk mitotic activity (up to ten mitoses in ten
microscopic H.P.F.) and often permeate the tumor capsule and
vessels. In its pure form (more than 40% of insular pattern),
the tumor shows local aggressiveness (invasion of surrounding
tissues and vessels) and distant spread (regional lymph node
and lung metastases at diagnosis). The definition of anaplastic
carcinoma (ATC) refers exclusively to an uncommon (less
than 5% of all thyroid malignancies) large cell indifferentiated tumor which, for its advanced stage at clinical detection
frustrates every attempt to surgical approach. The smear from a
ATC shows, in a background of necrotic debris, large round or
spindle cells with hyperchromatic pleomorphic nuclei, sometimes exhibiting prominent nucleoli. This cytological diagnosis
is quite important because often the tumor cannot be resected
so that either external radiation therapy or chemotherapy must
be used. In conclusion the cytologic evaluation of a thyroid
nodule is a diagnostic essential procedure in order to define a
pre-surgical risk stratification of thyroid cancers. The application of additional techniques such as immunocitochemistry and
molecular biology improve the overall diagnostic efficacy of
the fine-needle aspiration cytologic technique.
References
1
Fadda G, Basolo F, Bondi A. et al. Cytological classification of thyroid
nodules. Proposal of the SIAPEC-IAP Italian Consensus Working
Group [Classificazione citologica dei noduli tiroidei. Proposta del
Consensus Working Group italiano della SIAPEC-IAP]. Pathologica
2010;102:405-8.
2
Bishop-Pitman M, Abele J, Ali SZ, et al. Techniques for thyroid FNA: a
synopsis of the National Cancer Institute thyroid fine-needle aspiration
state of the science conference. Diagn Cytopathol 2008;36:407-42.
3
Rossi ED, Raffaelli M, Zannoni GF, et al. Diagnostic efficacy of
conventional as compared to liquid-based cytology in thyroid lesions.
Evaluation of 10,360 fine needle aspiration cytology cases. Acta Cytol
2009;53:659-66.
4
Rossi ED, Raffaelli M., Minimo C, et al. Immunocytochemical evaluation of thyroid neoplasms on thin-layer smears from fine-needle aspiration biopsies. Cancer Cytopathology 2005;105:87-95.
5
Baloch ZW, Gupta PK, et al. Follicular variant of papillary carcinoma. Cytologic and histologic correlation. Am J Clin Pathol
1999;111;216-22.
Citologia cervicovaginale
M. R. Giovagnoli
Roma
L’esame citologico cervico-vaginale trova la sua collocazione
in contesti clinici e di medicina preventiva tra loro profondamente diversi.
Può essere utilizzato come test primario in un percorso di
screening per il cervico-carcinoma per l’identificazione di
lesioni precancerose e di forme neoplastiche iniziali, come
83
relazioni
anche come test di triage in donne identificate come a rischio
in quanto portatrici di infezioni virali con ceppi di HPV potenzialmente trasformanti.
Ma fin dai tempi della sua introduzione il paptest è stato utilizzato come punto di inizio o come completamento dell’iter diagnostico delle alterazioni e delle lesioni della portio uterina, come
pure nel follow-up delle donne operate per tali patologie.
Data la molteplicità di contesti è estremamente importante
da una parte attenersi rigorosamente al dato morfologico, per
rendere tale esame, pur gravato da una certa quota di soggettività, massimamente riproducibile, e dall’altra conoscerne le
caratteristiche in termini di sensibilità, specificità e accuratezza, per poterne utilizzare appieno le grandi potenzialità nelle
diverse situazioni.
Verranno fatti una serie di esempi sia per meglio caratterizzare il dato morfologico sia per specificare quali informazioni trarne in risposta a quesiti clinici di volta in volta
differenti.
Tavola Rotonda
Quale evoluzione per il pap test oggi
Moderatori: Guido Collina (Bologna), Leonardo Resta (Bari)
Quale evoluzione per il pap test oggi: Il punto di
vista del Virologo
F.M. Buonaguro, M.L. Tornesello, L. Buonaguro
Istituto Naz. Tumori “Fond. Pascale”, Napoli
mente associato a due componenti: a) il tipo di HPV (peculiare
per affinità delle proteine oncogene E6/E7 agli oncosoppressori
p53 e pRb e per capacità di evadere la risposta immunitaria); b)
il tipo e l’efficacia della risposta immunitaria individuale.
Introduzione
Il test citologico introdotto nel 1943 dal Dr Papanicolau ha
il gran pregio di poter rapidamente screenare la presenza di
lesioni del collo dell’utero ed ha avuto il merito eccezionale di
ridurre del 74% l’incidenza di lesioni invasive, permettendo la
diagnosi di lesioni precoci e del loro trattamento.
I nuovi progressi scientifici nel campo dell’eziopatogenesi
delle patologie della cervice uterina verosimilmente in un prossimo futuro ne modificheranno drasticamente le strategie di
prevenzione. Di fatto, l’identificazione dell’HPV quale agente
eziopatogenetico di tale lesioni, la messa a punto di metodiche
biomolecolari virologiche per la identificazione dei diversi
ceppi virali (n=30), la determinazione del ruolo oncogeno dei
rispettivi ceppi, verosimilmente modificherà le modalità e le
metodiche alla base dei programmi di screening di popolazione.
Donne negative per ceppi di HPV o positive per ceppi a basso
rischio saranno monitorate meno frequentemente, mentre le
donne positive per ceppi ad alto rischio saranno sottoposte a
controlli più frequenti con l’impiego di tecniche ad alta specificità e sensitività per identificare lesioni precoci e poterne
valutare il grado di progressione neoplastica. Inoltre metodiche
virali permetteranno di valutare l’efficacia dei programmi vaccinali che saranno di volta in volta proposti ed effettuati.
2. I cofattori implicati nella cronicizzazione/progressione dell’Infezioni da HPV
La caratterizzazione biomolecolare dei ceppi di HPV prevalenti
nelle diversi stadi clinici e gli studi di biologia cellulare hanno
permesso di acquisire notevoli conoscenze sui meccanismi
eziopatogenetici implicati nella induzione e nella progressione
neoplastica; gli studi immunologici, d’altro canto, hanno messo
in evidenza il ruolo cruciale dell’immunità cellulare nella storia naturale dell’infezione. L’infezione genitale da HPV, che
interessa circa l’80% delle donne nel corso della vita sessuale,
determina generalmente delle infezioni transitorie che nel 90%
dei casi regrediscono nei dodici mesi successivi all’esposizione.
Poiché la trasmissione è da cellula a cellula e non si associa a
lisi cellulare, la risposta umorale nel corso della storia naturale
dell’infezione è modesta, presente solo nel 50% dei soggetti,
limitata nel tempo (<36 mesi) e non protettiva. Pertanto è prevalentemente la risposta Th1 a determinare un contenimento
efficace dell’infezione e delle lesioni displastiche/neoplastiche
ad esse associate. Tale condizione è evidenziato inoltre dall’effetto favorente della progressione neoplastica di co-fattori che
alterano il pattern citochinico e la risposta Th1, quali il fumo e
la contemporanea infezione da HIV, che si associa più frequentemente alla persistenza di infezioni di HPV, anche a bassa
patogenicità, e alla presenza di infezioni multiple.
1. Le Infezioni da HPV
Le infezioni da papillomavirus umani (HPV) sono associate
all’insorgenza di lesioni benigne e maligne della cute e delle
mucose. Gli HPV che interessano la regione anogenitale vengono suddivisi, a seconda della minore o maggiore frequenza
di associazione con lesioni displastiche di alto grado e carcinomi, in genotipi a basso e ad alto rischio oncogeno (di cui i
più rilevanti sono l’HPV16 e 18 che si associano a >70% delle
neoplasie invasive della cervice uterina).
Gli HPV ad alto rischio determinano più frequentemente infezioni persistenti, che costituiscono il maggiore fattore di rischio per
lo sviluppo di lesioni intraepiteliali squamose (SIL) della cervice
uterina ad evoluzione neoplastica, e questo è stato prevalente-
3. La prevenzione delle neoplasie della cervice uterina
3.1 La prevenzione primaria
I modelli vaccinali anti-HPV attualmente disponibili rientrano
negli approcci vaccinali sviluppati sinora e basati prevalentemente sull’induzione di una risposta umorale, dovuta sia alla tipologia
dell’immunogeno utilizzato (non in grado di infettare una cellula
ospite, e quindi di far esporre i propri antigeni in associazione ai
complessi di immuno-istocompatibilità di classe I) che agli adiuvanti utilizzati (quali l’allume che inibiscono l’immunità cellulare). Questa tecnologia limita la possibilità di sviluppare vaccini
terapeutici, ma permette la produzione di vaccini preventivi che,
se somministrati prima dell’esposizione ai ceppi di HPV presenti
nelle preparazioni vaccinali, assicurano una ottima efficacia di
84
Fig.1.
prevenzione sia di trasmissione, che di infezioni persistenti e di
sviluppo delle lesioni neoplastiche associate.
3.1.a Studi clinici: immunogenicità delle VLPs in trials umani
Un modello vaccinale mucosale preventivo deve essere in grado
di indurre un buon livello di anticorpi nella mucosa cervicovaginale, determinato dalla produzione in loco di immunoglobuline
secretorie polimeriche-IgA2 o dalla trasudazione di immunoglobuline sieriche monomeriche (prevalentemente IgG1). Queste
ultime sembrano svolgere un ruolo determinante protettivo nel
tratto genitale femminile umano, a differenza degli altri comparti mucosali, ed il livello ottenuto in corso di vaccinazione è
stato preso come indicatore di efficacia protettiva 1, tenuto anche
conto che alti livelli sierici di IgG1 si associano ad alti livelli
locali di IgA2 2. Alti livelli di IgG sieriche anti-HPV sono stati
ottenuti dal vaccino quadrivalente anti-HPV della Merck con
l’adiuvante tradizionale di allume, che è un adiuvante di tipo
TH2. La GSK, invece, ha incluso nella prepara-zione vaccinale
l’adiuvante ASO4, contenente allume e l’MPL, che agisce attraverso il binding del TLR4 inducendo una forte risposta umorale
e cellulare, prevalentemente associata all’attivazione TH1, che
risulta in un livello di anticorpi neutralizzanti sierici di 2-3 log
(100-1000 volte) più alto di quello presente nel corso della infezione naturale e per periodi superiori ai 50 mesi 3.
L’alto livello della risposta umorale è necessario inoltre per assicurare una efficiente protezione a livello genitale dove i titoli
anticorpali mostrano nel corso del ciclo mestruale una notevole
variazione, peculiare rispetto ai livelli costanti delle IgG ematiche. La variazione di IgG a livello della mucosa cervicale, la
cui ampiezza è fino a nove volte, tra gli alti livelli della fase
proliferativa e quelli bassi della fase peri-ovulatoria 4, potrebbe
far variare il livello di protezione vaccinale, se anche il livello
minimo non fosse sufficientemente protettivo 5.
3.1.b Studi clinici: Sicurezza ed efficacia delle VLPs nei trials
umani
Entrambi le preparazioni hanno mostrato una buona tollerabilità
e presentato modesti effetti collaterali. Per entrambi la valutazione dell’efficacia dei vaccini si basa sulla quantificazione
della riduzione dell’incidenza dell’infezione da HPV vaccinale
(due rilevazioni positive a distanza di almeno 4-6 mesi) e sulla
riduzione dell’incidenza delle lesioni precancerose da genotipi di
HPV vaccinali. Nessun RCT ha finora potuto valutare l’efficacia
di prevenzione delle lesioni cancerose dal momento che il vaccino è stato somministrato a donne giovani ed il follow up delle
sperimentazioni al massimo è stato di 5 anni, mentre normalmente il tempo che intercorre fra l’infezione da HPV e lo sviluppo
delle lesioni cancerose è mediamente di 20-30 anni ed il picco di
incidenza del cervicocarcinoma si registra intorno ai 45 anni.
Gli RCT hanno dimostrato che la vaccinazione in tre dosi è
efficace nel prevenire l’infezione da HPV16/18 e le lesioni
precancerose correlate. Il vaccino quadrivalente si è dimostrato
efficace anche nel prevenire le lesioni condilomatose da genotipi
di HPV contenuti nel vaccino. Gli eccellenti risultati (~100%) riportati sono stati ottenuti nel gruppo di giovani donne (<26 anni)
che hanno aderito pienamente al protocollo (per protocol), con
una vita sessuale non sregolata (numero medio di partner limitato), con un’anamnesi negativa per precedenti lesioni cervicali
e negative per i tipi di HPV contenuti nel vaccino al momento
dell’arruolamento. Risultati più modesti (<80%) sono stati ottenuti nel gruppo di donne che hanno deviato dal protocollo o che
hanno ricevuto solo parte delle dosi vaccinali programmate per
la detezione di positività all’HPV (intention to treat).
3.2 La prevenzione secondaria
L’uso di metodologie virologiche per l’identificazione di infezioni virali e della loro persistenza nei diversi distretti dell’apparato genitale femminile riveste una notevole rilevanza sia
per valutare l’efficacia dei protocolli vaccinali, che per poter
monitorare l’andamento di una infezione in corso. Diversi
sono i metodi attualmente disponibili per la identificazione di
infezioni da HPV e la successiva identificazione degli HPV:
(A) metodi basati sull’amplificazione del segnale, che utilizzano come sistema rivelatore substrati cromogeni che in caso
di positività danno luogo a prodotti che assorbono a specifiche
lunghezze d’onda [in particolare l’HC2 HPV DNA- test];
(B) metodi basati sull’amplificazione del genoma virale, che
utilizzano la Taq polimerasi per reiterare un miliardo di volte
(1x109) nel corso di 30 cicli di amplificazione la sequenza virale presente nel campione [in particolare l’AMPLICOR HPV
test]. L’incremento di specifiche sequenze virali, permette poi
la caratterizzazione del tipo di HPV presente mediante tecniche
di ibridazione o di sequenza nucleotidica (con o senza previo
clonaggio). Le recenti metodologie biomolecolari permettono
inoltre la determinazione dei livelli di espressione dei singoli
geni, dello stato fisico dell’HPV nelle cellule interessate (HPV
episomale vs integrato), delle caratteristiche molecolari e dei
livelli di espressione di oncosoppressori (p53 e pRb) e dei target
(inclusa la p16) dei loro rispettivi pathway molecolari.
L’insieme di tali metodiche, pertanto, permettono al momento
di identificare non solo la presenza di infezione da HPV, ma di
poter con buona approssimazione predire il rischio di progressione delle lesioni in atto e di poter programmare il più idoneo
tipo di monitoraggio e/o di trattamento 6 7.
Conclusioni
L’armamentario medico si è certamente arricchito nel corso
degli ultimi anni di metodologie biomolecolari in grado di identificare con maggiore accuratezza i soggetti affetti da lesioni
da HPV a rischio di progressione neoplastica. La novità più
recente e più innovativa è però il poter disporre di un valido
strumento per prevenire ed eventualmente debellare una delle
principali (in particolare nei paesi in via di sviluppo) patologie
neoplastiche della donna. Alla prevenzione secondaria fornita
dal Pap-test, che nei Paesi dove è stato incluso nei programmi di
prevenzione nazionali ha determinato il contenimento delle neoplasie genitali ad una incidenza inferiore ai 4 casi per 100’000
abitanti per anno, si è affiancato uno strumento di prevenzione
primaria: il Vaccino anti-HPV. Sebbene le preparazioni attuali
siano specificamente contro i due ceppi (HPV16 ed HPV18)
prevalentemente associati alle neoplasie invasive (>70% delle
neoplasie della cervice uterina), e sebbene non ci siano ancora
i dati definitivi dell’efficace protettiva nei confronti di tali neo-
85
relazioni
plasie, i promettenti dati riportati recentemente dalla GSK sulla
durata della efficacia protettiva (>50 mesi) e su una protezione
crociata nei confronti di ceppi correlati all’HPV16 e 18, fanno
ritenere che nei prossimi anni assisteremo a radicali cambiamenti sia nella frequenza e nella distribuzione dei ceppi virali delle
patologie ad essi associati, che nei programmi di prevenzione
primaria, con l’eventuale diffusione della vaccinazione anche ai
partner maschili, e negli schemi di prevenzione secondaria, con
la sistematica introduzione del test dell’HPV DNA in sostituzione/ complementazione del Pap-test.
Inoltre, l’identificazione e l’uso di nuove classi di adiuvanti 8,
potrebbe permettere in un futuro non molto lontano lo sviluppo
di modelli vaccinali preventivi e terapeutici, capaci di contrastare infezioni croniche in atto ed il decorso delle patologie ad
esse associate.
Bibliografia
1
Brandtzaeg P. Mucosal immunity in the female genital tract. J Reprod
Immunol 1997;36:23-50.
2
Ho GY, Studentsov Y, Hall CB, et al. Risk factors for subsequent
cervicovaginal human papillomavirus (hpv) infection and the protective role of antibodies to Hpv-16 virus-like particles. J Infect Dis
2002;186:737-42.
3
Zhou J, Gissmann L, Zentgraf H, et al. Early phase in the infection of
cultured cells with papillomavirus virions. Virology 1995;214:167-76.
4
Nardelli-Haefliger D, Roden R, Balmelli C, et al. Mucosal but not
parenteral immunization with purified human papillomavirus type 16
virus-like particles induces neutralizing titers of antibodies throughout
the estrous cycle of mice. J Virol 1999;73:9609-13.
5
Toka FN, Pack CD, Rouse BT. Molecular adjuvants for mucosal immunity. Immunol Rev 2004;199:100-12.
6
Tornesello ML, Duraturo ML, Botti G, et al. Prevalence of alphapapillomavirus genotypes in cervical squamous intraepithelial lesions
and invasive cervical carcinoma in the Italian population. J Med Virol
2006;78:1663-72.
7
Barzon L, Giorgi C, Buonaguro FM, et al. Guidelines of the Italian society for virology on HPV testing and vaccination for cervical cancer
prevention. Infect Agent Cancer 2008;3:14.
8
Buonaguro FM, Tornesello ML, Buonaguro L. New adjuvants in
evolving vaccine strategies. Expert Opin Biol Ther 2011;11:827-32.
What evolution for the Pap Smear?
L. Viberti
Struttura Complessa Anatomia Patologica Ospedali Martini e Valdese di Torino, ASLTO1
In recent years, the role of cervicovaginal cytology as the
primary test for the cervical screening has been matter of discussion because of the higher sensitivity of the new molecular
HPV-tests.
Although the HPV test is able to determine the presence of
a viral infection of the cervical epithelium, it cannot distinguish between a transitory infection and a already developed
pre-malignant or malignant lesions. Thus, the positivity of
molecular tests in the context of primary screening imply the
need for further diagnostic investigations.
In this context, the morphology may provide new values,
which may be useful for the management of patients with a
known HPV infection.
Therefore, we must consider and answer many important
questions:
Can we assert that in this setting the problem of false negative
cytological reports is solved?
Is the distinction between a diagnostic and a screening pap
smear meaningful?
Do we need a new training program for cytologists involved
in second-level cytology?
Are the diagnostic categories as defined in Bethesda System
still applicable if the Pap smear becomes a second-level diagnostic procedure?
Would it be reasonable to avoid reporting morphological abnormalities that probably do not lead to a clinically relevant
diagnosis?
How would in this setting a doubtful report affect the patient’s
management?
The debate with experts is opened and some solutions will be
proposed and discussed.
Quale evoluzione per il Pap Test oggi.
Il punto di vista del patologo molecolare
G. Giuffrè
Dipartimento di Patologia Umana, Università di Messina
Cervical cancer is the second most frequent malignant neoplasia
among women worldwide and human papillomavirus (HPV)
infection is the necessary cause for its development. Cervical
HPV infection is a common sexually transmitted disease and
most women are infected shortly after beginning their first sexual
relationship, with the highest prevalence seen in women under 25
years of age. HPV infections are usually transient and the concurrent or sequential detection of different HPV types represents a
common event. However, only a very small fraction of infected
women develop cervical cancer and its immediate precursors.
Cervical cancer is characterized by a well-defined pre-malignant
phase that can be suspected on cytological examination of
exfoliated cervical cells (Pap test) and successively confirmed
by histology in colposcopically obtained cervical biopsy. Premalignant changes represent a spectrum of cytological abnormalities ranging from low-grade squamous intraepithelial lesion
(LSIL) to high-grade squamous intraepithelial lesion (HSIL);
however, 70-80% of LSIL regress spontaneously as well as
a consistent percentage of HSIL, specially in young women.
Therefore, cytological and histological examination cannot
reliably distinguish the few women with abnormal smears who
will progress to invasive cancer from the vast majority of those
whose abnormalities will spontaneously regress. However, Pap
test based screening programs in developed countries has led to
a substantial reduction in the incidence of cervical cancer.
In the last twelve years, the recognized importance of highrisk HPV infection in the onset of cervical cancer has introduced a role for HPV-DNA testing in cervical screening
programmes. Combining HPV testing with primary cytology
has been demonstrated to be useful in the triage of minor cytological abnormalities (ASC-US) in order to select women to
send to colposcopy. In addition, the high negative predictive
value of HPV-DNA test has hallowed to increase intervals
between screening rounds.
Several studies have shown that HPV testing is more sensitive
but less specific than cytology for detecting SIL as well as
invasive cancer. Therefore, HPV-DNA test has been proposed
as primary cervical screening test, while Pap test has been
utilized in the triage of positive cases and some controlled
trials have supported this new screening approach. Recently,
the National Comprehensive Cancer Network has suggested
the combination of cytology plus HPV DNA testing as an appropriate screening test for early detection of cervical cancer
in women older than 30 years.
HPV screening is usually performed using Hybrid Capture 2 DNA
test, a molecular assay based on hybridization of RNA probes
with viral DNA, that allows to diagnose an high- or low-risk HPV
infection, without the identification of a specific genotype.
However, persistent infection with a high-risk HPV is the
86
most important risk factor for cervical cancer, so to understand genotype-specific HPV persistence is essential for
elucidating the natural history of HPV infection. Utilizing
PCR-based genotyping procedures, association between persistence of specific high-risk HPV infection and high absolute
risk for progression to high-grade cervical lesions has been
documented. Therefore, HPV genotyping could play a role
in cervical cancer screening, especially in women affected
by SIL. Moreover, HPV genotyping provides information in
HPV circulation in different geographic area and this aspect
may represent an important indication in the development of
new vaccination strategies.
Aula Orione – ore 9.00-10.30
Linee guida e coni d’ombra in patologia gastrointestinale
Moderatore: Roberto Fiocca (Genova)
MicroRNAs dysregulation in gastrointestinal
tumors
M. Fassan*,**, M. Rugge*,***
Department of Medical Diagnostic Sciences & Special Therapies,
Surgical Pathology & Cytopathology Unit, University of Padova,
Padova-Italy; ** Department of Oncological and Surgical Sciences;
General Oncology Unit, University of Padova, Padova-Italy; *** Istituto Oncologico del Veneto - IOV-IRCCS, Padova, Italy
*
In the last two decades, no biomarker has generated the excitement that has accompanied the interest in the potential in
human carcinogenesis of microRNAs (or miRNAs).
miRNAs are a class of endogenous small non-coding RNAs
that modulate gene expression by causing either target mRNA degradation or translation inhibition. Since their initial
discovery in Caenorhabditis elegans in 1993, an enormous
body of research has been published supporting miRNAs
biological function as critical in most cellular processes. In
humans, aberrant miRNAs expression is a hallmark of different diseases, including cancer. In contrast, due to their
molecular structure, to most mRNAs, miRNAs are longlived in vivo and very stable in vitro. This uniqueness is
indispensable in allowing the analysis of FF-PE samples,
underlying a potential central role for miRNAs in the molecular study of preneoplastic lesions.
Several reports have already demonstrated the excellent reproducibility and accuracy of miRNA expression profiling
in archived FF-PE specimens and, in gastroenterology as in
other fields of human pathology, integrating genome-wide
profiling with the functional characterization of miRNAs
(their over-expression or down-regulation) and the identification of miRNA-specific gene targets currently represents the
approach most likely to yield advances in the new field of
non-coding RNA research.
In FF-PE specimens, miRNA expression could also be
visualized at cellular/sub-cellular level (in situ hybridization)
and this particular characteristic makes miRNAs potentially
suitable for supporting routine diagnostic surgical pathology
practice.
Aberrant miRNA expression signatures have been extensively investigated in GI diseases and several key oncogenic
miRNAs have consistently been found dysregulated. In some
cases, specific miRNA expressions have been linked to
cancer-associated pathways, indicating a role for them in GI
carcinogenesis. The systematic molecular evaluation of the GI
mucosa (always supported by the advanced histological and
clinical characterization of the specimens) not only provides
new basic biological information, but can also pave the way
to risk-stratified patient management programs and innovative
therapeutic measures.
In fact, miRNAs and miRNA-related gene expression and
polymorphism have a central role in determining individual
(patient-specific) cancer susceptibility and cancer progression. The upcoming challenge lies in the reliable identification
of disease-specific targets of dysregulated miRNAs, to enable
miRNA testing in clinical practice. The miRNA revolution is
only just beginning!
Selected references
Baffa R, Fassan M, Volinia S, et al. MicroRNA expression profiling of
human metastatic cancers identifies cancer gene targets. J Pathol
2009;219:214-21.
Croce CM. Causes and consequences of microRNA dysregulation in cancer. Nat Rev Genet 2009;10:704-14.
Dong Y, Wu WK, Wu CW, et al. MicroRNA dysregulation in colorectal
cancer: a clinical perspective. Br J Cancer 2011;104:893-8.
Faber C, Kirchner T, Hlubek F. The impact of microRNAs on colorectal
cancer. Virchows Arch 2009;454:359-67.
Fassan M, Volinia S, Palatini J, et al. MicroRNA expression profiling in
human Barrett’s carcinogenesis. Int J Cancer 2011;129:1661-70.
Gruppo Italiano Patologi Apparato Digerente (GIPAD); Società Italiana
di Anatomia Patologica e Citopatologia Diagnostica/International
Academy of Pathology, Italian division (SIAPEC/IAP). Dig Liver Dis
2011;43 Suppl 4.
Iorio MV, Croce CM. MicroRNAs in cancer: small molecules with a huge
impact. J Clin Oncol 2009;27:5848-56.
Kan T, Meltzer SJ. MicroRNAs in Barrett’s esophagus and esophageal
adenocarcinoma. Curr Opin Pharmacol 2009;9:727-32.
Lehmann U. MicroRNA-profiling in formalin-fixed paraffin-embedded
specimens. Methods Mol Biol 2010;667:113-25. [PMID: 20827530]
Matsushima K, Isomoto H, Inoue N, et al. MicroRNA signatures in Helicobacter pylori-infected gastric mucosa. Int J Cancer 2011;128:361-70.
O’Hara SP, Mott JL, Splinter PL, et al. MicroRNAs: key modulators of
posttranscriptional gene expression. Gastroenterology 2009;136:1725.
Petrocca F, Visone R, Onelli MR, et al. E2F1-regulated microRNAs
impair TGFbeta-dependent cell-cycle arrest and apoptosis in gastric
cancer. Cancer Cell 2008;13:272-86.
Tsujiura M, Ichikawa D, Komatsu S, et al. Circulating microRNAs in plasma of patients with gastric cancers. Br J Cancer 2010;102:1174-9.
Visone R, Petrocca F, Croce CM. Micro-RNAs in gastrointestinal and
liver disease. Gastroenterology 2008;135:1866-9.
Wu F, Zhang S, Dassopoulos T, et al. Identification of microRNAs associated with ileal and colonic Crohn’s disease. Inflamm Bowel Dis
2010;16:1729-38.
Wu WK, Lee CW, Cho CH, et al. MicroRNA dysregulation in gastric
cancer: a new player enters the game. Oncogene 2010;29:5761-71.
Wu WK, Law PT, Lee CW, et al. MicroRNA in colorectal cancer: from
benchtop to bedside. Carcinogenesis 2011;32:247-53.
87
relazioni
Problems of pathological staging in colorectal
cancer
P. Greco
Azienda Ospedaliero-Universitaria “Policlinico- Vittorio Emanuele”,
Catania
There are two critical issues with a strong impact on the
prognosis of colorectal cancer that are still controversial: i)
the number of lymph nodes to be recovered for an adeguate
staging; and ii) the classification of tumor deposits (TDs).
Regarding the number of lymph nodes to be recovered for an
adeguate staging it should be stressed that an ideal number
does not exist, being only a statistical concept that cannot
be applied to each single patient. The tumor-node-metastasis
(TNM) system recommends that at least 12 lymph nodes is
a sufficient number for an accurate identification of regional
metastases. In a prospective study, we first compared the
mean number of lymph nodes recovered in the same surgical
specimen at the routine sampling and at a resampling performed by a second expert gastrointestinal pathologist. This
study was performed on 50 cases of pT2N0 and pT3N0 rectal
cancer with a minimum number of 12 lymph nodes recovered
at first sampling, histologically negative for metastases. Resampling retrieved a variable number (1 to 24) of nodes missed
at first sampling.
The final pN0 status was maintained in the pT2 patients,
whereas in 18,7% of pT3 patients, metastatic lymph
nodes were detected if the mean number of lymph nodes
increased from 17.8 to 26.8 after the second sampling. As
we have shown that most missed metastatic lymph nodes
were detected in specimens in which a maximum number
of 19 lymph nodes had been originally recovered, we
strongly suggest a resampling of pT3N0 rectal specimens
if less than 20 lymph nodes have been recovered. However, as the number of lymph nodes recovered in a single
surgical specimen greatly varies and largely depends on
multiple factors, the recovery of only a few lymph nodes
in a single surgical specimen does not necessarily imply
that sampling has been insufficient provided that the pathologist has a good experience in the manual dissection
of lymph nodes from the pericolonic/perirectal adipose
tissue.
Tumor deposits (TDs) are macroscopic and/or microscopic
metastatic foci of variable size within pericolorectal fibroadipose tissue, which are not associated with a recognizable
lymph node structure and are not contiguous with the mural
component of invasive carcinoma. Change of classification
of TDs in different editions of TNM creates poor reproducibility and stage migration. TDs >3 mm are classified as
regional lymph nodes metastases in TNM5, whereas if their
size is ≤ 3 mm they are regarded as discontinous T3 adenocarcinoma.
In the TNM6, TDs are viewed as lymph nodes metastases
or venous invasion if they exhibit the form and smooth contours of a lymph node or an irregular contour, respectively.
We believe that neither TNM5 nor TNM6 is accurate in
classification of TDs. In TNM5 the creation of the 3-mm
rule was not based on trial or survival data and in TNM6
the definition of a rounded versus irregulary shaped tumor
deposit is quite arbitrary and poor reproducible. In TNM7
TDs are classified as N1c in otherwise node-negative cases
of T1-T2 colorectal cancer, similary with TNM staging of
skin melanoma. However, there is no cancer, but the final interpretation of the lesions is left to the individual pathologist,
essentially reverting to the TNM4 staging, although recent
data have shown that the best prognostic model predictive
of survival outcome is obtained by including all TDs in the
N-stage, regardless of T-stage size and shape, except for
TDs defined as “ vascular type” or intravascular TDs, which
would be better classified as extramural vascular invasion.
Also, there is still no general consensus about classification
of residual tumor foci that are morphologically similar to
TDs in patients undergoing neoadjuvant therapy for rectal
cancer. In daily practice, we believe that TDs should be included in the pathology report, specifying their total number,
size (the largest diameter if there are multiple lesions), and
association, or not, with large vessels and /or nerves, in order
to create more homogeneous groups of patients for enrolment in clinical trials.
Tavola Rotonda
Necessità di codifica ed esperienze locali
Moderatore: Domenico Ientile (Palermo)
Nomenclatore sistematico per l’Anatomia
Patologica in Italia (NAP Italia)
A. Bondi, P. Crucitti
Anatomia Patologica Ospedale Maggiore – A. USL Bologna
Tutti i software per la gestione delle prestazioni di Anatomia
Patologica prevedono un nomenclatore sistematico per la codifica diagnostica. Alcuni programmi impiegano i codici solo
per la rappresentazione simbolica delle diagnosi una volta
completato l’esame, altri utilizzano le informazioni codificate
per indirizzare le varie fasi di lavorazione 1-3.
Il sistema di codifica diagnostica più diffuso nel mondo è
l’ICD (Classificazione Internazionale delle Malattie e delle
Cause di Morte) adottato dall’Organizzazione Mondiale della
Salute (WHO) che ne promuove la traduzione, la diffusione
e gli aggiornamenti periodici. Per usi di sanità pubblica, e
comunque senza fini di lucro, il codice è liberamente utilizzabile. Il Servizio Sanitario Nazionale Italiano ha ufficialmente
adottato l’ICD per la raccolta e l’analisi dei dati epidemiologici relativi allo stato della salute degli Italiani, per programmazione ed analisi sanitarie.
L’ICD, anche nelle varianti adattate per uso clinico (CM,
Clinical Modifications) 4, è però difficilmente applicabile alla
88
casistica pratica di Anatomia Patologica, dove sono necessari
dettagli non inclusi in questa classificazione. Il sistema di
codifica più diffuso fra i Patologi è lo SNOMED (Nomenclatore Sistematico della Medicina) che deriva dal Nomenclatore
Sistematico di Patologia (SNOP), messo a punto da Coté nel
1965 5 6.
La semiotica (rappresentazione simbolica dei concetti) usata
nello SNOP, che si basa sulla scomposizione nei termini
elementari che costituiscono un’informazione, è risultata innovativa ed efficace, tanto che è stata mutuata nell’ICD per
scorporare le malattie tumorali e realizzare l’ICD-O con le
“regole” SNOP che hanno profondamente modificato il sistema monodimensionale di ICD.
La prima versione dell’ICD-O è stata tradotta in lingua italiana
da F. Rilke 7, la terza versione, ICD-O 3, è disponibile in italiano grazie all’impegno di S. Ferretti ed altri 8. Con l’ultima
edizione di ICD-O si è realizzato un ottimo aggiornamento dei
termini ed una perfetta coincidenza tra SNOMED CT 2002 e
ICD‑O 3, ovviamente solo per le morfologie tumorali.
Dallo SNOMED International (seconda edizione) è stato
estratto il raggruppamento dei codici usati più frequentemente dai Patologi ed è stato pubblicato il “Microglossary
for Pathology” 9. Di questo ne esiste una versione in lingua
italiana 10, realizzata col patrocinio di SIAPEC (Società Italiana di Anatomia Patologica e Citodiagnostica). Purtroppo
il College of American Pathologists (CAP), titolare del
copyright di SNOMED aveva affidato la distribuzione nel
nostro Paese ad una casa editrice che ha interrotto le attività
e qualunque forma di vendita e assistenza per lo SNOMED
in italiano.
Il microglossario è un estratto non esaustivo del nomenclatore generale e talora ai Patologi mancano termini per
codificare nuove entità o casistiche complesse. Per sopperire
alla mancanza di una uniforme strategia di distribuzione e di
manutenzione, i Patologi Italiani lo hanno adattato il microglossario alle diverse realtà locali. Si sono così prodotti dei
nomenclatori parziali, dialetti codificati fra loro assolutamente
incompatibili e disallineati.
Nel 2006 SIAPEC ha promosso un gruppo di lavoro con l’incarico di uniformare le molteplici versioni dei nomenclatori in
uso in Italia e di realizzare una struttura in grado di manutenere, aggiornare e distribuire le tabelle dei codici diagnostici.
Hanno aderito al progetto anche AIRTum, la Società Scientifica degli Epidemiologi che si occupano dei Registri Tumori,
il Centro Collaboratore Italiano del WHO per le Codifiche
Sanitarie (CC-WHO) ed alcune Agenzie Sanitarie Regionali
(Friuli Venezia Giulia e Liguria, con disponibilità di Emilia
Romagna e Lombardia).
Nel frattempo il CAP aveva raggiunto un accordo con la National Library of Medicine (un’agenzia del National Institutes
of Health del governo statunitense) per permettere l’uso dello
SNOMED CT nell’ambito delle Università Americane e dei
sistemi di raccolta dati legati alle varie Agenzie Governative per la Salute; accordi di collaborazione erano già stati
realizzati anche con il Servizio Sanitario Nazionale Inglese
(NHS). Nel giugno 2006 i rappresentanti dei governi di USA,
Australia, Canada, Danimarca, Lituania e Gran Bretagna
hanno promosso la formazione dell’IHTSDO (International
Standards Development Organization) una organizzazione
senza fini di lucro, che ha ottenuto dal CAP i diritti di distribuzione di SNOMED RT: è in via di definizione un accordo
fra questa nuova organizzazione e il WHO per studiare le
possibilità di convergenza fra SNOMED ed ICD. Nell’aprile
2009 l’IHTSDO ha perfezionato un accordo con Healt Level
Seven (HL7 Inc.) la principale agenzia internazionale di stan-
dardizzazione per l’informatica in sanità, per una completa
compatibilità fra SNOMED CT ed HL7 11.
Il gruppo di realizzazione del NAP non intende perdere di
vista lo sviluppo internazionale del settore: nello sforzo di
uniformare il sistema di codifica per l’Anatomia Patologica Italiana si manterranno le compatibilità con SNOMED,
ICD-O, ed ICD e si consolideranno i rapporti col CC-WHO
con l’obiettivo di convergere verso una piena compatibilità
con gli standard IHTSDO.
Bibliografia
1
Bondi A, Ghidoni D, Spagnuolo A. Codifiche internazionali in medicina: lo SNOMED. In: Bondi A, Facibeni G, eds. Informatica in
Anatomia Patologica. Ravenna: Celcoop 1987, pp. 24-37.
2
Pierri C, Bubani C, Zani J, et al. Correlazioni cito-istologiche automatiche, fra codici SNOMED, per verifica della accuratezza diagnostica. Pathologica 2000;92;473.
3
Bondi A. La codifica delle diagnosi: una complicazione legata all’uso
del computer. Pathologica 1996;88:69-72.
4
Levitsky S. Using ICD-9-CM and CPT in the nineties. Ann Thorac
Surg 1990;50:519-20.
5
College of American Pathologists. SNOP - Systematized Nomenclature of Pathology. Ed 1. Skokie, Ill., USA: CAP 1965.
6
Cote’ RA. The SNOP-SNOMED concept: evolution towards a common
medical nomenclature and classification. Pathologist 1977;31:383-98.
7
ICD-O. Classificazione Internazionale delle Malattie per Oncologia.
Edizione italiana della prima edizione inglese del 1976 - Traduzione a
cura di F. Rilke. Milano, Edi Ermes, 1983.
8
Giacomin A, Ferretti S. ICD-O Classificazione Internazionale delle
Malattie per l’Oncologia. Terza Edizione. Traduzione Italiana di
“International Diseases Classification for Oncology” (C) WHO 2000.
Milano: Inferenze Scarl 2005.
9
Rothwell DJ, Cote’ RA, Brochu L. Microglossary for pathology The systematized nomenclature of human and veterinary medicine
SNOMED International. Northfield, IL, USA: College of American
Pathologists 1996.
10
Bondi A, Nesti P, Rossi Mori A. SNOMED International. Microglossario di patologia (Lingua italiana). Ed 1. Udine: Pubblicazioni
Medico Scientifiche, 1995.
11
HL7 and IHTSDO Sign Agreement: http://www.ihtsdo.org/fileadmin/user_upload/ Docs_01/News/HL7_IHTSDO_agreement_release_
final__2_.pdf. 2009.
Il “minimum dataset diagnostico” in Anatomia
Patologica
S. Ferretti
Associazione Italiana Registri Tumori
Il referto diagnostico anatomo-patologico ha negli anni progressivamente ampliato la sua valenza epidemiologica, per la
sempre maggiore ricchezza di informazioni contenute e per
l’importanza di queste ultime (stadiazione, caratterizzazione
biopatologica) nella impostazione delle strategie terapeutiche
rivolte al paziente.
Il ruolo del patologo, oltre a dirimere i singoli quesiti diagnostici, ha così via via assunto un ruolo chiave anche in una logica di Sanità pubblica, nella gestione dei percorsi assistenziali
e nelle loro valutazioni di impatto.
Rispetto ad altre serie di dati sanitari che i flussi informativi
comprendono ormai da alcuni decenni (scheda nosologica
individuale, ambulatoriale, farmaceutica etc.), l’enorme ricchezza della diagnosi anatomopatologica, non contenuta in
nessun altra fonte corrente, si trova così spesso relegata all’interno di sistemi informativi locali, destinati più che altro alla
gestione diagnostica di un’Unità Operativa, senza la possibilità delle molteplici utilizzazioni che potrebbero avvalersene
con enormi vantaggi come ad esempio:
ricostruzione dei percorsi diagnostici di un Paziente indipendentemente dalla U.O. di diagnosi;
89
relazioni
disponibilità allargata di dati di terapia chirurgica, stadiazione
e caratterizzazione dei tumori, utile alla ricostruzione e verifica dei percorsi assistenziali;
disponibilità di dati diagnostici relativi ad indagini con esito
negativo rispetto a sospetti diagnostici presenti in altra documentazione (es. SDO), con possibile risoluzione di casi “falsi
positivi”;
disponibilità di dati diagnostici per la ricostruzione di comorbidità o percorsi diagnostici a bassa tracciabilità da parte
dei sistemi informativi correnti (tumori bilaterali e multipli,
malattie non oncologiche o a scarso ricorso di ospedalizzazione);
disponibilità di caratterizzazioni biologiche complesse.
Al momento l’attività di recupero e collegamento delle informazioni relativa ai tumori, è condotta dai Registri tumori di
popolazione attraverso l’integrazione dei sistemi informativi
sanitari correnti (SDO, ASA, Mortalità etc.), prevedendo per
l’Anatomia Patologica un’attività di consultazione manuale
o semi-automatica molto gravosa, spesso limitata alla sede
territoriale del Registro (per l’assenza di collegamenti informativi tra le varie UU.OO.) e alle diagnosi oncologiche, per
l’impossibilità di indagare nel dettaglio enormi moli di dati
riguardanti ad es. le lesioni preneoplastiche, la diagnostica
citopatologica o le anamnesi remote precedenti la diagnosi
tumorale.
Da ciò, proprio per il valore imprescindibile di questo livello
diagnositico, deriva in gran parte il ritardo nella produzione
dei dati di incidenza da parte dei Registri Tumori, che non
hanno a disposizione per l’Anatomia Patologia flussi informatizzati e standardizzati per la consultazione in remoto
delle informazioni per ogni paziente, così come avviene, con
limitazioni proprie di ogni sistema, per SDO e altre fonti
informative.
In questa logica prende avvio la proposta di un minimum data
set diagnostico anatomo-patologico, da condurre progressivamente al livello di informatizzazione e fruibilità proprio degli
altri flussi informativi sanitari correnti, con grandi ricadute
anche nel campo della patologia non oncologica e in tutto il
novero di approfondimenti e verifiche possibili (clinici, organizzativi, di Sanità pubblica) conseguenti all’integrazione di
questi dati con l’attuale corredo informativo.
Le caratteristiche fondamentali di questo minimun data set
devono comprendere l’essenzialità (non ridondanza con altri
sistemi informativi), l’accuratezza, l’universalità (uso delle
codifiche sanitarie e diagnostiche) e la trasparenza rispetto
alla pratica diagnostica (non introducendo ulteriori compiti
per il patologo diagnosta)
Notevoli strumenti ed esperienze propedeutiche sono già a disposizione, dal know-how dei Registri tumori (che dispongono
anche di strumenti informatici per la codifica automatica),
all’imponente impegno di SIAPEC nella stesura di un Nomenclatore diagnostico nazionale e nella promozione di sistemi di
codifica e di gestione informatica degli archivi in linea con i
più avanzati livelli tecnici.
La proposta di un tracciato-record particolarmente focalizzato
sulla patologia tumorale, attualmente oggetto di sperimentazioni regionali, prende le mosse dalle consolidate esperienze
del College of American Pathologists, proponendo tre livelli di
checklist informative, costituiti da un primo livello comprendente tutte le diagnosi anatomo-patologiche (istopatologiche,
citopatologiche, autoptiche, biotecnologiche) uniformate per
informazioni, nomenclatura e codici diagnostici, un secondo livello riguardante la caratterizzazione patologica e la stadiazione
dei principali tumori ed un terzo livello contenente variabili di
caratterizzazione biologica, ormai organiche alla diagnostica in
oncologia.
Patologia del fegato e delle vie biliari
Moderatore: Oscar Nappi (Napoli)
Il ruolo del patologo nella diagnosi delle
malattie biliari
L. Terracciano
Department of Pathology, University Hospital, Basel, Switzerland
Liver biopsy is helpful in the diagnosis of most cholestatic
liver diseases in infants as well as in adult patients
1. Features of cholestasis in general.
Such features include parenchymal changes and periportal /
architectural changes.
A. Parenchymal changes comprise bilirubinostasis (hepatocellular, canalicular and in Kufffer cells)in the early stage of
complete cholestasis; cholate stasis of periportal hepatocytes
in later stages represented by ballooning, expression of cytokeratin 7, coarse cytoplasmic granularity, accumulation of
copper and copper-binding protein, and development of Mallory bodies; cholestatic liver cell rosettes corresponding to
a switch of hepatocytes from normal plate arrangement into
tubular structures with or without bilirubin concrements in the
lumen; occurrence of single and clustered xanthomatous cells;
so-called feathery degeneration (resembling cholate stasis) in
intralobularhepatocytes; and in later stages of severe cholestasis: development of paraportal bile infarcts.
B. Periportal / architectural changes gradually develop in
later stages of chronic cholestatic conditions and comprise:
ductular reaction recognized as an increase in ductular structures, associated with oedema and neutrophil infiltration in the
periportal region; development of periductular fibrosis which,
together with wedge-like extension of the ductular reaction,
results in development of periportal septa that eventually connect adjacent portal tracts, thus creating the pattern of biliary
fibrosis and, with addition of nodular parenchymal regeneration, finally biliary cirrosis.
2. Features indicative of specific disorders
A. Primary Biliary Cirrhosis
For patients with typical biochemical and serological features of primary biliary cirrhosis (PBC), histological confirmation may not be required. However, liver
90
biopsy can still provide useful information about disease severity and ist important fort he diagnosis atypical cased (e.g. AMA-negative PBC, overlap syndromes)
B. Primary Sclerosing Cholangitis
For many cases of primary scerosing cholangitis (PSC),
a diagnosis can be made on the basis of cholangiographic
findings, without the need for histological confirmation.
Liver biopsy may be useful to diagnose cases with clinical or
radiological fetaures (e.g. small duct PSC) and to determine
disease stage. Histological and cytological assessments are
also important in diagnosing cholangiocarcinoma and precursor lesions complicating PSC
C. Drug Induced Liver Disease (DILI)
Drugs may cause several overlapping syndromes of cholestasis, the pathophysiological syndrome resulting from impaired
bile flow. These reactions comprise approximately 17% of all
hepatic adverse drug reactions (ADRs) and they may be severe. Causes of ‘pure’ (bland) cholestasis include oestrogens
and anabolic steroids; rarer associations are with antimicrobials and NSAIDs. ‘Cholestatic hepatitis’ is a common drug
reaction in which liver injury and inflammation cause significant elevation of serum alanine aminotransferase (ALT)
as well as cholestasis.
Chlorpromazine and ketoconazole are classic examples, but it
is now exemplified by amoxycillin-clavulanate and other oxypenicillins. Chronic cholestasis results from small bile duct
injury leading to the vanishing bile duct syndrome (VBDS),
a disorder mimicking primary biliary cirrhosis, or from injury
to larger bile ducts causing secondary sclerosing cholangitis.
Whilst there is increasing evidence of a genetic predisposition
to cholestatic drug reactions, there are currently no pretreatment tests to predict drug safety. Prevention of severe reactions therefore relies on early detection of liver injury and
prompt drug withdrawal.
La valutazione della biopsia epatica nel pre
e post trapianto
R. Gentile
Palermo
Valutazione della biopsia epatica in fase di pretrapianto.
La biopsia del donatore viene effettuata a giudizio dell’equipe chirurgica e dipende dalle condizioni organizzative del
Centro in cui avviene il prelievo o del Centro che eseguirà il
trapianto. Alcuni Centri Trapianto effettuano comunque un
prelievo bioptico di tutti gli organi scartati su base macroscopica. È comunque consigliabile eseguire la biopsia epatica in
tutti potenziali donatori che presentano un qualsiasi fattore di
marginalità:
• Steatosi ecografica in soggetti con BMI > 25
• Anamnesi positiva per abuso alcolico acuto o cronico
• Presenza di criteri diagnostici per sindrome metabolica.
La biopsia del donatore “marginale” è anche indicata per
definire i i comuni criteri di ccettazione/esclusione e il corretto matching donatore ricevente per valutare l’incidenza
di primary non function, delayed non function e sugli esiti a
lungo termine.
Per sopperire alla crescente necessità di organi e alla scarsa
disponibilità degli stessi sono stati ampliati i criteri per la
selezione degli organi, pertanto, l’Associazione Italiana per lo
Studio del Fegato (AISF) ha dettato i criteri per l’inclusione
di donatori non ottimali: soggetti >50 anni; permanenza in terapia intensiva > 5gg. e con instabilità emodinamica; presenza
di malnutrizione o di steatosi epatica macrovescicolare > 25%
o micro vescicolare > 60%; ipersodiemia; infezione da virus
epatitici (HCV+, HBcAb+, HBsAg+).
Il fegato viene definito marginale o non ottimale quando il
suo uso si associa ad un aumento significativo del rischio di
complicanze dopo trapianto. Le complicanze post trapianto
possono essere raggruppate in:
• Primary non-function(PNF) mancata ripresa funzionale
con necessità di ritrapianto in 3-7 gg.
• La Delayed Non Function (DFN), cioè le condizioni che
conducono a ritrapianto entro il primo mese.
Le complicanze dipendono dalle caratteristiche del donatore, del ricevente, dai tempi di ischemia, dall’utilizzo di un
organo marginale. Inoltre, poiché la DFN aumenta in modo
proporzionale all’aumento dei numero dei fattori di rischio,
la biopsia è consigliabile come parte della valutazione del
donatore. Il sistema risulta efficace e consente l’utilizzo di donatori marginali, con una percentuale di Primary non-function
accettabile (circa 3%).
Raccomandazioni tecniche: occorre
• Eseguire una biopsia cuneiforme di 1 cm. o agobiopsia di
cm. 2 di lunghezza, ma non sottocapsulare, viene sconsigliato l’uso di aghi sottili e sarebbe preferibile ottenere 2
agobiopsie da sedi differenti (lobo dx e sx).
• Trasportare immediatamente il campione in Anatomia
Patologica ponendolo su garza inumidita con il liquido di
preservazione.
• Eseguire l’esame intraoperatorio, colorare più sezioni con
E.E., (PAS)
• Valutare semiquantitativamente la steatosi, espressa in %
di tessuto esaminato, separando la steatosi macro e la microvescicolare.
Il Patologo esaminatore deve avere una preparazione specifica
per patologia epatica e patologia del trapianto e deve essere
informato su dati clinici del donatore, caratteristiche macroscopiche dell’organo o delle lesioni campionate e dei quesiti
specifici.
Valutazione della biopsia epatica di donatore vivente (5%)
La valutazione deve essere medica e chirurgica (screening per
malattie gravi, obesità, precedenti interventi chirurgici addominali, compatibilità anatomica, malattie infettive, instabilità
psicosociale, alterazioni della funzionalità epatica, o malattie
che potrebbero mettere a rischio il donatore). Poiché la procedura chirurgica è a rischio per il donatore (mortalità circa
2/700) la biopsia può contribuire a rafforzare o minimizzare
il rischio della donazione. Il 20-50% delle biopsie presentano
anormalità (soprattutto NAFLD).
Una % di steatosi macrovescicolare > 30% può esclude il
donatore o prevedere una dieta o terapia a cui seguirà biopsia
di follow up.
La biopsia può evidenziare epatiti lievi ad eziologia indeterminata, granulomi, patologie epatiche inaspettate, depositi di
ferro.
Valutazione della biopsia epatica nel post trapianto
• Determinazione della causa di disfunzione epatica
• Esaminare lo status immunologico e architetturale epatico
• Valutazione dell’effetto della terapia e/o progressione della
malattia di base.
Considerazioni tecnico/diagnostiche: la biopsia post trapianto
viene allestita con processazione di routine, allestendo 2 vetrini con 2-4 sezioni colorate con E.E., Reticolo, PAS, PAS-D,
Tricromica, Perls, (eventualmente rame e CK 7 e CK19).
Al momento della biopsia il Patologo deve conoscere la
tipologia e la tempistica dell’intervento di trapianto, infatti
le procedure chirurgiche alternative e le manipolazioni chirurgiche del fegato del donatore prima del trapianto (split
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liver, riduzione della grandezza, fegato da donatore vivente)
aumentano il rischio di complicanze vascolari e biliari. Inoltre
deve conoscere la malattia di base, gli esami di laboratorio e la
diagnosi clinica. Sarebbe auspicabile sempre una discussione
clinico-patologica prima della refertazione, comparando eventuali biopsie precedenti.
Le patologie post trapianto sono:
• Preservation/reperfusion injury
• Rigetto anticorpo mediato
• Rigetto celulare acuto
• Rigetto cronico
• Trombosi dell’arteria epatica
• Stenosi o ostruzioni delle vie biliari
• Ostruzione del deflusso venoso
• Infezioni opportunistiche (virali o fungine)
• Epatite virale ricorrente o de novo
• Recidiva di epatite autoimmune, cirrosi biliare primitiva e
colangite primitiva sclerosante
• Abuso di alcool
• Steatoepatite non alcolica
La preservation/reperfusion injury è un danno dell’organo
donato che si verifica durante la fase agonica del donatore, il
periodo di conservazione (preservation solution) e dopo riperfusione del fegato nel ricevente.
La diagnosi di preservation/reperfusion injury deve avvenire
dopo esclusione di danno vascolare (trombosi arteriosa o
venosa), rigetto, reazione avversa a farmaci, esposizioni a
tossine, infezioni. I fattori contribuenti sono: ipotensione del
donatore e del ricevente, ischemia calda, anormalità metaboliche, ischemia fredda, danno da riperfusione.
Nel danno lieve si osserva steatosi microvescicolare, rigonfiamento citoplasmatico e aggregazione epatocitaria. Nel danno
severo: necrosi confluente, a distribuzione zonale, infiltrati
neutrofili, necrosi di singoli epatociti con architettura reticolinica intatta.
Possibili errori interpretativi potrebbero essere dovuti alla sede della biopsia (il parenchima sottocapsulare è maggiormente
vulnerabile al danno) e alla cosiddetta epatite chirurgica (neutrofili sinusoidali e intorno alla vena centrolobulare).
I processi riparativi sono caratterizzati da mitosi, aumento di
volume dei nuclei, ispessimento delle trabecole, rigonfiamento citoplasmatico e colestasi (epatocitaria e canalicolare) in
aree centro lobulari.
La necrosi confluente e periportale può esitare in neoformazione colangiolare, con formazione di ponti e distorsione
dell’architettura.
Rigetto cellulare acuto Viene definito come “infiammazione
del trapianto suscitata da una disparità genetica tra donatore e
ricevente” che interessa per primi i dotti biliari e gli endoteli
vascolari (vene epatiche e portali).
Istologicamente è caratterizzato da: A) Infiammazione portale: 1 (infiammazione prevalentemente linfocitaria, che interessa una minoranza di spazi portali, senza peraltro ingrandirli).
2 (espansione della maggior parte o di tutti gli spazi portali,
con infiltrato misto, contenente linfociti, blasti occasionali,
neutrofili, eosinofili) 3 (notevole espansione della maggior
parte o di tutti gli spazi portali, con presenza di numerosi
blasti e eosinofili nell’infiltrato; presenza di spillover nella
zona periportale).
B)Infiammazione e lesioni dei dotti biliari: 1 (solo una parte
minoritaria dei dotti sono circondati e permeati da cellule
infiammatorie, con presenza di lievi alterazioni, rappresentate
prevalentemente da incrementato rapporto nucleo/citoplasmatico delle cellule epiteliali) 2 (la maggior parte o tutti i dotti
sono infiltrati da cellule infiammatorie; presenza di frequenti
alterazioni degenerative delle cellule epiteliali, rappresentate
da pleomorfismo nucleare, vacuolizzazione citoplasmatica
alterazioni della polarità) 3 (come il grado 2, ma con presenza
delle alterazioni degenerative in quasi tutti i dotti e focali distruzioni dell’epitelio).
C)Infiammazione dell’endotelio venoso: 1(Infiammazione
sub endoteliale linfocitaria che interessa solo una parte non
maggioritaria delle venule portali o epatiche) 2 (Infiammazione sub endoteliale che interessa la maggior parte o tutte
le venule portali o epatiche) 3 (come nel grado 2, ma con
infiammazione che si espande nell’area periportale, con focale
necrosi epatocitaria periportale).
Graduazione del grado complessivo del rigetto: /9 (Valutazione secondo RAI (Rejection Activity Index) 1
Il rigetto cronico è un danno immunologico che evolve da
rigetti acuti gravi e/o persistenti, e risulta in un potenzialmente irreversibile danno dei dotti biliari, arterie e vene. Insorge
dopo parecchi mesi dal trapianto e può portare all’insuccesso
del trapianto anche nel giro di un anno. È più frequente in
pazienti “non compliant”, riceventi HCV+ trattati con alfa interferone e riceventi che hanno un basso dosaggio di immunosoppressione a causa di Post Transplant Lymphoproliferative
Disorder. I fattori di rischio possono essere raggruppati in due
grandi categorie: Immunologici e non immunologici.
Secondo lo Schema Banff il rigetto cronico può essere classificato in:
Fase iniziale: A) Lieve flogosi nello spazio portale (linfociti,
plasmacellule e mast cell); lieve colangite linfocitaria, perdita
dei dotti biliari in < 50% degli spazi portali; modificazioni degenerative degli epiteli duttali della maggioranza dei dotti. B)
infiammazione subendoteliale e luminale delle vene centrolobulari, lieve necrosi litica della zona 3, accumulo di macrofagi
con pigmento, lieve fibrosi perivenulare. C) occasionale perdita <25% delle arterie epatiche; spotty necrosi degli epatociti.
D) arteria epatica (periilare) infiammazione dell’intima, depositi di macrofagi schiumosi senza compromissione del lume.
E) Dotti biliari periilari con infiammazione, danno epiteli e
deposito di macrofagi schiumosi.
Fase tardiva: A) perdita dei dotti biliari in > 50% degli spazi
portali; modificazioni degenerative degli epiteli duttali di
tutti i dotti. B) infiammazione intimale e luminale delle vene
centrolobulari, focale obliterazione dei lumi, fibrosi a ponte
centro-centrale o porto-centrale, severa fibrosi perivenulare.
C) perdita >25% delle arterie epatiche; accumulo di cellule schiumose nei sinusoidi, marcata colestasi. D) arteria
epatica (periilare) restringimento del lume per iperplasia
fibromiointimale e depositi di macrofagi schiumosi. E) Dotti
biliari periilari con fibrosi della parete. La diagnosi finale
di rigetto cronico dovrebbe comunque essere basata su una
combinazione di dati clinici, radiologici, di laboratorio ed
istopatologici.
Inoltre si raccomandano le riunioni clinico-patologiche periodiche per avere feed-back con clinici e controllo di qualità.
Bibliografia
1
Banff Schema for Grading Liver Allograft Rejection: An International
Consensus Document. Hepatology 1997;25:658-63.
Tumori epatici delle vie biliari, classificazione
WHO, diagnosi
L. Tornillo
Istituto di Patologia, Università di Basilea
Il termine “colangiocarcinoma” può essere applicato a qualsiasi tumore epiteliale con differenziazione ghiandolare origi-
92
nantesi dai dotti biliari. Esso si riferisce a due differenti entità
clinico-patologiche:
Colangiocarcinoma intraepatico, che prende origine dai dotti
biliari intraepatici, sia interlobulari che dai rami maggiori.
Colangiocarcinoma extraepatico, che prende origine dai dotti
epatici, dal coledoco o dalla colecisti.
Si tratta di tumori relativamente rari. Tuttavia, per quanto riguarda il colangiocarcinoma intraepatico, l’incidenza dipende
dalla collocazione geografica, con un massimo nel Sud-Est
asiatico (Laos, Thailandia). Fattori eziologici noti sono:
• L’infestazione da Clonorchis sinensis e da Opistorchis
viverrini, endemiche in Asia;
• Epatolitiasi, piuttosto frequente in Oriente, e colelitiasi;
• Colite ulcerosa e colangite sclerosante primitiva;
• Giunzione coledoco-pancreatica anormale;
• Cisti del coledoco;
• Cirrosi;
• Infezione da HCV;
• Diabete mellito.
Il colangiocarcinoma intraepatico si presenta macroscopicamente come una massa intraepatica solida, di colorito biancastro, con necrosi e/o cicatrice centrale, talvolta con crescita
intraduttale polipoide. Il colangiocarcinoma extraepatico al
contario può avere differenti aspetti: nodulare, polipoide,
scirroso e infiltrativo diffuso.
L’istologia è rappresentata nella stragrande maggioranza dei
casi da adenocarcinomi “classici”. Altre forme sono il carcinoma squamoso e adenosquamoso, il carcinoma colangiolocellulare, l’adenocarcinoma mucinoso e quello “signet”. Rari
sono i carcinomi a cellule chiare.
Nonostante le somiglianze istologiche (si tratta di adenocarcinomi originantisi da epiteli morfologicamente identici), i colangiocarcinomi intraepatici e quelli extraepatici
differiscono sia nel profilo immuofenotipico che in quello
molecolare. Un ulteriore problema è rappresentato dalla
differenziazione fra i colangiocarcinomi intraepatici e le
lesioni metastatiche. L’immunoistochimica è naturalmente
di aiuto nella diagnosi differenziale, che si basa sull’uso
delle citocheratine 7 e 20, delle mucine MUC1 e MUC2, di
markers di tipo emopoietico come il CD5 e il CD7, tipicamente positivi nel colangiocarcinoma e di markers di differenziazione tissutale come CDX2, TTF1, Mammoglobina,
Hep-Par, GCDFP-15, Tireoglobulina. Naturalmente esistono
dei (per fortuna rari) casi in cui la diagnosi differenziale è
virtualmente impossibile.
Un’altra importante (e spesso difficilissima) diagnosi differenziale è quella fra dotti biliari con atipie reattive e dotti
francamente neoplastici. In questi casi può essere di aiuto la
tecnica FISH. I dotti neoplastici sono caratterizzati da aneuploidie che possono essere facilmente rivelate con test multicolor (per esempio Urovysion®).
Esistono almeno due tipi di lesioni precursore:
Neoplasia intraepiteliale biliare (BIIN), ulteriormente suddivisa in BIIN-1, BIIN-2, BIIN-3, in base al grado di atipia
citologica
Neoplasia intraductale papillare (IPN), caratterizzata da dilatazione dei dotti biliari per la presenza di una proliferazione
papillare e /o villosa. In un terzo dei casi è presente secrezione
di muco. A parte la differenziazione fenotipica (pancreaticobiliare, oncocitica, intestinale e gastrica), simile a quella dei
precursori dell’adenocarcinoma pancreatico, è importante
distinguere il grado di atipia. Il sistema utilizzato è in tre
gradi (basso, intermedio e alto grado), analogamente a quanto
avviene per le neoplasie pancreatiche.
Noi abbiamo studiato una serie di 128 colangiocarcinomi
(42 intraepatici, 70 extraepatici e 16 carcinomi della colecisti) mediante la tecnica dei “tissue microarray”. La nostra
atttenzione si è concentrata sulle proteine di controllo del
ciclo cellulare (p16, p21. p27, p53), dell’’apoptosi (bcl-2 e
bax) e sul COX-2, di cui abbiamo determinato l’espressione immunoistochimica. In confronto con i tessuti normali,
abbiamo osservato livelli più elevati di COX-2, p53, bcl-2
e bax nei colangiocarcinomi. Bcl-2 e p16 erano poi più frequentemente espressi nei tumori intraepatici e p53 in quelli
extraepatici. Una ridotta espressione di p16 è associata con
ridotta sopravvivenza. Questi dati confermano le differenze
esistenti fra colangiocarcinomi intra- ed extraepatici. Un
panel immunoistochimico comprendente questi markers
potrebbe essere suggerito nei casi di difficile inquadramento
diagnostico. Nello stesso tempo la p16 sembra avere valore
prognostico positivo.
La patogenesi del colangiocarcinoma è ancora lontana dall’essere chiarita. In collaborazione con il Dipartimento di Biomedicina dell’Università di Basilea abbiamo studiato lo sviluppo
di neoplasie biliari in topi con attivazione costitutiva del
gene NOTCH2 nel fegato (N2ICD/AlbCre). Abbiamo potuto
dimostrare lo sviluppo di tumore di tipo colangiocarcinoma
e talvolta epato-colangiocarcinoma a diverse età. L’immunofenotipo delle cellule tumorali ha dimostrato una dedifferenziazione (espressione di HNFA4) rispetto alle proliferazioni
biliari non neoplastiche di tipo cistico. Lo stesso immunofenotipo si ritrova nei colangiocarcinomi umani. Questo ceppo
di topi potrebbe quindi rappresentare un buon modello per lo
sudio dei colangiocarcinomi.
Role of biopsy in HCC and the use of biomarkers
M. Roncalli
Milano
Small hepatocellular nodules measuring between 1-2 cm are
increasingly detected during the surveillance of cirrhotic
patients. These nodules include either large regenerative
and dysplastic nodules (15-30%) and small HCC (70-85%).
International guidelines have proposed the use of noninvasive imaging techniques as first screening tool of small
HCC 1, with an overall sensitivity in the detection of malignancy around 30% 2. Liver biopsy is therefore increasingly
used in the detection of small HCC, which is at an earlier
stage and well differentiated. As such the diagnostic distinction between malignant and dysplastic small hepatocellular
nodules is a challenge as it requires the strict cooperation of
pathologists, radiologists and clinicians. When approaching
the liver biopsy of a 1-2 cm nodule in cirrhosis pathologists should be aware in which clinical setting the lesion
was sampled (cirrhotic under surveillance, with or without
previous HCC, number of nodules, US pattern, AFP values
etc) and then ascertain sampling adequacy. Today the study
of neoangiogenesis and of a number of morphological features which are mainly architectural and less cytological
are of help in the distinction between malignant and nonmalignant/dysplastic nodules. This explain why cytology
alone is not recommended in this subtle diagnostics. Notwithstanding a careful analysis of all the features, a number
of cases still remain equivocal particularly in the grey area
between high grade dysplastic nodules and early well differentiated HCC 3.
Recent advances have shown that the most reliable indicator
of malignancy in early and well differentiated HCC is socalled stromal invasion which can be indirectly ascertained
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through the analysis of surrogate markers such as the so-called
ductular reaction, which takes place around non malignant
nodules.
While we are today still focusing on classical morphological
markers of malignancy (architectural abnormalities, neoangionesis, cell invasion) novel, less explored but more exciting
markers are on view. They come from expression profiling
studies where they have been shown to be able to distinguish
between early HCC and dysplastic nodules 4-6. Among them
Glypican 3 is the most studied and valuable and its use in
combination with other markers in a diagnostic panel is going
to become a major source of diagnostic information of the
liver biopsy. More recently we have shown that the diagnostic
accuracy in the recognition of small HCC can be improved
using a panel composed by 4 immunomarkers, namely GPC3,
Heat Shock Protein 70 (HSP70), Glutamine Synthetase (GS)
and Chlatryn Heavy Chain (CHC) 7.
The introduction in the routine pathology of immunomarkers
able to predict the tumor’s behaviour in individual patients
has been proposed but not yet validated in longitudinal studies. Among these markers CK19 reflecting progenitor cell
phenotype, has been correlated with poor outcome of HCC 8.
Additional molecular markers predictive of tumor behaviour
such as miRNA and trascriptome patterns of expression and
epigenetic alterations have been proposed but required an
external validation 9.
References
1
Bruix J, Sherman M. Management of hepatocellular carcinoma. Hepatology 2005;42:1208-36.
2
Forner A, Vilana R, Ayuso C, et al. Diagnosis of hepatic nodules
20 mm or smaller in cirrhosis: prospective validation of the noninvasive diagnostic criteria for hepatocellular carcinoma. Hepatology
2008;47:97-104.
3
International Consensus Group on Hepatocellular Neoplasm (ICGHN)
Group. International Consensus on the pathologic Diagnosis of Early
Hepatocellular Carcinoma: a report of the International Consensus
Group for Hepatocellular Neoplasia. Hepatology, in press.
4
Paradis V, Bièche I, Dargère D, et al. Molecular profiling of hepatocellular
carcinomas (HCC) using a large-scale real-time RT-PCR approach: determination of a molecular diagnostic index. Am J Pathol 2003;163:733-41.
5
Llovet JM, Chen Y, Wurmbach E, et al. A molecular signature to
discriminate dysplastic nodules from early hepatocellular carcinoma
in HCV cirrhosis. Gastroenterology 2006;131:1758-67.
6
Seimiya M, Tomonaga T, Matsushita K, et al. Identification of novel
immunohistochemical tumor markers for primary hepatocellular carcinoma; Clathrin Heavy Chain and Formiminotransferase Cyclodeaminase. Hepatology 2008;48:519-30.
7
Di Tommaso L, Destro A, Fabbris V, et al. Diagnostic accuracy of
chlatryn heavy chain staining in a markers panel for the diagnosis of
small HCC. Hepatology 2011;53:1549-57.
8
Ding SJ, Li Y, Tan YX, et al. From proteomic analysis to clinical
significance: overexpression of cytokeratin 19 correlates with hepatocellular carcinoma metastasis. Mol Cell Proteomics 2004;3:73-81.
9
Villanueva A, Hoshida Y, Toffanin S, et al. New strategies in hepatocellular carcinoma: genomic prognostic markers. Cancer Res.
2010;4688-94.
Aula Mizar – ore 9.00-10.40
Ragion d’essere, rilevanza clinica, utilità medico-legale
della diagnostica feto-placentare
Moderatore: Gaetano Bulfamante (Milano)
La patologia feto-placentare nelle gravidanze
a rischio
M. D’Armiento
Dipartimento di Scienze Biomorfologiche e Funzionali, Sez. di Anatomia Patologica e Citopatologia, Università di Napoli “Federico II”
La problematica della mortalità perinatale (MP) è costantemente attuale poiché i suoi tassi, in quanto indice della qualità
dell’assistenza pre e perinatale, entrano nel computo della
“vita media” di una popolazione che è espressione del livello
socio-economico di una società. La MP ritorna emergente
quando, nonostante le continue nuove acquisizioni nell’ambito delle tecnologie/strategie utili alla diagnosi e cura, i tassi di
mortalità perinatale permangono, alti; infatti al 2002 sono:4,5
x1000 nati vivi il NMR (neonatal mortality rate), 3,1 x1000
nati vivi PMR (perinatal mortality rate) e 4,5 x1000 nati vivi
+ morti fetali FMR (fetal mortality rate) pressoché invariati
rispetto al 1994! Contemporaneamente si assiste ad un aumento del contenzioso medico-legale, in quanto, proprio alla
luce del continuo aumento delle conoscenze, diventa sempre
meno accettabile un’evoluzione infausta di una gravidanza,
talvolta imprevedibile, ma che comunque, non può rimanere
inspiegabile. Pertanto si è assistito ad un’impennata delle
richieste di esami soprattutto delle placente, specialmente per
le gravidanze a rischio e/o complicate che spesso esitano in
un parto pretermine, talvolta associato ad una morte fetale o
neonatale o ad esiti permanenti. La patologia dell’unità fetoplacentare è una fonte inesauribile di informazioni interessanti
da un punto di vista diagnostico e scientifico, sottovalutata e,
quindi, sottoutilizzata, non sempre adeguatamente insegnata, inadeguatamente considerata nell’ambito delle patologie
subspecialistiche, soprattutto dagli stessi anatomopatologi;
soprattutto per quanto concerne la placenta, organo a struttura
apparentemente semplice che presenta delle lesioni apparentemente semplici e costanti che sottendono una patologia complessa (La cosiddetta complessa semplicità della palcenta!?).
La placenta è l’unico organo multifunzionale e perciò diverso da ogni altro campione patologico! La placenta è connessa, attraverso differenti circolazioni, a due differenti persone. I problemi che colpiscono la placenta potrebbero colpire
ogni persona in maniera differente; inoltre, quando l’evento
patologico si verifica, la madre e il bambino verranno seguiti
da due (o più) specialisti, con veramente differenti competenze ed approcci, interessati alle implicazioni della patologia
placentare per uno specifico dei soggetti. Pertanto l’esame
della placenta deve prevedere l’interpretazione della patologia
da entrambi gli individui. Un diagnosi completa ed esaustiva
94
può diventare una significativa sfida per il patologo! Infatti
un altro tratto distintivo di questa patologia è che le notizie
cliniche sono spesso poche e, spesso, richieste a distanza dal
parto (in caso di eventi patologici post-natali). Appare chiaro
che un’analisi ottimale prevede collaborazione fra patologo,
ostetrico e pediatra (ma a volte l’approccio multidisciplinare
è molto più ampio) e la diagnosi sarà, spesso, un work in progress e potrebbe essere definitiva anche dopo molti giorni dal
parto! È opportuno adottare, per la valutazione della patologia
placentare, delle “linee guida di sopravvivenza”, soprattutto
per le placente di gravidanze complicate. Linee guida, standardizzate, che prevedano indicazioni per un corretto esame
macro/microscopico, i tempi e il tipo di conservazione, la tipologia ed il numero dei prelievi e, uno schema che sia di guida per la compilazione della diagnosi che tenga conto di tutti
i parametri macro/microscopici rilevati atti ad una corretta
interpretazione della patologia. L’algoritmo di valutazione di
dette lesioni è cambiato negli anni anche grazie ad un aumento
delle competenze dei patologi ed alla loro capacità di applicare tutte le tecnologie disponibili (immunoistochimica, M.E,
PCR) alla valutazione dei reperti. Storicamente l’attenzione
dello studio istopatologico della placenta è stato orientato al
comparto materno, più recentemente molta attenzione è stata
posta anche al comparto fetale la cui istopatologia potrebbe
fornire molte informazioni in termini predittivi o esplicativi
di morbidità.
Di entrambi i numerosi gruppi di patologie, viene fatta una
messa a fuoco su quelle condizioni che si è osservato essere
correlate a sequele per il feto e/o a rischio di ricorrenza. Le
lesioni patologiche note della placenta si osservano in associazione a numerosissime differenti condizioni e la loro rilevanza
nel determinismo di un evento avverso è relativa all’epoca
della gravidanza in cui insorge, all’estensione delle lesioni,
alle condizioni materne. Le patologie da alterata perfusione
materna (legate più comunemente ad ipertensione, anche
esclusivamente gestazionale, diabete) esitano nell’infarto che,
per essere patologico ed avere un impatto sull’ossigenazione
fetale, deve superare il 5% della massa placentare; la gamma
di lesioni morfologiche associate all’ischemia è ampia e si associa ad alterazioni apparentemente strutturali. La ipossemia
materna, invece (correlata a patologia cardiopolmonare, alle
alte altitudini, alcune patologie ematologiche, etc.) è caratterizzate dall’ ipervascolarizzazione. La trombofilia congenita
(proteina C o S deficit) o acquisita (Ab antifosfolipidi) è caratterizzata dai depositi di fibrina intervillosi fino all’infarto del
letto materno. Questa patologia è ad alto rischio di ricorrenza
e per il feto le sequele dipendono dalla riserva placentare e
vanno dal FGR, al parto pretermine, alla morte fetale. Le patologie derivanti dai difetti di perfusione fetale sono da correlare
alla patologie del cordone e del piatto coriale e del circolo
intravilloso; per molti anni la terminologia fetal thrombotic
vasculopathy (FTV) ha abbracciato una gamma di lesioni
morfologiche correlate al danno della parete vascolare (cfr.
studio degli endoteli e dei mediatori dell’infiammazione); ma
anche le cellule dell’infiammazione associate a una patologia
sottovalutata come la corionamnionite (di origine materna) o
a villite cronica sono responsabili di detta patologia, patologia
che spesso prevede immediate cure neonatologiche così come
un danno tossico alla parete dei vasi da meconio. Gli esiti di
queste patologie sono asfissia, esiti neurologici e, talvolta la
morte fetale. L’edema massivo dei villi e l’emorragia intravillosa sono altri due eventi drammatici di questo gruppo che
correlano con la morte fetale. La maggior parte delle patologie del comparto fetale può provocare un parto pretermine,
a termine,una morte endouterina per cui bisogna allertare i
clinici e minimizzare le paure dei genitori; va notato, però,
che quando c’è un significativo danno vascolare si può avere
compromissione neurologica. Fondamentale è la diagnosi
differenziale di etiologia! Anche alcune malattie specifiche
(metaboliche, sindromi che) possono essere diagnosticate
dalla patologia placentare. Pertanto si può comprendere che la
gravidanza a rischio non è solamente quella che deriva da patologia materna pregravidica: anomalie strutturali dell’utero,
anemie, malattie congenite, malattie autoimmuni, diabete etc.,
ma l’ipertensione, anche esclusivamente gestazionale, che è
in aumento data l’età avanzata in cui molte donne affrontano
la gravidanza, “tormenta” il percorso di gravidanze pseudofisiologiche che possono esitare in preeclampsie ed in FGR, o
in un parto pretermine grave fino alla morte endouterina. Le
infezioni materne così come le neoplasie dovrebbero essere
diagnosticate prima della gravidanza. Appare chiaro che
molto del contenzioso medico-legale sarebbe ridotto se la patologia placentare assumesse il ruolo che le compete e se i patologi offrissero diagnosi esaustive per l’identificazione delle
patologie e delle loro etiologie da cui trarre informazioni per
il monitoraggio di successive gravidanze e/o delle patologie
materne e del bambino. Le diagnosi andrebbero discusse con i
vari specialisti periodicamente e, successivamente, con i genitori per una serena pianificazione concezionale. Con l’augurio
che con l’approccio multidisciplinare si possano istituire non
solo delle working conference ma anche delle teaching conference sulla patologia dell’unità feto-placentare,eccezionale
materiale didattico interdisciplinare.
The pathological investigation on specimen
from early and late spontaneous abortion
G. Ottoveggio, G. Becchina, F. Genovese, B. Giacalone, C.
Nagar, V. Tralongo
U.O.C. di Anatomia Patologica, Distretto Ospedaliero 3, P.O. “G.F.
Ingrassia”- A.S.P., Palermo
Abortion is generally defined the premature spontaneous
expulsion of a nonviable human conceptus. It is a common
event in human reproduction. It has been estimated that up
to 70% of all conceptions fail to complete their development.
Early spontaneous abortion (ESA) describes pregnancy loss
occurring in the first trimester (12 weeks and 6 days). Late
spontaneous abortion (LSA) includes pregnancy loss within
180th day. In many histopathology laboratories, embryo-fetal
pathology is poorly performed compared to other research
fields of pathology, this, despect the high probability of
pathological findings (malformations, maternal illness, etc.)
prognostically important for the management of subsequent
pregnancies. In the last ten years, it has increased interest
and knowledge to this field of pathology, through programs
of training and professional updating. Although the specimen related to ESA are commonly submitted to pathological examination, these are often not subject to systematic
studies and the amount of time spent by most pathologists
to these samples and their reporting is poor. Clinicians often
receive non-diagnostic reports from the pathologists, who
only provide some answers: confirmation of early intrauterine
pregnancy and the identification of gestational trophoblastic
disease. We believe that abortion specimen should be studied
in detail and that histopathological examination is the starting
point of a collaboration network among different specialists
(gynecologist, geneticist, immunologist, hematologist, etc.).
This integrated analysis should start from anamnesis and history of pregnancy, underlining maternal risk factors. In this
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regard, we use a data sheet to collect these information. We
also have an interview with the couple for more details. In the
diagnostic work up, is essential to follow the procedural steps.
The first stage is a careful macroscopic examination on large
Petri dish. There are different macroscopic types of ESA specimens. Approximately one-half of samples are incomplete,
which means that they contain only fragmented chorionic sac
and no embryo. The insufficient specimens are those with
only decidua and blood clots. The complete specimens consist
of either an intact chorionic sac or rupted sac with an embryo.
Gross examination of complete specimens can yield several
types of useful findings: embryo with normal morphology,
embryo with growth disorganization, embryo with localized
defect(s), macerated, damaged or unclassifiable embryo. The
presence of an intact but empty sac or a grossly disorganized nodular embryonic structure suggests that an abnormal
karyotype is likely. Curetted tissues of early abortions often
includes a pale spongy mass (the villi) attached to a glistening
translucent membrane (the chorionic plate with amnion). The
decidual component is folded sheets or stripes of soft pink
tissue with a smooth surface. Careful inspection of the gross
fragments ensures adequate villous sampling. Evidence of
hydropic villi requires more complete sampling. Vescicular
villi are more readily appreciated when floating in water. The
presence of a large hematoma and its location on the surface
of the sac should be documented. Microscopic examination of
ESA specimens follows a diagnostic flow chart. The first step
is to identify the structures in histological sections (deciduas,
villi, embryo/fetus, cord, amniotic sac, yolk sac). It is essential
to define the adequacy of specimen: it is important not only
the quantity but the representativeness of the structures. It is
essential the presence of the basal and parietal decidua and
chorionic villi. The diagnostic algorithm proceeds through the
identification of pathological changes (primary lesions) and
their location; that allows the pathologist to make a diagnosis indicative of major categories of disorders, directing the
gynecologist to further clinical and laboratory investigations.
Generally, the identification of lesions and their anatomical
location predict the pathophysiological consequences in the
pathology of organs. In the study of ESA, the individual lesions are not always related to a well-defined or known pathogenetic pathway. In a few situations, the observed lesions
are markers for specific genetic, infections, immunologic,
thrombophilic or vasodestructive process. The probability that
a recognizable underlying pathologic basis for ESA can be
unequivocally demonstrated is small, but the identifiable aspects are potentially significant in the evaluation of abortion.
Although in most cases, ESA has chromosomal etiology, it
can recognize other groups of injures in its pathogenesis. The
first group includes disorders involving the decidua, decidual
vessels and trophoblast of implant site. The use of a panel of
histochemical (Masson’s trichrome stain, Weigert’s stain for
fibrin, PAS) and himmunoistochemical (alpha smooth muscle
actin and hPL) stains is useful in the evaluation and interpretation of lesions of decidual vessels. The study of decidual vessels can reveal: the presence of unconverted decidual vessels,
suggestive of inadequate progestinic support; intimal thickenings/sclerosis of vascular walls and perivasculitis, suggestive
of autoimmune maternal disease (latent or known); fibrinoid
necrosis and/or hypertrophy of vascular walls, acute atherosis
and diffuse thrombosis of decidual vessels, suggestive of
decidual vasculopathies. The second group includes lesions
mostly limited to the villi with regard to morphological and
structural findings; these represent peculiar or suggesting
aspects of karyotype abnormalities. The third group is rep-
95
resented by decidual and/or villous and/or intervillous space
and/or membranous lesions consequent to acute or chronic
inflammatory states due to infections. The fourth group
comprises retroplacental massive hemorrhage, responsible
for unexpected chorionic detachment, or large blood clots
in the intervillous space, or infarction; these, in some cases,
may be related to maternal diseases. Specimens from LSA
consist of the fetus and placenta; both must examined together
as constituting the fetal-placental unit. The most common
causes of LSA are intrauterine infections. The protocol for
the examination of the fetus is the same as perinatal autopsy.
The fetus should be inspected for abnormalities of phenotype
that may suggest chromosomal defects. The incidence of
cytogenetic abnormalities among LSA is substantially lower
than among ESA. The most frequently detected chromosomal
abnormalities are specific autosomal trisomies (13-18-21), sex
chromosome monosomy (45X0) and triploidy. The morphological investigation aims to identify specific malformations.
Single malformations are more frequent among spontaneously
aborted previable fetus, than among stillbirths or live births.
Common abnormal morphologic findings in LSA are neural
tube defects, posterior cervical cystic hygroma, abdominal
wall defects, facial clefts, renal anomalies, obstructive uropathies and heart defects. A single lesion may have several
pathogenetic mechanisms (multifactorial, caused by a single
gene defect, chromosomal defect or non genetic mechanism).
The distinction of a specific mechanism responsible for a
defect provides guidance for future pregnancies, as the recurrence risk for each of these pathogenetic mechanisms differs
substantially.
Therefore detection of a specific defect and determination
of its cause provide valuable informations for genetic counseling and allows specific prenatal investigations in future
pregnancy. Fetal-placental examination may reveal patterns
indicative of specific maternal, subclinical or latent, pathologic disorders responsible for LSA. For example, maternal
thrombophilia, either of genetic origin or acquired, may be
implicated in the pathophysiological process underlying both
ESA and LSA. Congenital thrombophilic disorders are associated with placental lesions such as infarct, retroplacental or
subchorial hematomas, massive perivillous fibrin deposits and
fetal thrombotic vasculopathy. Thromboembolic events may
be also present in the fetal compartment. The placenta may
show specific morphological findings in maternal autoimmune diseases, such as massive perivillous fibrin deposition/
maternal floor infarct, decidual vasculopathy and villitis of
unknown etiology. In conclusion, the prognosis for the future
pregnancies is critically dependent on recognizing an accurate
pathogenesis of pregnancy loss. The pathological investigation on specimens from ESA and LSA has great importance
both clinically and socially, because it provides answers to the
gynecologist, the couple, helping them in this experience, and
guide clinical management of future pregnancies.
References
1
Fulcheri E, Bulfamante G, Resta L, et al. Embryo and fetal pathology in routine diagnostics: what has changed and what needs to be
changed. Pathologica 2006;98:1-36.
2
Fulcheri E, Musizzano Y. Pathologica 2010;102:211-3.
3
Musizzano Y, Fulcheri E. Decidual vascular patterns in first-trimester
abortions. Virchows Archives 2010;456:543-60.
4
Kraus FT, et al. AFIP Atlas of nontumor pathology. N. 3 Placental
Pathology 2004.
5
Kalousec DK, Oligny LL. Potter’s pathology of the fetus, infant and
child. (2nd Ed.). Pathology of abortion: the embryo and the previable
fetus, 2007.
96
Istopatologia della placenta: fattori di rischio
materni ed esiti perinatali
G. Bartoloni
Dipartimento Ingrassia; Patologia Diagnostica Fetale Malformativa
e Perinatale, Università degli Studi di Catania - ARNAS Garibaldi
La comprensione del ruolo svolto dalla placenta e dai suoi
annessi nei singoli casi di danno neurologico feto-neonatale
non è sempre agevole, poiché ancora oggi le lesioni placentari
non sono definite in modo univoco e non sempre è chiara la
loro eziopatogenesi ed il loro significato clinico. Tuttavia,
l’attenta indagine della placenta e lo studio delle relazioni tra
le sue patologie e le caratteristiche anatomiche e cliniche del
danno neurologico appaiono essere tra gli elementi di maggiore significato per definire se la patologia del SNC derivi da
una condizione acuta o cronica, da uno stato di anossia/ipossia
insorta durante e/o a causa del travaglio di parto e se questo
danno si sarebbe potuto evitare curando la madre o il feto.
La placenta è un organo che non viene esaminato routinariamente in Anatomia Patologica, vuoi per mancanza di specifico interesse, vuoi per legittima economia di risorse, tendenza
che sempre piu’ viene ad affermarsi.
Attualmente nella maggioranza dei reparti ostetrici la placenta
viene esaminata macroscopicamente dal personale ostetrico
che ha assistito al parto ed inviata all’esame istopatologico
solo nel caso in cui vengono riscontrate anomalie.
La placenta viene inoltre inviata per l’esame quando sono presenti patologie materne o fetali note. Con tali criteri si identificano la maggior parte delle patologie (che vengono riscontrate
in circa il 15% dei parti). Resta tuttavia aperta la problematica
relativa a quei neonati in salute alla nascita, che manifestano
tardivamente sequele patologiche. D’altra parte esaminare
routinariamente tutte le placente è non solo improponibile
(alto costo di materiale, impegno tecnico e sanitario) ma assai
poco gratificante perché esito di parti del tutto fisiologici.
Si puo’ pertanto affermare che le esigenze diagnostiche devono da una parte essere mantenute entro ragionevoli criteri
di razionalizzazione e contenimento della spesa, ma tuttavia
senza trascurare tutte le possibili informazioni clinico-patologiche atte a scongiurare esiti avversi perinatali o a lunga
distanza.
Indicazioni all’invio per esame istologico della placenta
In un ottimale “flow chart” di un Dipartimento MaternoFetale, è assolutamente necessario delineare le indicazioni al
suddetto esame macroscopico e istopatologico che devono essere individuate nell’anamnesi pre-parto. Esse possono essere
suddivise intanto in due grandi categorie:
A) patologie materne;
B) patologie fetali e neonatali.
Tra le prime vanno considerate ad esempio il parto pretermine
o le sospette infezioni ed inoltre la rottura prematura delle
membrane
Per quanto riguarda patologie fetali si ricordano solo a titolo
di esempio, un basso indice di APGAR o il ritardo della crescita.
Patologie placentari: in sala parto deve essere messo in atto un
ottimale screening pre-anatomo-patologico dei casi, atto a individuare ad esempio placente di volume assai aumentato o assai
diminuito rispetto a età gestazionale, placente con anomalie
all’esame ecografico,associate a patologie fetali.
Il patologo da parte sua deve utilizzare un algoritmo procedurale riproducibile e condiviso in ambito culturale generale e
specialistico. Schematicamente, esso si muovera’ vagliando e
suddividendo le diverse patologie in: infiammatorie, circolatorie, maturative, entrando pertanto nel merito delle patologie
emorragiche o occlusive vascolari e al tempo stesso dismaturative vascolari.
In conclusione si puo’ affermare che l’esame accurato della
placenta ha notevoli ricadute cliniche; esso puo’ infatti dirimere i dubbi relativi al processo patologico (acuto o cronico)
che ha comportato una complicanza al parto. Possono inoltre
essere acquisite informazioni utili per gestire successive
gravidanze ed infine riconosciute patologie antenatali e intrapartum che possano determinare sequele nello sviluppo
neurologico del nascituro.
Neuropatologia oncologica
Moderatori: Felice Giangaspero (Roma), Salvatore Lanzafame (Catania)
Pitfalls nella diagnostica intraoperatoria
delle lesioni del SNC
M. Antonelli
Roma
La diagnostica intraoperatoria in neuropatologia mediante
esame citologico, ha un ruolo importante per la definizione
delle lesioni del sistema nervoso centrale, poichè numerose
lesioni possono simulare radiologicamente e clinicamente
una neoplasia intracranica. Nella maggior parte dei casi, il
patologo può facilmente distinguere tra neoplasia e lesioni
non neoplastiche, da cui dipende l’entità dell’asportazione
chirurgica.
Tuttavia una serie di lesioni non neoplastiche, quali la lesioni
demielinizzanti, le lesioni infettive e le alterazioni post-tratta-
mento mostrano caratteristiche morfologiche che possono mimare una neoplasia. Altre condizioni in cui la diagnosi intraoperatoria è di fondamentale importanza per il neurochirurgo è
la distinzione, a livello del midollo spinale tra un ependimoma
ed un astrocitoma. In tali condizioni, lo striscio citologico e la
conoscenza dei dati clinico-radiologici aumentano l’accuratezza diagnostica durante un esame intraoperatorio.
Meningeal non-meningothelial neoplasias
V. Barresi
Department of Human Pathology, University of Messina, Italy
According to the WHO Classification of Tumors of the
Central Nervous System (CNS), meningeal neoplasias can
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be subdivided into meningiomas and non-meningothelial tumors, comprising mesenchymal non-meningothelial tumors,
haemangiopericytoma, melanocytic lesions and haemangioblastoma 1. Mesenchymal non-meningothelial tumors include a
variety of benign and malignant tumors originating in the CNS
and histologically corresponding to tumors of soft tissues and
bone 1. The most frequent meningeal non-meningothelial
tumor is haemangiopericytoma 1. Clinical and radiological
features of haemangiopericytoma maybe indistinguishable
from those of meningioma, but differently from meningioma,
haemangiopericytoma shows a high tendency to recur and
metastatize outside the CNS 2.
Other meningeal tumors showing dural adhesion may radiologically mimic meningioma. Among these, rare cases of
primitive meningeal gliomas have been reported 3 4. These
tumors have been supposed to derive from heterotopic astroglial nests in the meninges 5. Metastases from carcinomas may
also display a meningeal localization and mimic meningioma,
especially when the existence of a primitive malignancy is
unknown 6. Lymphomas may also represent primitive meningeal neoplasias 7 and rare cases of intradural chordoma 8 and
germinoma 9 have been reported.
In conclusion, as a number of benign and malignant tumors
may localize at meninges and mimic meningioma, the histological diagnosis is of striking importance for the correct
diagnostic and therapeutic approach to meninegal neoplasms.
References
1
Perry A, Louis DN, Scheithauer BW, et al. Meningiomas. In: Louis
DN, Ohgaki H, Wiestler OD, Cavenee WK, eds. WHO Classification
of Tumors of the Central Nervous System. Lyon: IARCC Press 2007,
pp. 164-72.
2
Guthrie BL, Ebersold MJ, Scheithauer BW, et al. Meningeal haemangiopericytoma: histopathological features, treatment and long-term of
44 cases. Neurosurgery 1989;25:514-22.
3
Horoupian DS, Lax F, Suzuki K. Extracerebral leptomeningeal
astrocytoma mimicking a meningioma. Arch Pathol Lab Med
1979;103:676-9.
4
Wakabayashi K, Shimura T, Mizutani N, et al. Primary intracranial
solitary leptomeningeal glioma: a report of 3 cases. Clin Neuropathol
2002;21:206-13.
5
Cooper IS, Kernohan JW. Heterotopic glial nests in the subarachnoid
space; histopathologic characteristics, mode of origin and relation to
meningeal gliomas. J Neuropathol Exp Neurol 1951;10:16-29.
6
Portocarrero-Ortiz L, Garcia-Lopez R, Romero-Vargas S, et al. Thyroid
follicular carcinoma presenting as skull and dural metastasis mimicking a meningioma: a case report. J Neurooncol. 2009;95:281-4.
7
Sacho RH, Kogels M, du Plessis D, et al. Primary diffuse large B-cell
central nervous system lymphoma presenting as an acute spaceoccupying subdural mass. J Neurosurg 2010;113:384-7.
8
Bhat DI, Yasha M, Rojin A, et al. Intradural clival chordoma: a rare
pathological entity. J Neurooncol;96:287-90.
9
Biswas A, Puri T, Goyal S, et al. Spinal intradural primary germ
cell tumour-review of literature and case report. Acta Neurochir
2009;171:277-84.
Lymphomas of the central nervous system: rare
entities and differential diagnosis
M. Gessi
Inst. of Neuropathology, University of Bonn Medical Center, Bonn,
Germany
Primary central nervous system (CNS) lymphomas (PCNSL)
account for approximately 2% of all primary brain tumors.
According to the World Health Organization (WHO) classification about 95% of PCNSL are non-Hodgkin B-cell lymphomas, which are at histological level similar to systemic diffuse
large B-cell lymphoma (DLBCL). However, other B-cell and
97
T-cell lymphoma variants may involve the CNS as primary or
secondary lesions. Owing to their rarity and their sometimes
misleading features, they could be not easily be recognized
and confused with classical DLBCL, with other primary or
metastatic brain tumors, with neoplastic and non-neoplastic
histiocytic proliferation and with inflammatory lesions of the
CNS. Among B-cell lymphomas, various subtypes other than
DLBCL may involve the CNS. Moreover, uncommon systemic B-cell proliferations (intra-vascular large B-cell lymphoma
and lymphoid granulomatosis) may be also encountered in
routine neuropathology.Primary CNS marginal zone B-cell
lymphomas (CNS-MZBCL) of MALT type are the most common B-cell low-grade lymphoma in the CNS. These tumors
are typically dura-based, meningioma-like lesion. They occur
in adult patients (median age 40-50 yrs) with higher incidence
in women. Histologically, the tumors are composed by small
and medium sized lymphocytes, which may present clear
cytoplasm (monocytoid cells). The tumor cells express B-cell
markers (such as CD20 and CD79a) and are negative for CD3,
CD5, CD10, and cyclin D1. At cytogenetic level, trisomy 3,
a common cytogenetic finding in MZBCL of MALT type is
described only in a subgroup of CNS cases. No rearrangements of MALT1 gene have been reported in CNS cases.
Because surgical treatment combined with radiation and/or
chemotherapy usually leads to complete remission of disease,
the distinction with other B-cell lymphoma, such DLBCL,
is mandatory.CNS involvement in Burkitt´s lymphoma (BL)
occurs in about 15% of systemic cases and it is more frequent
among AIDS patients. However, rare cases of primary CNSBL have been described in pediatric as well as adult patients.
CNS-BL may affect cerebral hemisphere or basal ganglia and
presents at histological level the classic “starry-sky” appearance. In contrast to systemic BL, the prevalence of c-Myc
translocations t(8;14)(q24;q32) among primary CNS-BL is
not known. Among other B-cell lymphomas, nodal mantle
cell lymphoma (MCL) may present CNS involvement in
about 15% of cases but primary CNS MCLs are exceptional.
As MCL also primary follicular lymphomas (FCL) are very
uncommon in CNS and a limited number of meningeal cases
have been reported. Intra-vascular large B-cell lymphoma
(IVL) is a rare, aggressive lymphoma, characterized by selective growth of neoplastic large B-cells in the lumina of small
and medium vessels in various organs. The high affinity of
the lymphoid cells for small vessels endothelium is probably
dependent to a defect in the interaction between endothelial
cell surface ligands and lymphocyte homing receptors, such
as Beta-Integrin and ICAM-1. About 70% of patients have
symptoms reflecting involvement of brain, spinal cord or
nerve. The cytological features of tumor cells are similar to
DLBCL. Tumor cells express usually B-cell antigens such as
CD20 but occasionally CD5 expression can be also found in
tumor cells. Because IVL responds poorly to chemotherapy,
the prognosis of patients is usually poor. Lymphomatoid
granulomatosis is a systemic disease characterized by the
proliferation of atypical EBV-positive B-cells, with a peculiar
angiocentric and angiodestructive pattern of growth. The disease affects most frequently immune compromised patients.
Lungs, skin and brain are typically involved. Histologically
the brain lesion consisted in perivascular and intravascular
infiltration of a variable amount of atypical B lymphocytes
(with variable cytology), in a background of T-lymphocytes,
plasma cells, immunoblasts and histiocytes. Granulocytes are
uncommon. The infiltration of lymphoid cells compromises
the integrity of the vascular structures in the CNS leading to
brain infarcts and large areas of necrosis.
98
The atypical EBV-positive B-lymphocytes are commonly
CD20 and CD30 positive. The main differential diagnosis includes vasculitis, necrotizing encephalitis and other low- and
high-grade PCNSL. The outcome of the patients is dependent
from the amount of neoplastic B-cell in tumor tissue, the age
of the patient and the presence of a wide CNS involvement.
Primary T-cell CNS Lymphomas are rare and show a slightly
increased frequency in Far East Countries. They occur mainly
in not-immunocompromised patients and may show a wide
spectrum of histopathological features. They arise as solitary
and multiple lesions and may present supra or infratentorial
localization. The majority of cases fulfil the criteria for the diagnosis adult T cell leukemia/lymphoma (ATLL), peripheral
T cell lymphoma or anaplastic large cell lymphoma (ALCL).
However, some T-cell lymphomas may show bland cytology
and lack of perivascular growth pattern. The neoplastic Tcells may mimic normal lymphocytes and may also show aberrant positivity for B- and T-cell markers. In this cases clonal
analysis of TCR rearrangement is useful for the diagnosis.
Anaplastic large cell lymphomas (ALCL) can occur as intraparenchymal or meningeal lesion. They are composed of cells
that are generally larger than those of a DLBCL, with pleomorphic nuclei that may be single or multiple. Nucleoli are
usually prominent, sometimes multiple. Histological subtypes
including “classic,” “lympho-histiocytic,” and “small cell”
variants have also been described in CNS-ALCL. The immunohistochemical positivity of tumor cells with CD30 (Ki-1)
antibody is pivotal for the diagnosis. Tumor cells of ALCLs
are positive for T-cell markers (CD43, CD45 and CD45RO)
but CD3 could be negative. Some cases are negative for both
B- and T-cell markers. EMA and CD15 expression in tumor
cells has also been reported in CNS-ALCL. Metastatic tumors
as well as glial and embryonal tumors of the CNS may resemble ALCL. CNS-ALCL, like its nodal counterpart, often
shows immunopositivity with ALK-1 antibody. Expression
of ALK-1 correlates with a young patient age and a favorable
prognosis.
Primary CNS Hodgkin’s lymphomas (HL) as well as spinal
or cerebral involvement as first manifestation of a systemic
HL disease are uncommon. Usually CNS involvement can be
observed in advanced stages of disease or in AIDS patients.
Primary CNS-HL seems to affect mainly middle aged adult
patients, can be supratentorial or infratentorial, and may
present intracerebral as well as dural localization or both.
CNS-HLs do not show specific neuro-radiological features
and may resemble ischemic or inflammatory lesion as well as
primary brain or meningeal tumors. Histologically, CNS-HL
is similar to standard nodal HL: “mixed cellularity” or “nodular sclerosis” variants have been described in the CNS. The
prevalence of EBV infection in Reed-Sternberg is reported
as 50-60% in standard systemic HL. In CNS-HL the EBV
status of Reed-Sternberg has been partially investigated. The
incidence appears similar to that in systemic HL and seems
to depend on the immune-status of the patient.Natural killer
(NK)/T-cell lymphoma of nasal type (NKTCL) is a malignant
disorder of cytotoxic NK or, rarely, T cells, invariably associated with Epstein-Barr virus (EBV) infection. Although
secondary involvement of the central nervous system has been
reported, cases of primary CNS NKTCL are very rare and
affect mainly the cerebral hemisphere. The occurrence of NKTLC of CNS seems to be independent from the immune status
or the ethnicity of the patients. NKTCL cells are positive for
CD2, CD56 and negative for surface CD3. The histological
immune phenotypes of extra-nasal cases are similar to those
of nasal cases, except for a higher percentage of CD30 expres-
sion. A small proportion of cases show rearrangements of the
TCR genes which probably represents neoplasms of cytotoxic
T cells. Interestingly the T cells origin seems to be more common in NKTCL of the CNS than in other extranodal cases.
NKTCL of the CNS present an aggressive course, with poor
response and short survival with standard therapies.
Selected references
Commins DL. Pathology of primary central nervous system lymphoma.
Neurosurg Focus 2006;21:E2.
Deckert M, Paulus W. Malignant Lymphoma. WHO Classification of
Tumours of the Central Nervous System. 4th edn. Lyon: IARC Press
2007, pp. 188-92.
Dulai MS, Park CY, Howell WD, et al. CNS T-cell lymphoma: an underrecognized entity? Acta Neuropathol 2008;115:345-56.
George DH, Scheithauer BW, Aker FV, et al. Primary anaplastic large
cell lymphoma of the central nervous system: prognostic effect of
ALK-1 expression. Am J Surg Pathol 2003;27:487-93.
Gerstner ER, Abrey LE, Schiff D, et al. CNS Hodgkin lymphoma. Blood
2008;112:1658-61.
Ham MF, Ko YH. Natural killer cell neoplasm: biology and pathology.
Int J Hematol 2010;92:681-9.
Jahnke K, Korfel A, O’Neill BP, et al. International study on low-grade
primary central nervous system lymphoma. Ann Neurol 2006;59:75562.
Pittaluga S, Wilson WH, Jaff ES. Lymphomatoid granulomatosis. WHO
Classification of tumors of haematopoietic and lymphoid tissues. 4th
edn. Lyon: IARC Press 2008, pp- 247-249.
Ponzoni M, Ferreri AJ, Campo E, et al. Definition, diagnosis, and
management of intravascular large B-cell lymphoma: proposals and
perspectives from an international consensus meeting. J Clin Oncol
2007;25:3168-73.
Tu PH, Giannini C, Judkins AR, et al. Clinicopathologic and genetic profile
of intracranial marginal zone lymphoma: a primary low-grade CNS lymphoma that mimics meningioma. J Clin Oncol 2005;23:5718-27.
Molecular diagnostics of brain tumors
R. Caltabiano
Dipartimento G.F. Ingrassia, Anatomia Patologica, A.O.U. Policlinico-Vittorio Emanuele, Catania
The classification and grading of brain tumors is based on the
assessment of histopathological and immunohistochemical
features under the light microscope according to the criteria
defined in the World Health Organization (WHO) classification of tumors of the central nervous system. However, the
WHO criteria for typing and grading of tumors are not always
precise, mainly because of the biological variation that is difficult to capture in strict criteria. Unfortunately, this situation
causes substantial interobserver variation in the classification
of these tumors and may well have undesirable clinical consequences. Moreover, tissue sampling is often incomplete,
which for example may lead to underestimation of the true
malignancy in regionally heterogeneous tumors. In addition,
novel therapeutic modalities are available now that require
specific information about a tumors pathobiology. Often, this
kind of information cannot be assessed accumulating that
certain molecular changes are closely associated with therapy
response and/or patient survival, thus making them attractive
targets for molecular diagnostic testing aiming to improve
treatment stratification and prognostic assessment of the individual patient. The application of molecular tests in the diagnostic assessment of brain tumors has a number of immediate
implications for surgical neuropathology that need to be dealt
with. As these tests are performed on tumor tissue specimens,
we would strongly argue that not only the morphological but
also the molecular diagnostic tests should be performed or at
least be supervised by the responsible pathologist.
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relazioni
The goal is to give an update on the state of the art of molecular diagnostics for the CNS tumors in order to combine
the classic morphological approach (which undoubtedly will
remain an important basis for brain tumor diagnostics) with
molecular diagnostics in a smart way that provides the best
possible information for the individual patient.
Venerdì, 28 ottobre 2011
Diagnosi e linee guida nel carcinoma della mammella
Moderatori: Domenico Messina (Trapani), Anna Sapino (Torino)
Quali anticorpi per la determinazione dei
recettori ormonali (specificità e sensibilità)
G. Bogina
Dipartimento di Anatomia Patologica Ospedale Sacro Cuore di Negrar (Verona)
I livelli dei recettori ormonali sono uno dei più importanti
fattori prognostici ma soprattutto predittivi nel carcinoma
della mammella. Numerosi studi hanno infatti dimostrato
che i recettori per estrogeni (ER) e progesterone (PR) sono
potenti predittori sia di risposta all’endocrinoterapia che di
chemiosensibilità. Cruciale è, quindi, il ruolo del patologo
che deve garantire al clinico una corretta determinazione dei
recettori ormonali, attraverso metodiche affidabili, al fine di
evitare errati trattamenti terapeutici. A partire dagli anni ’70,
la metodica di elezione è stata di tipo biochimico, basata sul
legame tra recettore e ligando (estradiolo) radioattivo. Questa
metodica, che consente una valutazione quantitativa dei livelli
di recettore espressa in fentomoli per milligrammo di proteine, è stata utilizzata per una gran mole di studi che ne hanno
validato l’efficacia sul piano clinico 1. Tuttavia tale metodica
è laboriosa, costosa, implica l’utilizzo di materiale radioattivo,
è effettuabile solo su materiale congelato e non consente la
discriminazione tra tessuto neoplastico e non neoplastico nel
tessuto omogeneizzato. Con la messa a punto degli anticorpi
monoclonali, a partire da metà degli anni ’80, il riconoscimento e quantificazione del recettore non è più avvenuto tramite
ligando radioattivo ma con metodo immunoenzimatico. Contemporaneamente però l’utilizzo di anticorpi monoclonali ha
consentito la determinazione dei recettori ormonali mediante
metodica immunoistochimica (ICC) su materiale congelato,
con l’utilizzo dell’anticorpo H222 (Abbott). L’ICC si è poi
ulteriormente sviluppata con l’introduzione dei metodi di recupero dell’antigenicità, basate sul calore, e di nuovi anticorpi
utilizzabili su materiale in paraffina. Rispetto alla metodica
biochimica l’ICC ha il vantaggio di essere meno laboriosa,
meno costosa, di utilizzare materiale in paraffina e di consentire di discriminare tra componente neoplastica e non neoplastica nel tessuto analizzato; per contro non consente una
determinazione quantitativa ma semiquantitativa dei livelli di
recettori ormonali, espressa perlopiù in percentuale di cellule
marcate dall’anticorpo rispetto al totale delle cellule neoplastiche. Numerosi studi hanno dimostrato che l’ICC è altrettanto
valida o addirittura superiore sul piano clinico per predire la
risposta alla terapia ormonale 2. Una suggestiva differenza tra
l’ICC ed il metodo biochimico è la differente distribuzione
dei valori dei recettori ormonali, in particolare di ER. Mentre
con il metodo biochimico i valori di ER tendono a distribu-
irsi omogeneamente, con l’ICC i valori di ER assumono una
distribuzione bimodale, essendo o completamente negativi o
diffusamente positivi. Questo probabilmente in conseguenza
dell’utilizzo di metodiche sempre più sensibili, che hanno
consentito di dicotomizzare il dato (positivo versus negativo),
rendendo più agevole la decisione terapeutica, ma probabilmente con perdita di informazioni utili 3. Quindi, pur se la
concordanza, in termini di positività e negatività, tra il metodo
biochimico e ICC è alta, la relazione della distribuzione dei
valori fra le due metodiche non è di tipo lineare. Anche se
l’ICC è divenuta la metodica standard nella determinazione dei
recettori ormonali, alcuni studi hanno dimostrato significativi
tassi di discordanza e mancanza di riproducibilità tra i diversi
laboratori 4. Numerose sono infatti le variabili preanalitiche ed
analitiche che possono essere causa di scarsa riproducibilità.
Tra queste cruciale è la scelta di quale anticorpo utilizzare,
essendo assai numerosi quelli presenti in commercio. Inoltre,
recentemente, accanto agli anticorpi monoclonali di topo,
finora utilizzati, sono stati introdotti anticorpi monoclonali
ottenuti da coniglio transgenico che, rispetto a quelli di topo,
sono in grado di riconoscere antigeni umani non immunogeni
nel topo ed hanno un’affinità antigenica molto maggiore. Teoricamente gli anticorpi da utilizzare dovrebbero essere quelli
con miglior rapporto sensibilità/specificità. Non esistendo un
“gold standard” utilizzabile come “vero statistico”, il valore
della sensibilità (capacità di identificare i veri positivi) e
specificità (capacità di identificare i veri negativi) di ciascun
anticorpo non sono determinabili secondo un rigoroso criterio
statistico. In considerazione del valore predittivo della determinazione dei recettori ormonali, le linee guida ASCO-CAP
consigliano l’utilizzo di anticorpi di cui sia stata dimostrata
una correlazione con il beneficio clinico, inteso come risposta
alla terapia ormonale, che diventa il surrogato del “vero statistico”. Dalla revisione della letteratura gli anticorpi validati
clinicamente sono 6F11 (Vector Laboratories), 1D5 (Dako),
SP1 (LabVision) per quanto riguarda ER e 1A6 (Dako), 1294,
312 per quanto riguarda PR. In alternativa può essere considerato accettabile l’uso di anticorpi che mostrino una significativa concordanza con quelli validati clinicamente (90% dei casi
positivi, 95% dei casi negativi) 5. Esistono in letteratura alcuni
lavori che hanno confrontato fra loro i diversi anticorpi. 1D5 e
6F11, entrambi anticorpi di topo, non hanno mostrato significative differenze, con una concordanza di circa il 97%, anche
se complessivamente 6F11 colora una maggior percentuale di
cellule e con più forte intensità 6. Cheang et al. dimostrano una
superiorità di SP1, anticorpo di coniglio, rispetto ad 1D5, in
termini di una maggior concordanza con il metodo biochimico
e una migliore predittività di beneficio clinico 7. In run 76 di
100
UK NEQAS (Organismo nazionale della Gran Bretagna per il
controllo di qualità dei laboratori) 6F11 è risultato l’anticorpo
più utilizzato dai laboratori (58%) contro il 19%, 18% e 5% di
SP1 e 1D5 ed altri rispettivamente. Gli anticorpi con migliori
performance sono risultati 6F11 e SP1. Peraltro in run 75, UK
NEQAS ha dimostrato un elevato numero di falsi positivi con
l’utilizzo dell’anticorpo antiPR di coniglio SP2, che è stato
ritirato dal commercio.
È importante notare che, nei diversi lavori, i casi discordanti
rispetto all’utilizzo di differenti anticorpi sono quelli che
mostrano un valore inferiore e superiore rispetto alla soglia
dell’1%, ma raramente viene specificato di quanto effettivamente i valori si discostino da questa soglia. Per cui un
ipotetico tumore potrebbe avere lo 0,5% di positività con un
anticorpo antiER e l’1,5% con un altro, facendolo considerare
come negativo e positivo rispettivamente. Questa differenza
“sostanziale” sarebbe tuttavia, verosimilmente, di scarso impatto clinico.
Allo stato attuale delle conoscenze non vi sono evidenze sufficienti per considerare un anticorpo nettamente superiore agli
altri, tale da consigliarne l’uso esclusivo.
È opportuno pertanto attenersi alle linee guida ASCO-CAP
che considerano l’ICC come la metodica di elezione per la
determinazione dei recettori ormonali, essendo prematuro
l’utilizzo di metodiche molecolari che necessitano di ulteriore validazione e standardizzazione. La scelta dell’anticorpo
dovrà essere, possibilmente, tra quelli che abbiano avuto una
validazione su base clinica.
Compito di ciascun laboratorio utilizzare tutti gli accorgimenti
in grado di garantire la massima affidabilità del risultato:
• standardizzare le procedure preanalitiche ed analitiche;
• verificare sistematicamente la presenza di controlli interni:
eterogenea positività dei dotti ed acini (controllo postivo) e negatività di linfociti, cellule endoteliali e stromali
(controllo negativo) nel tessuto mammario circostante la
neoplasia;
• riconsiderare la validità del test in caso di risultato discordante da quello atteso in base all’istotipo: carcinoma lobulare, tubulare, papillare, mucinoso, duttale G1 generalmente positivi; carcinoma metaplastico, midollare, apocrino
generalmente negativi;
• verificare periodicamente che la percentuale dei casi positivi e negativi siano in linea con quanto riportato dai dati
della letteratura (70%-80% di casi positivi);
• raccomandabile la partecipazione a programmi riconosciuti
di “quality assurance”.
Bibliografia
1
Osborne CK, Yochmowitz MG, Knight WA, et al. The value of estrogen and progesterone receptors in the tratment of breast cancer.
Cancer 1980;46:2884-8.
2
Harvey JM, ClarK GM, Osborne CK, et al. Estrogen receptor status
by immunohistochemistry is superior to the ligand-binding assay for
predicting response to adjuvant endocrine therapy in breast cancer. J
Clin Oncol 1999;17:1471-81.
3
Collins LC, Botero ML, Schnitt. Bimodal frequency distribution of
estrogen receptor immunohistochemical staining results in breast
cancer. Am J Clin Pathol 2005;123:16-20.
4
Viale G, Regan MM, Maiorano M, et al. Prognostic and predictive
value of centrally reviewed expression of estrogen and progesterone
receptors in a randomized trial comparing letrozole and tamoxifen
adjuvant therapy for postmenopausal early breast cancer: BIG 1-98. J
Clin Oncol 2007;25:3846-52.
5
Hammond MEH, Hayes DF, Dowsett M, et al. American Society
of Clinical OncologyCcollege of American Pathologists guideline
recommendations for immunohistochemical testing of estrogen and
progesterone receptors in breast cancer. J Clin Oncol 2010;28:278495.
Kaplan PA, Frazier SR, Loy TS, et al. An immunohistochemical
comparison of two monoclonal antibodies for the evaluation of estrogen receptor status in primary breast carcinoma. Am J Clin Pathol
2005;123:276-80.
7
Cheang MC, Treaba DO, Speers CH, et al. Immunohistochemical
detection using the new rabbit monoclonal antibody SP1 of estrogen
receptor in breast cancer is superior to mouse monoclonal antibody
1D5 in predicting survival. J Clin Oncol 2006;24:5637-44.
6
Clinical impact of androgen receptor in breast
cancer
I. Castellano*, E. Allia*, A. Vandone*, L. Chiusa*, R. Arisio**,
G. Viale***, A. Sapino*
*
Dipartimento di Scienze Biomediche ed Oncologia Umana, Università di Torino; **Dipartimento di Ostetricia e Ginecologia, Ospedale
Sant’Anna, Torino; ***Dipartimento di Anatomia Patologica e Medicina di Laboratorio, Istituto Oncologico Europeo, Milano
About 75% of breast cancer grows because they are stimulated by steroid hormones, therefore, the expression of Estrogen (ER) and Progesterone Receptors (PgR) is the most
important prognostic and predictive factor. On the other
hand the role of the androgen receptor (AR) is still debated
and many studies are currently addressed to investigate its
prognostic value. A study performed in our institution 1 on a
retrospective series of 953 patients with ER positive breast
cancers showed that the immunohistochemical expression
of AR is closely related to small tumors (<2cm), with a
low proliferative index and without lymph node metastases
involvement, as previous described by other studies 2-4. In
addition, the follow-up analysis of these patients showed
that AR has a significant positive prognostic impact both
on overall survival (OS) and disease-free survival (DFS).
More specifically, subdividing the general population, based
on the type of treatment, we demonstrated that in patients
receiving only anti-hormonal treatment AR correlates with
DFS but not with OS, on the other hand in patients undergoing chemotherapy combined with endocrine therapy AR
showed a positive prognostic value both in DFS and OS. To
further explore the prognostic value of AR in patients with
poorer outcome, we selected a series of cases classified on
the basis of immunohistochemical markers as closely similar
to the genetically defined Luminal B, as stated by Cheang
M.C. et al. 5, considering as Luminal B HER2 positive tumors and HER2 negative tumors with Ki67 >14%. Univariate analysis showed that the positive prognostic value on OS
of AR was maintained within this category.
A significant lower AR expression was demonstrated for triple negative or ER negative as the non- triple negative or ER
positive breast cancer 6. The same study, that evaluated AR
expression by immunohistochemistry on a series of primary
breast cancer patients treated with neoadjuvant chemotherapy
6
, showed that patients with an AR-negative tumor have a
higher chance of achieving a pathological complete response
(pCR) than those with an AR-positive one, but, patients with
AR-positive tumors have a better survival especially if they
did not achieve a pCR.
All these data confirm the role of AR as a favorable prognostic
factor in patients with breast cancers. These results suggested
that AR could be an important regulator of chemo-endocrine
response and could pave the way for specific clinical trial that
will use AR to select patients with ER-positive breast cancer
to be treated with chemoendocrine therapy. Moreover AR
expression adds independent information for the possibility to
achieve pCR after neoadjuvant chemotherapy.
101
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References
1
Castellano I, Allia E, Accortanzo V, et al. Androgen receptor expression is a significant prognostic factor in estrogen receptor positive
breast cancers. Breast Cancer Res Treat. 2010;124:607-17.
2
Rakha EA, El-Sayed ME, Green AR, et al. Prognostic markers in
triple-negative breast cancer. Cancer 2007;109:25-32.
3
Ogawa Y, Hai E, Matsumoto K, et al. Androgen receptor expression
in breast cancer: relationship with clinicopathological factors and
biomarkers. Int J Clin Oncol 2008;13:431-5.
4
Park S, Koo J, Park HS, Kim JH, et al. Expression of androgen receptor in primary breast cancer. Ann Oncol 2009;21:488-92.
5
Cheang MC, Chia SK, Voduc D, et al. Ki67 index, HER2 status, and
prognosis of patients with luminal B breast cancer. J NatI Cancer Inst
2009;101:736-50.
6
Loibl S, Müller BM, von Minckwitz G, et al. Androgen receptor
expression in primary breast cancer and its predictive and prognostic
value in patients treated with neoadjuvant chemotherapy. Breast Cancer Res Treat 2011 Aug 12.
La valutazione dei margini chirurgici
F. Pietribiasi, J. Prestipino
Ospedale S. Croce Moncalieri (TO) ASL TO 5 Regione Piemonte
Il principale svantaggio della terapia chirurgica conservativa delle neoplasie mammarie è il rischio di recidiva locale
(RL), corrispondente all’8-10% a 10 anni di follow- up. Lo
stato dei margini chirurgici (MC) è uno dei principali fattori
predittivi di RL ed è un parametro indispensabile nel report
anatomopatologico sia delle lesioni invasive che in situ della
mammella.
L’accuratezza e la standardizzazione sia delle modalità di
invio che di trattamento macroscopico del pezzo operatorio
sono requisiti fondamentali per una valutazione precisa dei
MC che si basa in definitiva sulla misurazione della distanza
della lesione da tutti i margini chinati e sulla valutazione
dell’ estensione lineare del/i margini eventualmente coinvolti
(positivi). “Cellule di carcinoma sulla china” è la definizione
più diffusa di margine positivo nelle lesioni infiltranti; meno
condivisa invece è la definizione di margine negativo o di
margine a ridosso (“close”). Numerosi fattori sono in grado
di predirre la positività dei MC; tra questi i principali sono:
l’istotipo lobulare, il grado G3, la giovane età, la presenza di
estesa componente intraduttale, il diametro tumorale. Lo stato
dei MC va discusso in un contesto multidisciplinare, con il
Patologo, con il Chirurgo, ed il Radioterapista per selezionare
in modo accurato quelle pazienti da sottoporre ad ampliamenti
chirurgici o a sovradosaggi RT per ottenere una radicalità
oncologica senza trascurare gli esiti estetici. Anche nelle più
recenti tecniche chirurgiche di oncoplastica i MC hanno un
ruolo importante la cui valutazione è stata oggetto di protocolli specifici.
Tumor- and tumor-like lesions of the mammary
stroma: potential mimickers of invasive
carcinoma
G. Magro
Dipartimento G.F. Ingrassia, Università di Catania
Mammary stroma encompasses a wide spectrum of tumorand tumor-like lesions that may represent potential diagnostic
pitfall of invasive carcinoma. Among the different stromal
lesions, especially myofibroblastoma, spindle cell lipoma-like
tumor, desmoid-type fibromatosis and inflammatory (myofibroblastic) pseudotumor need to be distinguished from some
special histotypes of breast carcinoma, such as low-grade
fibromatosis-like spindle cell metaplastic carcinoma, invasive
lobular and apocrine carcinoma.
Desmoid-type fibromatosis is a low-grade mesenchymal
tumor that only rarely occurs in the breast parenchyma. This
lesion is similar, if not identical, to its counterpart occurring
in soft tissues. Interestingly, there is a low-grade spindle
cell metaplastic breast carcinoma that looks like a desmoidtype fibromatosis. A correct differential diagnosis between
these two lesions is crucial because of different treatment
and prognosis. Inflammatory pseudotumor is a reactive lesion that only rarely occurs in the breast parencyhyma. It is
mainly composed of spindle cells arranged in a fascicular pattern, with a variable degree of nuclear pleomorphism. Apart
from many malignant spindle cell tumors, including leiomyosarcomas, fibrosarcoma/malignant fibrous histiocytoma,
spindle cell liposarcoma, peripheral nerve sheath tumors and
myoepithelioma, the differential diagnosis of inflammatory
pseudotumor also revolves around spindle cell metaplastic
breast carcinoma. Myofibroblastoma is the prototypic benign
tumor of the mammary stroma, composed of cells showing
a variable degree of fibro-myofibroblastic differentiation at
different levels (morphologic, immunohistochemical, and ultrastructural levels). Although myofibroblastoma is a spindle
cell tumor, there is increasing evidence that it encompasses
a wide morphologic spectrum. This benign tumor should be
distinguished by the low-grade fibromatosis-like spindle cell
metaplastic carcinoma, especially when it exhibits infiltrative margins or contains a large amount of intratumoral fatty
component (so-called lipomatous myofibroblastoma) that
intermingles with the spindle cell component. Similarly, spindle cell lipoma-like tumor, a benign tumor of the mammary
stroma, closely resembling soft tissue spindle cell lipoma,
may exhibit a pseudo-infiltrative growth pattern mimicking a
malignant lesion. Epithelioid cell variant of myofibroblastoma
may represent a diagnostic challenge. In this tumor variant,
variably-sized epithelioid cells are arranged in clusters or in
alveolar, solid, trabecular or single cell growth patterns, and
they are embedded in a myxoid to fibrous stroma. Due to its
morphology, epithelioid cell myofibroblastoma is closely
reminiscent of invasive lobular carcinoma and sometimes
invasive apocrine carcinoma.
In the present lecture morphological and immunohistochemical features helpful in distinguishing the above mentioned
stromal lesions from peculiar histotypes of invasive carcinoma are discussed, providing an update on the topic.
102
Problematiche in patologia uterina
Moderatori: Michele De Nictolis (Ancona), Gaetano De Rosa (Napoli)
Smooth muscle tumors of uterine body
G. F. Zannoni
Anatomia Patologica, Policlinico A. Gemelli, Università Cattolica del
Sacro Cuore, Roma
Leiomyomas are the most common uterine tumor, occurring
in up 75% of hysterectomy specimen. The clinical manifestations are related to their number, size and location and typically include pelvic pain, abnormal vaginal bleeding and uterine
enlargement. These tumors are multiple in about 75% of cases
and are typically confined to the corpus of uterus. Leyomiomas are typically round, well circumscribed, no encapsulated
myometrial mass. They are constituted by elongated spindle
cells, with appreciable eosinophilic cytoplasm and central,
pale, fusiform nuclei forming intersecting fascicles. Variable
amounts of collagen, which typically increase with age, separate the tumor cells. In most cases there is sharp border with
the surrounding myometrium. Immunonostaining is rarely
required for diagnosis: leyomiomas typically stain for desmin
and h-caldesmon, smooth muscle myosin heavy chain.
It is important recognize the different leyomiomas variants.
They may cause problems in diagnosis as they share one or
more histological features with leyomiosarcoma, such as high
cellularity, striking cytologic atypias and increased mitotic
activity.
Cellular and highly cellular leyomiomas: this variant show
high cellularity comparable to that encountered in endometrial
stromal tumors, thus the cells have small oval to spindled nuclei with very scant cytoplasm. The cells often form fascicles
and at the periphery of the tumor, the neoplastic cells merge
imperceptibly with the surrounding myometrium. This variant
has has very low mitotic count and are citologically bland;
rarely they can show increased mitotic rate and thus are defined as mitotically active highly cellular leyomiomas.
Leyomioma with bizarre nuclei (pleomorphic or symplastic or atypical): the histological marker is the presence of
atypical pleomorphic nuclei, frequently multinucleated but
sometimes mononucleated, associated with abundant eosinophilic cytoplasm. Typically the atypical cells have spotty
distribution within the tumor, only occasionally the cells may
be seen uniformly throughout. The cells can show prominent
nucleoli. The mitotic count, although can reach 7x10HPF by
the highest mitotic count method, is typically 1-2x10HPF. No
coagulative necrosis is seen.
Mitotically active leiomyoma: this tumor show increased
mitotic activity that ranges from 5 to 15 mitosesx10HPF. The
single most important criterion to separate this entity from
leiomyosarcoma is the absence of cytological atypia. These
tumor are not infrequently associated with areas of hemorrhage or show degenerative changes. Probably the increased
mitotic activity in this tumor is related to mitogenic effect of
progesterone in the myometrium during the menstrual cycle.
Leyomioma with hormonal-related changes: these changes
are related to pregnancy or progestin and include hemorrhage,
edema, mixoid change, focal hypercellularity, nuclear pleomorphism and increased mitotic activity. Red degeneration,
which tipically occurs in pregnant woman or less commonly
in those on oral contraceptives, result in a beefy red appearance and is due to infarction and hemorrhage with subsequent
hemolysis. Microscopic examination characteristically reveals
densely cellular proliferation of bland, occasionally mitotically active smooth muscle cells surrounding stellate zones of
recent hemorrhage.
Myxoid leiomyoma: this variant is very uncommon. A gelatinous gross appearance is present in some cases. Microscopic
examination show spindle and stellate tumor cells separated
by abundant, weakly basophilic, alcianophilic material. The
border are typically uniform and regular. No mitotic activity
is seen.
Leiomyoma with vascular invasion: this variant represents
a typical leiomyoma or leyomioma variant with microscopic
intravascular growth within the tumor
Intravenous leiomyoma: in this variant the histological
marker is represented by the presence of endothelium-covered
protrusions of smooth muscle resembling either a typical leiomyoma with spindle cells arranged in intersecting fascicles or
the appearance of any leiomyoma variant, showing clefted or
lobulated contours.
Uterine leiomyosarcoma constitutes 1% of all uterine malignancies, it is the most common uterine sarcoma and represents
approximately 40% of all uterine sarcomas. It occurs most
commonly as a single nodule in almost 90% of cases. Leiomyosarcomas typically forms an intramyometrial mass with
either well-circumscribed or irregular infiltrative growth into
the surrounding myometrium. The diagnosis of malignancy
in a smooth muscle tumors is based on three histological features: 1) tumor cell necrosis; 2) moderate to severe cytological
atypias; 3) mitotic activity. Tumor cell necrosis is defined by
the finding of an abrupt transition between the nonviable and
viable tumor. The viable tumor frequently grows around vessels. Pleomorphic cells may still be identified in viable and
devitalized areas. In most case tumor necrosis is accompanied
by tumor cells showing increased mitotic activity and marked
cellular atypias. Moderate to severe atypia is defined by cellular pleomorphism, nuclear enlargement and/or irregular outlines, hyperchromatism, as well as prominent nucleolus. Cytological atypia should be identified at medium power (10x).
Finally, counting mitotic activity in smooth muscle tumors
may be difficult but it is important not to misinterpret apoptototic cells as mitotic figures. Apopototic cells are typically
characterized by refractive dense eosinophilic cytoplasm and
coarse clumped chromatin, which contrasts with the delicate
and thin appearance of the dividing chromatin. Even though
mitotic activity had been considered the most important
criterion to establish a diagnosis of malignancy in a smooth
muscle tumor, it has been demonstrated that mitotic activity in the absence of one of the other two histologic features
previously described is insufficient to establish the diagnosis
of leiomyosarcoma. Leiomyopsarcormas are divided in three
main categories depending on their morphological appearance: spindled, epitheliod and mixoid.
Spindle cell leiomyosarcoma is composed of fusiform cells
showing central elongated nuclei with blunted end occasionally indented by a clear vacuole. The cytoplasm is deeply eosi-
relazioni
nophilic due to the presence of myofilaments that are disposed
parallel to the cell axis.
Epitheliod leyomiosarcoma is composed of sheets, nests, or
cords of the cells with abundant cytoplasm. The criteria to
establish the diagnosis of malignancy in epitheliod smooth
muscle tumors are not well established. However as a general
rule the diagnosis of epitheliod leyomiosarcoma is warranted
when there are >5 mitoses x 10HPF and diffuse moderate to
severe cytologic atypia or tumor cell necrosis.
Mixoid leiomyosarcoma is a rare variant of uterine leiomyosarcoma. It is characterized by the presence of abundant
myxoid matrix that is positive for Alcian Blue. The tumors are
typically hypocellular in contrast to most spindled and epitheliod leiomyosarcomas. Most tumors show infiltrative growth
into the surrounding myometrium.At higher magnification,
the degree of cytologic atypia and mitotic activity is quite
variable. The diagnosis of mixoid leiomyosarcoma is maid
when either marked cytological atypia or tumor cell necrosis
is identified. In their absence, the finding of 2 or more mitosesx10HPF separates myxoid leiomyosarcoma from myxoid
leiomyoma.
Uterine smooth-muscle tumors that show some worrisome
histological feautures but not fulfill the diagnostic criteria of
leiomyosarcoma fall into the category of STUMP (Smooth
Muscle Tumors of Low or uncertain malignant potential).
They include 1)banal leiomyoma with tumor cell necrosis, 2)
necrosis of uncertain type with 10 or more 10 mitoses x10HPF,
3)marked diffuse atypia and borderline mitotic counts.
Endometrial hyperplasia: hot topics
C. Mignogna
Anatomia Patologica, Ospedale Buccheri La Ferla “Fatebenefratelli”
Palermo
Defining the “normal” endometrium is virtually impossible,
because innumerable variables have an influence on the endometrial mucosa.
First of all the age of the patient: thisi is a fundamental information for the pathologist, orienting to a premenarcal, reproductive, pregnancy, perimenopausal and postmenopausal
condition.
Secondarily, at a cycle of life, we have to add the physiological mestrual cycle, with related endometrial morphological
variations. Clinical use of steroid hormones and/or oral contraceptive and other therapies sophisticate the situation.
Endometrial biopsies and curettings are the most common
tissue specimens received in a pathology lab, specimens those
are often irregularly oriented, with “disturbing” blood and
mucus and with sampling artefact that can make the normal
patterns difficult to interpret.
A rational approach that considers the overall clinical history of the patient, clinician/gynecologist diagnostic informations, in association with a specimen adeguancy, appropriate
fixation, sectioning of the tissue is mandatory for a correct
diagnosis.
In this complex scenario we try to analyze endometrial hyperplasia, defined as non invasive proliferation of the endometrium, that results in a spectrum of morphological alterations
ranging from benign changes to premalignant disease.
Many classifications have been used over the past years,
the one that is currently used was proposed by Kurman and
Norris in 1986, and now is sanctioned by the World Health
Organization that classifies the endometrial hyperplasia by
their degree of architectural complexity as simple or complex
103
and by their the cytological features as hyperplasia or atypical
hyperplasia.
So, the diagnosis of endometrial hyperplasia it’s a combined
analysis of glandular/stromal ratio, glands proliferation pattern,
cytological features, blood vessels and presence of other cells.
Simple hyperplasia
The histological appearance of simple hyperplasia is characterized by an increased volume of the endometrium, qualitatively different from normal cycling endometrium. Both glandular and stromal component are involved into the process,
glands are not really crowed, are tubular, although frequently
cystic or angular. Their lining epithelium is pseudostratified to
modestly stratified, with nuclei that maintains their orientation
to the underlining basement membrane. Cells are columnar
with amphophilic cytoplasm, and elongated nuclei lacking
atypia. Mitotic activity can be quite variable, but mitotic rate
do not have influence on the diagnosis of simple hyperplasia.
Stromal component is cellular, may be mitotically active, and
contains small blood vessels resembling the spiral arterioles
of the late secretory endometrium. This small blood vessel
are different from those thick-walled blood vessel that we can
observe in endometrial polyps, that are characterized also by
polipoid fibrotic stroma.
A further entity that goes in differential diagnosis with simple hyperplasia is cystic atrophy, were glandular component
is cystically dilated too, but the glands are lined by reduced
rather than proliferated epithelium and the stroma is dense and
appears atrophic.
Chronic endometritis could be source of overdiagnosis of endometrial hyperplasia, because of glandular reactive changes
which can result in glandular crowding, abnormal gland
shapes and occasional variable degree of cytological atypia.
It’s fundamental searching stromal plasma cells and, neutrophils in the surface epithelium and stromal spilling and
edema.
Fragmentation is frequent during biopsy or courettage, associated with active bleeding with stromal collapse and poor
orientation. In this circumstances glandular component can
appear irregular and crowded.
The telescoping artifact frequently occurs iIn association to
fragmentation frequently occurs the telescoping artifact with
a “gland within a gland” effect that can be mistaken for hyperplasia.
Disordered of proliferative phase are difficult to define and to
differentiate on biopsy specimens, thus some authors 1 suggest
to define a quite normal proliferative endometrium with focally cystic glands, should be better defined as “proliferative
disorder” rather than simple hyperplasia.
Complex hyperplasia
In contrast to simple hyperplasia, the complex hyperplasia
shows a more densely crowded glandular proliferation, with
marked variability in size and shape, with more out-pouchings
and infoldings. Glands are closely packed back-to-back,
although a small amount of intervening stroma is always
present. Cellular component is similar to simple hyperplasia,
with pseudostratification, lacking of atypia and variable mitotic activity.
The differential diagnosis is mainly with atypical complex
hyperplasia, but in this case nuclear atypia is absent.
Atypical hyperplasia
Atypical hyperplasia combines a simple and/or complex architectural pattern with features of cytological atypia.
104
Even if atypical hyperplasia could be associated to simple or
complex pattern, the most frequent pattern observed is the
complex one (complex atypical hyperplasia).
The gland are closely spaced with little intervening stroma,
cells shows loss of axial polarity, there is an irregular stratification, with unusual nuclear shapes accompanied by nuclear
rounding, nucleomegaly, hyperchromatism, irregularity of
nuclear membranes, and prominent nucleoli.
In many cases there is also marked cytoplasmic eosinophilia;
this eosinophilia it’s a helpful feature when present, but is not
specific for atypical hyperplasia.
In that cases were the atypia is focal, the presence of cytoplasmic eosinophilia may alert the pathologist to search the
atypical glands.
Atypical polipoid adenomioma (APA) is an entity that goes
in differential diagnosis with complex and atypical complex
hyperplasia.
APA is a solitary polypoid lesion characterized by an intimate admixture of glandular and stromal component. Glands
shows a complex architectural pattern, with cytological
atypia. Morular/squamous metaplasia is frequently found in
the epithelial component, however cytological atypia of the
squamous epithelium is unusual 2.
Gland are separated instead of endometrial stroma, by a
smooth muscle component composed of intersecting and
swirling fascicles.
The worrier mistake is confusing APA with an endometrial
adenocarcinoma invading the myometrium:. To distinguish it
it’s important to remember that myoinvasion is rarely seen in
courettage specimens, and that glands of APA lack cytological and architectural features of malignancy, and the smooth
muscular component exhibits a swirling pattern not seen in
normal myometrium without desmoplastic stromal response
to invasive cancer.
The differential diagnosis between atypical complex hyperplasia and well differentiate endometrioid adenocarcinoma is
a crucial point.
The diagnostic clue for a well differentiate endometrial adenocarcinoma is the presence of myometrial invasion, but this is a
rare finding in curettings.
There is not a single diagnostic criteria useful alone, also in
this case the diagnosis is composed of many considerations.
Cyologic features are a little help, also even because in atypical hyperplasia is frequent to observe cytological malignancy,
also more than in low-grade adenocarcinomas, so nuclear
atypia alone, is not a fundamental distinguishing feature.
More important is the so called “stromal disappearing” indicating the presence of back-to-back gland growing. This features is in association with stromal desmoplasia and stromal
necrosis with necroinflammatory debris replacing the stroma
are fundamental findings for the diagnosis of low-grade adenocarcinoma. A papillary pattern
Some authors assert that the gold standard for validation the
diagnosis of adenocarcinoma is the presence of myometrial
invasion in the subsequent hysterectomy specimen3. Obviously this standard it is virtual and often unreliable just for the
fact that not all patient with a diagnosis of atypical hyperplasia
undergo immediately to a hysterectomy. So, as Sirvelberg
teaches, the real goalslas that may be achievable include
never havingavoiding a myoinvasive lesion underdiagnosed
as hyperplasia, or never having a hysterectomy performed for
a lesion that is not myoinvasive 1.
It is important to conclude with a last consideration that is
thereupon the risk “progression” / behaviour of the endometrial hyperplasia.
An interesting study of Lacey 2008 and a previously study
of Kurman 1985 4 concludes that the presence of cytological
atypia, and not the simple or complex pattern is the most important parameter for cancer progression.
Other studies also find that 17-25% of patient undergo a
hysterectomy soon after the diagnosis of atypical hyperplasia
at biopsy have a well-differentiated adenocarcinoma in the
uterus5. So it is evident the importance and all the clinic and
prognostic implication related to the diagnosis of endometrial
hyperplasia.
Some researchers proposed a new classification for hyperplasia composed of two group of pathologies the first one with
little to not risk of evolution to carcinoma called “endometrial
hyperplasia” and a second called “endometrial intraepithelial
neoplasia (EIN) used to describe true adenocarcinoma precursor lesions 6.
Until now the WHO classification it is still in use.
Diagnosis of endometrial hyperplasia hot points- topics:
1. Clinical data – histeroscopic appearance.
2. Dialogue and cooperation with the gynaecologist.
3. Light microscope analysis.
• Glandular/stromal ratio.
• Glands proliferation pattern .
• Cytological features.
• Blood vessels - Presence of other cells.
• Myometrial invasion.
References
1
Silverberg SG. Problems in differential diagnosis of endometrial hyperplasia and carcinoma. Mod Pathol 2000;13:309-27.
2
Longacre TA, Chung MH, Rouse RV, et al. Atypical polypoid adenomyofibromas (atypical polypoid ademomyomas) of the uterus: a clinicopathological studi of 55 cases. Am J Surg Pathol 1996;201:1-20.
3
Hendricskson MR, Ross JC, Kempson RL. Toward the development
of morphologic criteria for well-differentiated adenocarcinoma of the
endometrium. Am j Surg Pathol 1983;7:819-38.
4
Kurman RJ, Kamisnski PE, Norris HJ. The behaviour of endometrial
hyperplasia. A long term behaviour of endometrial hyperplasia. A
long term study of “untreated” hyperplasia in 170 patients. Cancer
1985;56:403-12.
5
Mazur MT, Kurman RJ. Diagnosis of endometrial biopsies and curettings. A pratical approach. Second Edition. Springer 2005, pp. 193.
6
Dietel M. The histological diagnosis of endometrial hyperplasia. Is
there a need to simplify? Virchows Arch 2001;439:604-8.
105
relazioni
Diagnostica delle lesioni melanocitarie
Moderatore: Nunzia Scibetta (Palermo)
Tumori melanocitici “borderline” della cute:
quali novità per tumori di difficile diagnosi e di
prognosi incerta?
C. Clemente
Anatomia Patologica Casa di Cura San Pio X e IRCCS Policlinico
San Donato, Milano
Alcune aree della patologia dei tumori melanocitici della cute
sono ancora oggetto di dubbi e controversie e non tutti gli
autori concordano sulla terminologia di alcune entità ed in
particolare poco è ancora conosciuto sulla loro caratterizzazione clinico-biologica. Anche i criteri diagnostici istopatologici non sono ancora ben definiti e spesso si dimostrano
scarsamente riproducibile anche tra esperti. Negli ultimi anni
un grande aiuto per la diagnosi di nevi e melanomi è venuto
dalla dermatoscopia con l’analisi delle immagini, esame che
si colloca in posizione intermedia tra la diagnosi clinica/
macroscopica e la diagnosi istopatologica. Tutta la procedura
diagnostica per la definizione di una lesione melanocitica
(esame del campione, descrizione macroscopica, prelievo e
diagnosi istologica) deve iniziare e tener conto dal quadro clinico/macroscopico e dermatoscopico. È quindi importante che
il patologo riceva dal dermatologo/dermatoscopista, insieme
al materiale escisso, anche le immagini cliniche/dermatosco-
piche in quanto sono informazioni essenziali ed irrinunciabili
per la diagnosi conclusiva. La classificazione istologica dei
tumori melanocitici è scarsamente correlata con la prognosi
ma è comunque importante individuare le differenti entità con
un nome riproducibile e ben identificabile. Nella tabella 1 ed
infine quello delle melanocitosi dermiche suddivise anch’esse
in melanocitosi dermiche benigne, atipiche e maligne.
Il gruppo di tumori melanocitici atipici e le melanocitosi dermiche atipiche sono caratterizzati istologicamente da quadri
morfologici ed biologici (immunoistochimici e molecolari)
che differiscono dai nevi e dai melanomi anche se ancora ben
poco definiti. Anche il comportamento biologico di tali entità
è ad oggi difficilmente prevedibile. Non di meno queste nuove
entità devono essere identificate e differenziate dai tumori melanocitici benigni e da quelli maligni per far sì che si abbiano
maggiori possibilità di raccoglierle e studiarle, soprattutto
per cercare di indicare al paziente una corretta definizione
terapeutica e prognostica. I tumori melanocitici atipici sono
stati descritti in letteratura con differenti nomi: melanocytic
tumor of uncertain malignant potential, severely atypical
melanocytic proliferations, borderline melanocytic tumor,
nevomelanocytic tumors of undetermined risk,ecc. Riteniamo
che si possano raccogliere tali tumori sotto una definizione
generale di tumori melanocitici atipici, riconoscendo in
Proposta di classificazione dei tumori melanocitici (Clemente, 2011)
Nevi:
Melanoma acrale lentigginoso
Lentigo simplex
Melanoma mucoso lentigginoso
Nevo giunzionale, composto e dermico
Melanoma nodulare
Nevo congenito
Melanoma, varianti rare
Nodulo di proliferazione in nevo congenito
Melanoma in nevo congenito
Nevo alonato
Melanoma nevoide
Nevo dei genitali
Melanoma a deviazione minima
Nevo ricorrente
Melanoma spitzoide
Nevo pigmentato a cellule fusate (nevo di Reed)
Altre varianti rare
Nevo a cellule epitelioidi e fusate (nevo di Spitz)
Melanocitosi dermiche:
Nevi, varianti rare
Nevo blu
Nevo displastico
Displasia melanocitica intraepiteliale epitelioide
Nevo blu cellulato
Tumori melanocitici atipici (borderline)
Nevo desmoplastico
Tumore di Reed atipico
Nevo penetrante profondo
Tumore di Spitz atipico
Melanocitosi dermiche atipiche (borderline)
Tumori melanocitici atipici, varianti rare
Nevo blu cellulato atipico
Melanoma
Melanocitoma epitelioide pigmentato
Melanosi premaligna (melanoma in situ)
Tumore penetrante profondo atipico
Lentigo maligna
Melanocitosi maligne
Melanoma a diffusione superficiale
Melanoma desmoplastico
Melanoma tipo lentigo maligna
Nevo blu maligno
106
questo gruppo varianti che si ricollegano alle corrispondenti
entità benigne e maligne. I caratteri morfologici principali che
debbono essere presi in considerazione per l’identificazione
di una tumore melanocitico atipico sono: l’architettura della
lesione ed in particolare i bordi e simmetria, la presenza di mitosi, in genere superiori a quelle riscontrabili in nevo, la irregolarità dei nidi con moderato polimorfismo citologico, scarsa
o irregolare maturazione in profondità. Utile per la diagnosi
istologica soprattutto per escludere un melanoma spitzoide in
diagnosi differenziale con un tumore di Spitz/Reed atipico è
l’utilizzo del siero anti p16 e l’analisi con FISH interfasica.
È un dato già consolidato da casistiche, anche se non così
numerose, riportate in letteratura, che in una percentuale non
indifferente dei casi di tumori melanocitici atipici l’esame del
linfonodo sentinella risulta positivo, pertanto è utile che tale
indagine venga eseguita. È auspicabile che si possano identificare anche ulteriori criteri per selezionare gruppi di pazienti
con tumori melanocitici atipici ad alto o basso rischio da sottoporre o meno all’esame del linfonodo sentinella. Pur non
essendo ancora comprovata da casistiche adeguate riportate in
letteratura sembra tuttavia che la prognosi, anche in presenza
di metastasi linfonodali, non sia comparabile ed equivalente
ad un analogo melanoma. Solo con la raccolta di casistiche più
ampie potremo rispondere ai tanti quesiti ancora aperti.
Stato dell’arte e linee guida nelle applicazioni
immunocitochimiche del melanoma primitivo
e del linfonodo sentinella
S. Staibano
Dipartimento Scienze Biomorfologiche e Funzionali Università Federico II, Napoli
L’utilizzo di tecniche ancillari risulta spesso prezioso nella
discriminazione fra alcuni nevo e melanoma, fra melanoma
e tumori di origine non melanocitica, fra varianti non ordinarie di melanoma, nonché in alcuni casi di difficile diagnosi
differenziale fra nevo ricorrente e melanoma. L’immunoistochimica riveste un ruolo importante a tal riguardo. Pur
considerando che attualmente non esistono singoli marcatori
immunoistochimici, o specifiche combinazioni di marcatori,
in grado di assicurare in tutti i casi dubbi la diagnosi differenziale inequivocabile fra melanoma e nevo melanocitico, e
sottolineando che è necessario analizzare attentamente il pattern di espressione e la localizzazione del segnale nel contesto
dei caratteri morfologici delle singole lesioni,questa tecnica
risulta di indubbia utilità nella definizione diagnostica della
maggior parte dei casi dubbi, soprattutto in assenza di dati
relativi alla storia clinica dei pazienti. Verranno presentati i
dati relativi allo stato attuale ed alle tendenze internazionali
concernenti il ruolo dell’immunoistochimica nella diagnostica
routinaria del melanoma e del linfonodo sentinella, mediante
utilizzo dei marcatori tradizionali (proteina S100, HMB45,
MART-1/Melan-A, tirosinasi, MITF, Ki67), evidenziando
inoltre i possibili pitfalls della metodica (legati, ad esempio,
anomala espressione di CD34, citocheratine, EMA, associata
ad assente espressione di proteina S100 e markers specifici
delle linea melanocitaria).Verrà inoltre fornito un breve cenno
al trend attuale internazionale di ricerca di nuovi marcatori di
possibile ausilio per la diagnosi e la valutazione prognostica
delle lesioni melanocitiche.
Molecular-genetics in melanoma diagnosis
D. Massi
Sezione di Anatomia Patologica, Dipartimento di Area Critica Medico-Chirurgica, Università degli Studi di Firenze
There have been great advances in our understanding of the
molecular pathogenesis of melanoma during the past decade
and these are already contributing to new ways to diagnose
and classify melanoma and treat patients with metastatic
disease.
A novel sensitive and specific molecular tool for the diagnosis of non-ambiguous melanocytic lesions is represented by
fluorescence in situ hybridization (FISH) performed by using
a panel of four probes, including three locus specific (RREB1,
MYB and CCND1) genes. It should be underscored that FISH
is not a replacement for light microscopy, but can be considered as a powerful ancillary diagnostic tool, and additional
work is required to develop new probes directed to identify
of specific subsets of melanocytic lesions. Improvement of
FISH sensitivity and specificity should be researched through
a continuous scrutiny of the FISH-probes. Prognostic data
predicting metastasis in a manner independent of tumor has
been recently obtained by the FISH test. Emphasis should be
placed on quality control and monitoring for proper clinical
validation as these tests may affect patient care.
Aberrant activation of the mitogen-activated protein kinase
(MAPK) pathway have been found in over 80% of primary
melanomas, which result from mutations in proteins along
the RAS-RAF-MEK-ERK pathway are thought to be mutually exclusive. All subtypes of melanoma seem to show such
mutations, including cutaneous (50% BRAF, 15% NRAS, up
to 17% CKIT chronic sun damage), mucosal (11% BRAF,
5% NRAS, 21% CKIT) and uveal (50% GNAQ) melanomas.
BRAF mutant melanomas typically occur on skin sites intermittently exposed to the sun and histologically they show
features of superficial spreading melanomas. Associatiation
has been documented also with a high degree of pagetoid
spread, prominent nesting, heavy melanin pigmentation, large
epithelioid cells, circumscription, epidermal thickening, and
minimal solar elastosis. In contrast, tumors with histological
features acral lentinginous and mucosal lentiginous melanomas usually show KIT mutations.
Oncogenic mutations in melanoma are now beginning to be
used as therapeutic targets, and in particular four mutations
(BRAF, GNAQ, CKIT, NRAS) of the MAPK pathway represent targets for systemic therapies. An updated melanoma
classification should take in consideration these new discoveries and data to integrate molecular marker with tumor
outcome and therapy.
Current investigation on RNA expression, proteomic and
microRNA analysis of melanomas is adding important information on mutation status. Investigating small RNAs, including miRNAs, may significant impact on the improvement of
melanoma prognostication by providing a series of biomarkers
associated with a specific clinical behavior. However, major
challenges for the future will be represented by the definition
of the clinical relevance of this information and how to utilize
it for melanoma classification and personalized patient care.
107
relazioni
Microenvironment and melanoma
G. Botti, A.M. Anniciello, M. Cerrone, G. Scognamiglio, M.
Cantile, G. Liguori, M. Farina, O. Sacco, R. Franco, S. Ferrone*
Dept of Pathology and Cytopathology, National Cancer Institute,
Fondazione G.Pascale, Naples - Italy; * Department of Immunology,
University of Pittsburgh School of Medicine - USA
Background
Skin melanoma is a cancer arising from the melanocytes of
the epidermis and occasionally from hair follicles. Areas of
the body primarily affected by melanoma are the trunk (chest
or back) in men and the legs of women, but it, also, can origines in other places (mucous membranes, eye, inner ear and
the meninges).
The incidence of melanoma is steadily growing around the
world, with an increase particularly among young individuals. The out-of-control of cell growth and the loss of cellular
homeostasis play a crucial role in the genesis of the tumor and
in progression. These mechanisms control the proliferation,
differentiation and apoptosis of melanocytes at the junction.
In particular, interactions between transformed cells and
transformed cell with extracellular matrix play a key role in
the progression of melanoma.
Ulceration, the most important adverse prognostic factors, is
defined histologically as the absence of an intact epidermis
overlying a significant portion of the primary lesion, with associated host response by inflammation, granulation tissue,
fibrin, and thinning, loss or reactive hyperplasia of the epithelium adjacent to ulceration. Survival rates for patients with an
ulcerated melanoma are lower than those of patients with a not
ulcerated melanoma of equivalent stage but are remarkably
similar to those of patients with a not ulcerated melanoma of
the next highest stage.
The ulceration is associated, as demonstrated by numerous
authors, with changes in the cell microenvironment: proliferation of the tumor nearby of the epidermis may erode it by
contact and thus favor tumor expansion.
Melanoma can be good treated if early detected and removed,
but can become incurable when metastasizes. Melanoma has
been refractory to most standard systemic therapy. Traditional
cancer treatments such as chemotherapy have proven inadequate to stop metastatic melanoma progression.
Chemotherapy has also proven ineffective at treating Stage
III melanoma. However, drug development in melanoma is
changing, developing therapies that target activating molecules and their pathways such as adjuvant therapy with
inhibitors of mutated B-RAF. Several studies suggest that
lack of response to immunotherapy and the development
of progressing metastases in cancer patients seem to be associated with immune selection of HLA (human leukocyte
antigen/ MHC-Major Histocompatibility Complex)-deficient
tumor cell variants.
Aim
Our main objective is precisely to study cell microenvironment modifications, in relation to different responses
(increased Disease Free Survival) to adjuvant therapy in
ulcerated melanoma patients in the third stage compared with
patients of the same stage but without ulceration. A major
focus of our research is investigate:
the expression of major ECM proteins (in particular, osteopontin, SPARC, tenascin-C and CCN) in relation to the
stage, in presence of ulceration and therapy with INF;
the role of matrix proteins in the deregulation of melanocytes
in neoplastic progression;
the mechanisms, autocrine and paracrine, by which these proteins interfere with the HLAI antigens;
the development of more effective and tolerable alternative
adjuvant therapies.
Methods
We selected, from our institutional tissue Archive, a large
group of intradermal and dysplastic nevi, pT1 melanomas,
pT2, pT3 and pT4, ulcerated or not ulcerated. Ulcerated pT1
was not possible to analyze because they had only small and
superficial ulceration. We began to analyze the immunohistochemical expression profile of matrix protein osteonectin/
SPARC (Novocastra-Leica,UK) and key molecules (Department of Immunology, University of Pittsburgh School of
Medicine, USA) involved in regulated proteolysis via the
proteasome/immunoproteasome.
RESULTS: Preliminary data show that Osteonectin/SPARC
has cytoplasmic immunoreactivity that increases with tumor
progression. The alpha chains of HLAI appear negative in the
intradermal nevus (Fig 1), while in melanoma cells there is
a strong membrane positivity in not-ulcerated (Fig 2) forms
(pT2, pT3). For ulcerated lesions, membraneus positivity
disappears (Fig 3) and in metastatic lesions we observed only
cytoplasmic immunoreactivity (Fig 4). HLAII is negative in
benign, dysplastic and malignant melanocytes, but it is typically positive in peri/intratumoral inflammatory cells.
The molecules of the proteasome, particularly delta and MB1
antigens, show very high nuclear and cytoplasmic positivity
in intradermal and dysplastic nevi. In melanoma cells, nuclear
immunoreactivity decreases and disappears completely in
pT3, pT4 and in metastasic lesions; cytoplasmic positivity is
fairly constant. For LMP7 and LMP10 immunoproteasome
molecules, nuclear and cytoplasmic positivity is low and decreases gradually from benign to malignant cells.
The family of proteins ABC, in particular TAP2, shows nuclear and perinuclear cytoplasmic positivity, which becomes
only cytoplasmic with increasing stage of melanoma.
Conclusions
As previously reported, important tumor escape mechanisms
in melanoma consist of dysregulation of ECM proteins and
HLA1 antigens.
Osteonectin/SPARC immunoreactivity may have prognostic
Fig. 1. Nevus 40X.
108
Fig. 2. pT3 not ulcerated melanoma 40X.
Fig. 3. pT3 ulcerated melanoma 40X.
significance. The molecular MHCI alterations are classificated as reversible and irreversible defects. The former are regulated by cytokines/gammaINF (soft lesions) and the latter are
structural (hard lesions). The nature of the preexisting MHCI
defects in the cancer cell would a crucial impact determining
the successfull of melanoma immunotherapy.
The first step of this study was to select a panel of ECM proteins (SPARC) and specific anti-HLAI antibodies and tested
on tumor samples, to determine potential protein expression
alterations of these molecules in patients with II and III
melanoma stage, ulcerated and not ulcerated.
The other objective of this study was to analyze the subcellular localization of these antibodies by immunohistochemistry.
We observed a membrane localization in samples of ulcerated
melanoma for HLAI and several molecules involved in HLA
synthesis, assembly, transport or expression on cell surface.
Fig. 4. Visceral metastasis 40X.
These molecules are mainly expressed in the cytoplasm of
ulcerated melanoma samples.
In patients with advanced melanoma there is no difference between
presence and absence of ulceration in terms of OS. Patients with
ulcerated melanoma, however, when treated with gamma/INF
obtain an improvement in terms of FDS. INF could increase new
cytokines production in the tumor microenvironment increasing
the HLAI expression only if the tumor cell has reversible defects.
In this case, the lesion will temporarily regress. On the contrary,
if tumor cell has irreversible defect, HLAI expression will remain
low or absent and the lesion will inexorably progress.
Our hypothesis is that ulceration could select cell clones with
reversible defects in HLAI system in melanoma advanced
stage patients, for which adjuvant immunotherapy could be
strongly recommend.
Selected references
Allen M, Louise Jones J. Jekyll and Hyde: the role of the microenvironment on the progression of cancer. J Pathol 2011; 223:162-76.
Baba T Sato-Matsushita M, Kanamoto A, et al. Phase I clinical trial
of the vaccination for the patients with metastatic melanoma using
gp100-derived epitope peptide restricted to HLA-A*2402. J Transl
Med 2010;8:84.
Eggermont AM Suciu S, Santinami M, et al. Adjuvant therapy with
pegylated interferon alfa-2b versus observation alone in resected
stage III melanoma: final results of EORTC 18991, a randomised
phase III trial. Lancet 2008;372:117-26.
Garrido F, Cabrera T, Aptsiauri N. “Hard” and “soft” lesions underlying
the HLA class I alterations in cancer cells: implications for immunotherapy. J Cancer 2010;127:249-56.
Ferrone S, Whiteside TL. Tumor microenvironment and immune escape.
Surg Oncol Clin N Am 2007;16:755-74.
Hemon P, Jean-Louis F, Ramgolam K, et al. MHC class II engagement
by its ligand LAG-3 (CD223) contributes to melanoma resistance to
apoptosis. J Immunol 2011;186:5173-83.
Spatz A, Batist G, Eggermont AM. The biology behind prognostic factors
of cutaneous melanoma. Curr Opin Oncol 2010;22:163-8.
Wheatley K, Ives N, Hancock B, et al. Does adjuvant interferon-alpha for
high-risk melanoma provide a worthwhile benefit? A meta-analysis of
the randomised trials. Cancer Treat Rev 2003;29:241-52.
109
relazioni
Aula Orione – 8.30-11.00
Topics in patologia neoplastica del polmone
Moderatore: Bruno Murer (Mestre - VE)
Anticorpi e regole nella diagnostica
differenziale delle neoplasie polmonari
G. Rossi
Modena
Immunohistochemistry belongs to the diagnostic armamentarium in the routine practice of pathologists, particularly in
the differential diagnosis of pulmonary neoplasms. In the
great majority of cases, pathologists do know the significance of every single marker used with diagnostic intent, but
the value of the immunoexpression depends on the morphologic scenario. For example, TTF-1 is considered a specific
marker of adenocarcinoma, but it is expressed also in a high
rate of small-cell lung cancer, as well as in other neoplastic
or non-neoplastic lesions, such as sclerosing hemangioma,
atypical adenomatous hyperplasia, tumorlets, multifocal
micronodular hyperplasia, peribronchiolar metaplasia. So,
the first key point relies on the correct use of immunostains
together with the adequate morphologic context. Another
important issue concerns the correct interpretation of immunohistochemical expression. Again, TTF-1 is a transcription
factor and only nuclear staining should be considered in
quoting positivity. It is evident that knowledge of the exact
cellular localization of immunostaining is mandatory in discriminating positive versus negative expression (see Cheuk
W, Chan JK. Features of the Antigens Helps Predict the Signal Localization and Proper Interpretation of Immunostains
Subcellular Localization of Immunohistochemical Signals:
Knowledge of the Ultrastructural or Biologic. Int J Surg
Pathol 2004;12;185). Third issue, how many tumor cells
should be stained to quote expression of a marker? This is in
open question without a clear-cut answer. By the way, there
are some practical points that can be helpful. It is mandatory
to premise that there are antibodies stronger than others, as
TTF-1. Based also on my own experience, any convincing
staining for TTF-1 in NSCLC subtyping, even when weak/
moderate and in few tumor cells, should be quoted as a positive signal addressing to adenocarcinoma histotype, while
scattered tumor cells showing a moderate/strong intensity
for p63 are not necessarily a absolute feature in considering
squamous cell differentiation.
This latter point introduces to the main problematic issue on
the use of immunohistochemistry, namely the presence of synchronous staining for different markers. Example: 43-year-old
woman; multifocal, nodular, epithelioid cell proliferation in a
myxoid and jaline matrix; CD31+, pan-cytokeratins+, CD34-.
In this peculiar clinical ground, although pan-cytokeratins+/
CD34- seems to favor an epithelial differentiation (carcinoma), CD31+ assumes a stronger value and a diagnosis of
epithelioid hemangioendothelioma is clearly at the top of
differentials.
Some practical rules:
Before using a marker, it is mandatory to know the significance, the cellular localization, the positive internal control,
the appropriateness of the selected clone and technical conditions.
Please, use immunohistochemistry only in the right morphological context and with at least a handled of differential
diagnosis in your mind.
Remember that the more you stain, the more difficult becomes
your diagnosis
When several antibodies are simultaneously expressed, keep
in mind what marker has a stronger diagnostic value along the
clinical background and correct morphology.
Horizons beyond EGFR: new therapeutic targets
A. Marchetti
Centro di Medicina Molecolare predittiva, Universtità-Fondazione
“G D’Annunzio”, Chieti
The advent of targeted therapies in the management of
NSCLC patients has greatly enhanced the interest for predictive molecular markers that could allow to select patients
maximising efficacy and avoiding toxic effects of treatments.
The identification of predictive biomarkers that can guide
treatment decisions is an important step for individualized
therapy and in ultimately improving patient outcomes.
Patients with lung adenocarcinoma and activating EGFR
mutations respond better to EGFR tyrosine kinase inhibitors
(Gefitinib, Erlotinib) compared with platinum-based therapy.
Therefore, molecular testing is now mandatory for patients
with lung adenocarcinoma and adequate biologic material is
now required to ensure a right histologic diagnosis and to have
sufficient DNA for molecular analysis. Patients with EML4/
ALK fusion gene treated with the ALK inhibitor Crizotinib
in any line of therapy also show a higher response rate and
longer progression-free survival compared with chemotherapy. Thus, advanced NSCLC patients should also have their
tumors tested for the EML4/ALK fusion.
A number of recent genetic studies have shown that nearly
50% of lung adenocarcinomas harbor driver mutations for
which there are now specific inhibitors. These mutations
include KRAS, EGFR, BRAF, HER2, PIK3CA, MEK1, and
AKT1. Most of these mutations are mutually exclusive events
and can be detected by a number of PCR-based mutationdetection tests, including multiplex analysis. Non–small cell
lung cancers may also be driven by amplification of MET and
fusion of ALK, with both being assessable by fluorescence in
situ hybridization (FISH). The presence of specific molecular
markers will increase, and may even replace in the future, the
role of histology in predicting responses as we move toward
an individualized approach.
110
Alterazioni del gene ALK nel carcinoma
polmonare non a piccole cellule
R. Franco*, F. Zito Marino*, G. Aquino*, G. Liguori*, E. La
Mantia*, M.P. Curcio*, M. Cerrone*, M. Cantile*, M. Scrima **,
G. Botti *
SC Anatomia Patologica, Istituto dei tumori “Fondazione G. Pascale”, Napoli; **Laboratori Biogem, Ariano Irpino (AV)
*
Non-Small Cell Lung Cancer (NSCLC) is the most common cause
of cancer-related deaths in the world, despite of improvements in
detection and therapeutic strategies. Currently molecular subtyping
of cancer is often required primarily for its therapeutic implications
because different subtypes may respond to treatment in a different
manner. In particular it is now ascertained that specific EGFR gene
mutations make NSCLC patients responsive to targeted therapy
anti-EGFR based. In fact recent phase III studies showed that
first-line treatment with gefitinib lengthened survival time in lung
adenocarcinoma patients with EGFR mutations 1.
Recently in NSCLC, translocations involving ALK gene have
been described. ALK is a critical protein for cell growth and
survival, being involved in the inhibition of apoptosis and the
promotion of cellular proliferation through activation of downstream PI3K/Akt and MAPK signaling pathways. Genetic
alterations involving ALK including gene fusion, amplification,
mutations and traslocations that lead to different genes fusion in
a variety of human malignancies. In NSCLC the most frequent
traslocation partner of ALK gene is EML4, followed by TGF and
KIF5. EML4-ALK traslocation has been described in 3-7% of all
NSCLC cases. Both ALK and EML4 are localized on the short
arm of chromosome 2 and are oriented in opposite directions.
The chimeric gene, which was generated by a small inversion
within the chromosome 2 short arm, encoded 1059 aa fusion protein, with N-terminal portion identical to the human echinoderm
microtubule associated protein like 4 (EML4) and C-terminal
portion corresponding to the intracellular domain of human
ALK. Thus, 13 variants of ELM4-ALK traslocation have been
described in NSCLC, involving 8 different EML4 exons (exon 2,
6, 13, 14, 15, 17, 18 and 20) and, invariably, exon 20 of ALK. In
the chimeric protein, EML4 preserves the N-terminal coil-coiled
domain (CC), responsible for the dimerization and the constitutive activation of EML4-ALK 2. ALK copy number changes and
amplification are not well characterized in NSCLC, whilst they
plays an oncogenic role in tumors such as neuroblastoma.
In a recently published series of 107 patients, eleven cases (10%)
exhibited ALK amplification and 68 (63%) copy number gains.
There was an association between ALK amplification and EGFR
FISH positivity (p= 0.0001) but not with prognosis 3.
Interest in NSCLC with ALK chromosomal arrangement has
been elicited by clinical trials based on the use of crizotinib
(PF-02341066), a dual MET/ALK inhibitor, responsible of significant clinical activity in patients with documented ALK rearrangement. Currently registration studies comparing crizotinib
to standard chemotherapy are ongoing, in order to accelerate
approval based on the initial phase I results. On the contrary
ALK amplification and copy gain are not characterized in relation to ALK inhibitors clinical response. The absence of association of such ALK gene alterations with ALK protein expression should suggest a not functional gene aberration, related to
high chromosomal instability observed in NSCLC 4.
The low rate of all NSCLC with ALK rearrangement, almost
4% in a recent meta-analysis, is slowing progression of ongoing
trials to get access to the drug. Moreover the rarity of this chromosomal aberration implies the need for a careful filtering of
patients to be examined through expensive diagnostic tests. Fi-
nally the optimal testing method to identify ALK rearrangement
is critical for correct management of patients with NSCLC 5.
The first filter for select NSCLC patient to submit to ALK rearrangement research is at clinical level. All published series underline that ALK rearrangement has been found in never smokers, more frequently in young man. The second level of filtering
is pathologic. ALK rearrangement has been found frequently
in adenocarcinoma. Some series have demonstrated that ALKrearranged NSCLCs were characterized by a conspicuous acinar
growth pattern and extracellular mucus production. On the
contrary, other series showed frequent signet-ring cell elements
associated with solid growth. This apparent contrast may be due
to the small number of ALK-positive cases in each study.
As often histologic features drive the choice of searching
specific biologic target, recently a comparison study of histomorphologic parameters between large series of 54 ALK rearranged NSCLCs and 100 ALK wild type NSCLCs has been
conducted. In ALK-rearranged cases solid or acinar growth
pattern, cribriform structure, presence of mucous cells (signetring cells or goblet cells), abundant extracellular mucus, lack of
lepidic growth, and lack of significant nuclear pleomorphism
were significantly more common than in ALK wild type cases.
In particular a solid signet-ring cell pattern and a mucinous
cribriform pattern, were observed in the majority (78%) of
ALK-rearranged cases, but were rare (1%) in ALK wild type
tumors. The combination of this two parameters represent the
most powerful indicator of ALK rearrangement in multivariate
analysis. Although these interesting data support that specific
histologic features are strictly correlated to specific molecular
anomaly, none of analysed histologic parameters were perfectly
sensitive or specific to ALK rearrangement. Thus, the diagnosis
of ALK-rearrangement need primarily confirmatory studies,
such as FISH, RT-PCR and immunohistochemistry 6.
FISH is the current gold standard method to identify patients suitable to crizotinib trials to date. Positive test is currently defined
if more than 15% of neoplastic cells show split signal, separated
by more than two signals diameter, or single 3’ signal. But more
other patterns of positive tests have been described as one fusion
+ one red signal only, one fusion + one green signal only, one to
two fusion signals + two to three green signals + two to three red
signals, one red + one green signal only (without fusion signal),
one to two fusions + one to four red signals, two fusions + one
BAP, three fusions + one BAP, and two to four fusions + one to
two red signals. Moreover different cut off have been proposed
for each one pattern, generating confusion in interpretation of
data. Thus, FISH is not only a relatively expensive test, but its
interpretation requires deep experience 7.
An alternative method with high sensibility and specificity for
ALK rearrangement detection is multiplex RT-PCR, but the
complexity of this test limits the use in the clinical practice 8.
The possibility to introduce a widely handle method, like immunohistochemistry (IHC) to identify ALK rearrangement in
lung adenocarcinoma could represent a valid alternative.
ALK protein expression is very low in normal tissue and it is
overexpressed in some human neoplasm, but few data have
been produced about relation of immunohistochemistry determination and ALK gene status in NSCLC. Thus a valid IHC
scoring algorithm to predict ALK rearrangement using FISH
has not yet produced.
In a series of 465 resected specimens from South Korea, an ALK
immunohistochemical scoring algorithm has been proposed,
based primarily on intensity of positiveness and then on percentage of positive cells. All the cases with score 3 showed ALK
rearrangement through FISH, and all the cases with scores 0 or 1
did not show ALK rearrangement through FISH. For cases with
111
relazioni
scores 2, 30% were FISH-positive and 70% (7/10) were FISHnegative. The interobserver agreement between pathologists
for both IHC and FISH was excellent, with high κ value. The
sensitivity of IHC test was 100%, and the specificity was 95.2%.
Thus a practical approach has been proposed, as for HER2 in
breast cancer: only cases with IHC score 2 (equivocal cases)
should require further assessment of ALK status through FISH.
At the same time an enriched series of 101 never smokers and
lung adenocarcinoma patients from Mayo Clinic has been tested
through ALK immunohistchemistry, in order to propose a valid
IHC scoring system. Cases have been stratified as score 0 (no
staining), 1 (faint cytoplasmic staining), 2 (moderate, smooth cytoplasmic staining), or 3+ (intense, granular cytoplasmic staining
in 10% of tumor cells). All IHC 3+ cases were FISH+, whereas 1
of 3 IHC 2+ and 1of 21 IHC1 + cases were FISH+. All 69 IHC 0
cases were FISH -. In this case sensitivity and specificity of IHC
test were 90 and 97.8%, respectively (7, 9).
In conclusion, identification of patients that benefit from ALK
inhibitors represents a challenge in the hands of pathologist.
Although ALK-rearranged NSCLCs seem to be histologically
distinct from ALK-wild-type cancer, confirmatory studies to
better select histologically these cases are required. In this set
surgical pathologists could play a critical role in triaging cases for
such confirmatory tests. FISH test and RT-PCR represent the gold
standard methods for ALK rearrangement identification. But different partner for ALK gene and different pattern of FISH results
have been described, rendering very challenging the interpretation
of ALK gene status in each one case. The use of a simple test, as
IHC could represent an appropriate alternative to the development
of a screening plan for ALK rearrangement in NSCLC. However
wide studies are still required to IHC screening test. Finally copy
number gains and amplifications are the most frequent ALK gene
aberration in NSCLC. Whether such aberration may have predictive significance to ALK inhibitor response represents a further
deepening of clinical trials on large series.
References
1
Maemondo M, Inoue A, Kobayashi K, et al. Gefitinib or chemotherapy
for non-small-cell lung cancer with mutated EGFR. N Engl J Med
2010;362:2380-8.
2
Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature
2007;448:561-6.
3
Salido M, Pijuan L, Martínez-Avilés L, et al. Increased ALK gene copy
number and amplification are frequent in non-small cell lung cancer.
J Thorac Oncol 2011;6: 21-7.
4
Kwak EL, Bang YJ, Camidge DR, et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 2010;363:1693-703.
5
Solomon B, Varella-Garcia M, Camidge DR. ALK gene rearrangements: a new therapeutic target in a molecularly defined subset of
non-small cell lung cancer. J Thorac Oncol 2009;4:1450-4.
6
Yoshida A, Tsuta K, Nakamura H, et al. Comprehensive histologic
analysis of ALK-rearranged lung carcinomas. Am J Surg Pathol
2011;35:1226-34.
7
Yi ES, Boland JM, Maleszewski JJ, et al. Correlation of IHC and
FISH for ALK gene rearrangement in non-small cell lung carcinoma:
IHC score algorithm for FISH. J Thorac Oncol 2011;6:459-65.
8
Takeucbi K, Choi YL, Soda M, et al. Multiplex reverse transcriptionPCR screening for EML4-ALK fusion transcripts. Clin Cancer Res
2008;14:6618-24.
9
Paik JH, Choe G, Kim H, et al. Screening of anaplastic lymphoma
kinase rearrangement by immunohistochemistry in non-small cell lung
cancer: correlation with fluorescence in situ hybridization. J Thorac
Oncol 2011;6:466-72.
Diagnosi differenziale tra carcinoma
polmonare e suoi mimi: un approccio pratico
per il patologo
A. Cavazza
Unità Operativa di Anatomia Patologica, Ospedale S. Maria Nuova,
Reggio Emilia
Nella maggior parte dei casi la diagnosi di carcinoma polmonare non pone alcun problema al patologo, ma occasionalmen-
Adenocarcinoma
Iperplasia dei pneumociti
Metaplasia peribronchiolare
Contesto clinico/
radiologico
Generalmente uno o più noduli/
masse, sono possibili opacità a vetro
smerigliato
Generalmente nell’ambito di
una pneumopatia diffusa
Nell’ambito di una
pneumopatia diffusa o come
reperto incidentale
Architettura
Complessità architetturale spesso
presente (ghiandole irregolarmente
affastellate e angolate/ramificate,
impilamenti nucleari che realizzano
strutture papillari o cribriformi)
Complessità architetturale
assente
Complessità architetturale
assente o minima,
localizzazione
bronchiolocentrica
Cellule
In genere colonnari, senza ciglia, con
affollamento dei nuclei; l’atipia può
essere modesta (o anche assente,
in particolare nell’adenocarcinoma
mucinoso) ma è uniforme/
monotona
Appiattite/cuboidali; l’atipia
può essere marcata ma
non è uniforme, con cellule
anche bizzarre fianco a
fianco con cellule blande
Blande, alcune hanno
lunghe ciglia (sempre assenti
nell’adenocarcinoma, da non
confondere con irregolarità
dell’orletto a spazzola che
sono invece spesso presenti
nell’adenocarcinoma)
In genere incospicuo;
occasionalmente flogosi e fibrosi
sono marcate e mascherano
l’adenocarcinoma
Danno acuto polmonare
Spesso fibrotico/infiammato
Assenti
Assenti
Presenti
Raramente positivi
Negativi
Negativi
Background
Cellule basali (evidenziate
ad esempio con p63 o
citocheratina 5)
Citocheratina 20 e/o CDX2
* Modificato da Cavazza et al. Pathologica 2010;102:75-81
112
te è difficile, soprattutto su piccola biopsia e in intraoperatoria
ma a volte anche su pezzo chirurgico. I motivi sono vari: più
spesso riguardano la biopsia, che può avere artefatti e contenere solo poche cellule neoplastiche, ma possono riguardare
anche la neoplasia stessa, che può essere ingannevolmente
blanda ed essere in parte mascherata dalla flogosi e/o dalla
fibrosi. A complicare ulteriormente le cose, alcuni processi
reattivi polmonari sono ingannevolmente atipici e simulano
molto da vicino un carcinoma.
Queste difficoltà possono complicare la diagnosi di qualunque
istotipo di carcinoma polmonare, ma più spesso riguardano
l’adenocarcinoma. La diagnosi differenziale tra l’adenocarcinoma del polmone e i suoi mimi si deve basare su un
insieme di reperti: nei casi difficili nessuno di tali reperti è
diagnostico di per sé e il patologo deve bilanciare con buon
senso e prudenza i dati in favore e contro la malignità. La
tabella riassume le principali caratteristiche morfologiche
dell’adenocarcinoma ben differenziato del polmone e le mette
a confronto con quelle dei suoi principali mimi.
Patologia infettiva
Moderatori: Antonino Carbone (Aviano - PN), Claudio Doglioni (Milano), Aroldo Rizzo (Palermo)
Approccio biomolecolare alla patologia infettiva
C. Parravicini , E. Longhi , C. Tonello , A.L. Ridolfo , S.
Antinori**
*
*
*
**
U.O. Anatomia Patologica, ** III Divisione-Clinica di Malattie Infettive, Ospedale Luigi Sacco, Azienda Ospedaliera-Polo Universitario,
Milano
*
La disponibilità di efficienti metodiche di estrazione degli
acidi nucleici da tessuti inclusi in paraffina 1 ha consentito di
modificare la diagnostica delle patologie infettive affiancando
alle tecniche di istochimica ed immunoistochimica di routine,
procedure di amplificazione genica in fase liquida (PCR,
LAMP 2) in grado di offrire sensibili vantaggi in termini di
sensibilità, specificità e tempi di risposta.
L’introduzione di metodiche di analisi molecolare in fase
liquida presenta tuttavia numerosi punti di potenziale criticità,
il più importante dei quali è costituito dalla rappresentatività
del campione. In questo contesto l’uso di sezioni seriate da
materiale incluso in paraffina offre la possibilità di verificare
istologicamente l’effettiva presenza di lesioni consentendo,
se necessario, una dissezione selettiva delle aree patologiche.
Necessaria al fine di una corretta interpretazione dei risultati, è una adeguata valutazione della qualità del DNA/RNA
estratto in termini di amplificabilità e competenza, così da
escludere l’eventuale presenza di inibitori o di una eccessiva
frammentazione degli acidi nucleici. La disponibilità online delle sequenze genomiche 3 consente di disegnare primer
specifici per singoli patogeni o per intere classi a generi di
microorganismi 4 5 6. In quest’ultimo caso l’amplificazione è
legata all’uso di primer complementari a sequenze genomiche
comuni a batteri, virus o funghi appartenenti ad un determinato ordine, famiglia o specie, mentre l’identificazione dello
specifico microrganismo richiede l’analisi di sequenza del
prodotto di amplificazione 7. Critica per l’implementazione di
metodiche di analisi molecolare in fase liquida è l’aderenza
ad un rigoroso disegno sperimentale che escluda possibili
problemi di contaminazione mediante l’analisi sistematica
di adeguati controlli positivi e negativi. L’interpretazione del
risultato e la sua rilevanza ai fini diagnostici, tuttavia, non
può basarsi unicamente su criteri formali di aderenza ad un
protocollo, ma richiede una attenta valutazione delle coerenza
tra risultato delle indagini molecolari, dati istopatologico e
valutazione clinica del paziente.
Bibliografia
1
Chan PK, Chan DP, To KF, et al. Evaluation of extraction methods
from paraffin wax embedded tissues for PCR amplification of human
and viral DNA. J Clin Pathol 2001;54:401-3.
2
Notomi T. Okayama H, Masubuchi H, et al. Loop-mediated isothermal
amplification of DNA. Nucleic Acids Res 2000;28:E63.
3
http://www.ncbi.nlm.nih.gov/sites/entrez?db=nucleotide
4
Greisen K. Loeffelholz M, Purohit A, et al. PCR Primers and probes
for the 16S rRNA gene of most species of pathogenic bacteria,
including bacteria found in cerebrospinal fluid. J Clin Microbiol
1994;32:335-51.
5
Johnson G, Nelson S, Petric M, et al. Comprehensive PCR-Based Assay for detection and species identification of human herpesviruses. J
Clin Microbiol 2000;38:3274-9.
6
White PL, Shetty A, Barnes RA. Detection of seven Candida species
using the Light-Cycler system. J Med Microbiol. 2003;52:229–38.
7
http://blast.ncbi.nlm.nih.gov/Blast.cgi
Interazione fra microorganismi e linfomi
C. Doglioni
Università Vita Salute San Raffaele Milano
L’infezione da Helicobacter pylori ed il linfoma gastrico
di tipo MALT rappresentano un modello ben noto di stretta
relazione etiopatogenetica fra una infezione batterica, infiammazione cronica e sviluppo di una neoplasia linfoide.
L’identificazione di questi nessi patogenetici ha permesso
l’efficace utilizzo della terapia antibiotica per il trattamento
di questa neoplasia, modificando radicalmente l’approccio
terapeutico. L’ipotesi patogenetica identifica nell’infezione da
Hp una stimolazione antigenica e forse anche auto-antigenica
cronica, che porta ad una proliferazione B policlonale e al
contemporaneo richiamo di elementi infiammatori, in particolare neutrofili nella mucosa gastrica con rilascio di Reactive
Oxygen Species (ROS) e la possibile induzione di aberrazioni
genetiche legate anche alla intrinseca instabilità genetica di
linfociti B durante fenomeni di mutazione ipersomatica e di
ricombinazione isotipica delle immunoglobuline. Alcuni di
questi eventi mutageni sono stati identificati, quali trisomie
dei cromosomi 3, 7, 12 e 18 e le specifiche translocazioni
cromosomiche (1;14)(p22;q32), t(14;18)(q32;q21), t(11;18)
(q21;q21). Alcune di queste trisomie e tutte le traslocazioni
colpiscono geni coinvolti in una stessa via di segnalazione
che porta all’attivazione del fattore di trascrizione nucleare
NFκB, fattore chiave nella mediazione di risposte immuno-
113
relazioni
logiche e nella sopravvivenza e proliferazione di cellule B: la
sua attivazione costitutiva può quindi portare all’incontrollata
proliferazione B ed al linfoma. Nel linfoma MALT gastrico
le osservazioni iniziali attribuivano all’infezione da Hp un
ruolo indiretto nella trasformazione neoplastica, quale stimolo antigenico cronica e alimentatore di flogosi cronica con
possibili danni genetici indotti da ROS. Recentemente è stata
individuata anche una possibile azione oncogene diretta della
proteina cagA, prodotta da alcuni ceppi di Hp e da questi iniettata nelle cellule della mucosa gastrica attraverso un sistema
di secrezione c.d. di tipo IV, dove agirebbe da oncoproteina
epigenetica, in grado di deregolare vie di segnalazione intracellulari, non solo in cellule epiteliali, ma anche in linfociti.
H. pylori può inoltre alterare l’integrità genomica inducendo
l’attivazione dell’enzima AID, una citidina deaminasi usualmente espressa nel centro germinativi, che può trasformare la
sua fisiologica attività di mutagenesi in eventi di tumorigenesi, portando a mutazione di TP53 ed a mutazioni e delezioni
di CDKN2b-CDKN2a. Il modello patogenetico rappresentato
dall’Hp ha aperto la strada all’identificazione di altri microorganismi con ruoli etiopatogenetici simili in altri tipi di linfoma
sia di tipo MALT che non-MALT. La Chlamyidophila psittaci
nei linfomi degli annessi oculari, la Borrelia burgdorferi in
alcuni linfomi cutanei ed il Campylobacter jejuni nella rara
IPSID ne rappresentano esempi, anche se i meccanismi molecolari utilizzati da questi microorganismi nella patogenesi dei
rispettivi linfomi sono ancora in gran parte sconosciuti.
Sometimes, the appearance of a new virus is the mutation of
an already existing one. This is particularly true for Influenza
viruses, where a recombination between animal and human
viruses may periodically give rise to a novel strain remarkably
different from the ones previously circulating, and therefore
not well contrasted by the host immune system. In this particular case, the patogenicity of the new viruse is impredictable and can abruptly change during the initial period of the
circulation in the human host.
New infectious clinical conditions can also arise as a consequence of medical practices. An example of this, beside all
the opportunistic infections that have been seen in transplant
and cancer patients, is for example the occurrence of JC-virus
encephalitis in patients with multiple sclerosis that underwent
treatment with human monoclonal antibody natalizumab. In
this case, an existing virus changes its virus-host relationship
due to a change in the host.
Both viral and host epidemiological factors may contribute to
the emergence of new pathogens, whose impact on the human
species is absolutely not predictable in its nature, timing and
severity The knowledge of the general principles of pathogenhost interplay is of crucial importance for the understanding,
the diagnosis, the treatment and the prophylaxis of these
expected but unforeseenable event that have the potential of
having a great impact not only in the medical field, but to a
greater extent in the human society.
Emerging infectious pathogens
Interazioni tra clinico e patologo nelle malattie
infettive
R. Burioni
S. Antinori
Università Vita-Salute San Raffaele, Milan, Italy
Dipartimento di Scienze Cliniche L. Sacco, Università di Milano
The emergence of new pathogens is a rare event, very important both from the medical and from the soscio-economical
point of views.
The major threat for the appearance of novel pathogens is
due to novel viruses. Usually, the appearance of a human
viral pathogen is related to the mutation of an animal virus
acquiring the capacity to replicate in the human host. The
features of its interplay with the human host are very hard to
foresee in details. As a matter of fact, a virus outside a host is
a simple nucleoprotein. Should in this moment exist viruses
able to infect an extinct species, they would not be viruses, but
simply proteins and nucleic acids. The possibility for a virus
of being a virus, and not a simple molecular aggregate, relies
on the availability of a suitable host able to support its replication. This fact has awkward consequences as the infection of a
given virus is usually detrimental for the host; but should the
virus be so detrimental to the host to cause its extinction, this
would lead its extinction as well.
For this reason all the virus that are able to survive have found
a “way of life” with the host; and all the hosts that survived
have found a “way of life” with their viruses. The host, as well
as the viruses, that were not able to find a mutually acceptable
virus-host interplay are not present anymore.
However, the finding of a mutually acceptable virus-host interplay is not straightforward, and sometimes it can be reached
only at the cost of the death of a great numbers of hosts; this
is the selection process making the host more resistant. At the
same time, thevirus has an advantage in decreasing its pathogenicity (a host dying in several days is a better “spreader” of
infection if compared to a host dying in a few hours); under
the effect of these two concurring actions, viral diseases usually evolve to forms that are clinically less severe.
The microbiologic confirmation of a suspected infectious
diseases relies on the successful relationship between the infectious diseases clinician with the microbiologist and the pathologist 1. In fact, with the possible exception of the malaria
diagnosis which is usually made by the infectious diseases
clinician itself by the examination of blood smears that also
allows the correct species identification, in all the other cases
laboratory and/or pathologic confirmation are needed.
Actually, the great challenge either for the infectious diseases
clinician and the pathologist is identifiable not only with the
continuous emerging of new pathogens but more importantly
with the increasing probability to encounter exotic infectious
diseases as the consequence of globalization and human
movement 2. Another important issue to be considered when
discussing the relationship between infectious diseases and
pathologist is related to the correct diagnosis of opportunistic
infections that affect the ever-growing population of patients
with iatrogenic, inherited or acquired immunodeficiencies. In
this regard, it should be highlighted that autopsy remains an
important tool that allows the physician to identify clinically
unsuspected disease processes and to correlate pre-mortem
clinical diagnosis with pos-mortem findings 3. It is well
known that autopsy data were key to understand the acquired
immunodeficiency syndrome (AIDS) when this disease unexpectedly appeared in 1981, and more recent examples of new
infectious agents for which autopsy data have been invaluable
include severe acute respiratory syndrome (SARS) and the
West Nile virus.
Histopathologic and cythopathologic studies are particularly
important for the definitive establishment of infectious diseases caused by such microorganisms that fail to grow in
culture (for instance Mycobacterium leprae) or that grow very
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slowly such as M.tuberculosis. Other settings include infectious agents for which conventional microbiologic tests are
insensitive, expensive or unavailable.
The diagnosis of any infectious disease require active and collaborative communication between clinicians and pathologists
that begin with the acquisition of an adequate clinical specimen and its transportation to the laboratory in an appropriate
container. On the other hand, the differential diagnosis generated at the bedside through epidemiologic clues, patient history and physical examination is particular useful for differentiating between morphologically similar microorganisms.
Several examples of timely and useful interactions between
the infectious diseases clinician in charge and the pathologist
will be presented.
References
1
Procop GW, Wilson M. Infectious disease pathology. Clin Infect Dis
2001;32:1589-601.
2
Olano JP, Walker DH. Diagnosing emerging and reemerging infectious diseases. The pivotal role of the pathologist. Arch Pathol Lab
Med 2011;135:83-91.
3
Mazuchowski EL, Meier PA. The modern autopsy: what to do if infection is suspected. Arch Med Res 2005;36:713-23.
Sicurezza del paziente e riduzione dell’errore in Anatomia Patologica
Moderatori: Lucia Borsellino (Palermo), Enrico Vasquez (Catania)
Il servizio di anatomia patologica nel percorso
diagnostico assistenziale
T. Mannone
A.O. Ospedali Riuniti Villa Sofia – Cervello di Palermo
L’analisi e la gestione degli errori, associati all’implementazione di attività di gestione del rischio sono componenti
fondamentali di una corretta politica di ricerca della qualità.
L’analisi degli errori porta ad evidenziare che generalmente
non più del 20% degli incidenti sono dovuti a responsabilità
dei singoli, mentre l’80% è ascrivibile a cause di natura sistemica, intesa come anomalie di organizzazione o carenza
di risorse.
Il Servizio di Anatomia Patologica rappresenta un punto di
riferimento essenziale all’interno di una azienda ospedaliera.
In una logica di sistema, interagisce ricevendo campioni ed
esportando diagnosi, inserendosi di fatto nel globale percorso
diagnostico-assistenziale che l’azienda ospedaliera offre ad i
propri clienti / pazienti. Da questo scaturisce la necessità che
la gestione del risk management dell’attività anatomopatologica venga affrontata con gli strumenti e le logiche proprie
dell’approccio sistemico alla gestione del rischio.
L’approccio sistemico, proteso al miglioramento del valore
complessivo della prestazione sanitaria, ricerca la massima
appropriatezza professionale ed organizzativa. Tale attenzione
ad una ottica di sistema consente di generare una cultura di
circolo virtuoso di relazioni, responsabilità e comunicazioni.
Uno dei momenti in cui il percorso diagnostico assistenziale
di un paziente si interseca con il servizio di anatomia patologica è rappresentato per esempio, ed ovviamente, dall’attività chirurgica. Per il buon esito degli interventi è necessario
il comportamento sinergico di tutti gli operatori coinvolti
nelle diverse fasi operative. Nel 2008 sono state pubblicate
le linee guida OMS “Guidelines for Safe Surgery” per la
sicurezza in sala operatoria, che si pongono come obiettivo
il miglioramento della sicurezza degli interventi chirurgici
tramite la definizione di raccomandazioni e standard di sicurezza. Oltre alle raccomandazioni l’OMS ha anche costruito
una check list per favorire l’adozione degli standard proposti. Il ministero della salute ha adattato al contesto nazionale
ed ulteriormente integrato le linee guida prodotte nel 2008
dall’OMS “Guidelines for Surgery” pubblicando il docu-
mento “Raccomandazioni per la sicurezza in sala operatoria
– 2009”. Le raccomandazioni legate a 16 obiettivi specifici,
comprendono al punto 3 la corretta identificazione dei campioni chirurgici, in quanto “la non corretta identificazione
dei campioni chirurgici può causare gravi conseguenze ai
pazienti e la prevenzione di tali errori è fondamentale per la
sicurezza dei pazienti”.
Da tale attenzione alla fase che precede direttamente l’invio
del materiale all’esame istologico, si evince come una corretta
politica di gestione del rischio non può assolutamente considerare i processi relativi all’anatomia patologica come avulsi
da un contesto più generale e complesso.
L’approccio sistemico alla gestione del rischio si traduce
quindi nel governare un processo le cui fasi fondamentali sono
l’identificazione del rischio, l’analisi del rischio, il trattamento
del rischio ed il monitoraggio (periodico o continuo) delle
azioni di miglioramento.
Il sistema di gestione del rischio si articolerà quindi lungo una
linea clinica, che vede gli operatori responsabili di segnalare
ed analizzare i processi critici, ed una linea manageriale,
lungo la quale è preciso compito della direzione aziendale garantire adeguati livelli di sicurezza e definire efficaci processi
di miglioramento.
SIAPEC-IAP per la qualità:
proposte e considerazioni
R. Giardini*, E. Tavani**
Anatomia Patologica Istituti Ospitalieri di Cremona; **Anatomia Patologica Ospedale di Circolo di Rho (Milano)
*
L’anatomia patologica e la medicina di laboratorio stanno
attualmente sperimentando svariati modelli di cambiamento,
che, con molta probabilità, saranno in grado di modificare
profondamente, in un futuro non troppo lontano, il modo di
praticare la specialità: svariate tecniche innovative in immunocitochimica, l’espansione della patologia molecolare e
l’estesa informatizzazione stanno portando all’acquisizione di
nuove ed eccitanti informazioni nel campo della diagnostica
anatomopatologica. Questo fenomeno, tuttavia, se da un lato
incrementa le finalità dell’informazione diagnostica, aggiungendovi dati di tipo prognostico e predittivo di risposta alla
relazioni
terapia, dall’altro impone la necessità di verificare, attraverso
meccanismi di convalida basati sull’evidenza, che le informazioni prodotte siano “di qualità”. L’applicazione sempre più
estensiva della tecnologia esige, ogni giorno di più, che anche
il patologo debba sforzarsi di abbandonare l’“eminence based
medicine”, sinora seguita in virtù del carattere eminentemente
interpretativo della propria disciplina, per basarsi su più robusti gradi di evidenza.
I manuali di procedure e le linee guida nascono come strumento che ha l’obiettivo di permettere all’operatore di fare scelte
“informate”, basandosi sull’analisi delle prove scientifiche e
sulla valutazione dei rischi e dei benefici di qualsiasi azione.
Di più, manuali di procedure e linee guida si sono rivelati uno
strumento di aggiornamento per i professionisti, d’ educazione ed informazione per i pazienti e di riferimento esterno, con
cui si rende possibile una verifica di quel che il professionista
è in grado di produrre. Tutto questo può essere riassunto nel
concetto di “qualità della prestazione professionale”.
Anche se l’anatomia patologica diagnostica è ancora relativamente lenta nell’abbracciare la pratica ed i principi
della medicina basata sull’evidenza, in parte perché storicamente il patologo è stato, da sempre, “l’evidenza” rispetto
all’elucubrazioni cliniche, la pratica operativa all’interno
d’una struttura d’anatomia patologica, dato l’alto livello
d’interscambio intra- ed extra-aziendale (consultazioni, centralizzazioni di casistiche, controlli di qualità, campagne di
screening) è improntata abitualmente e, per così dire, fisiologicamente, al conformarsi a protocolli e standard definiti
ed accettati, anche se, abbastanza spesso, non capillarmente
estesi a tutti i professionisti e talora conosciuti solo all’interno di una specifica branca di specializzazione, per organo
e/o per patologia. Questo, tuttavia, avviene spesso in modo
implicito, non formalizzato e, comunque, non come risultato
di procedure metodologicamente rigorose ed esplicitamente
dichiarate. Al contrario, sono prove di un implicito bisogno
di riferimenti consolidati quegli strumenti procedurali che,
nel tempo, sono stati messi a punto, a livelli quasi sempre
qualitativamente elevati, da parte dei gruppi di studio nati
in seno alla società scientifica o da parte di sezioni regionali
della stessa.
In effetti, da qualche tempo è stato avviato un confronto all’interno della SIAPEC-IAP sull’uso di strumenti di lavoro, come
manuali delle procedure, linee guida diagnostiche, indicazioni
di controlli di qualità, che, a nostro parere, sono sempre più
rilevanti nell’attività diagnostica e che esprimono una valenza
particolare, sia per l’accreditamento e la certificazione delle
strutture di anatomia patologica, sia per il ruolo che il loro uso
assume nella gestione del rischio nella nostra specialità.
D’altro canto, che si voglia, o non si voglia, considerare
l’anatomia patologica una branca delle medicine di laboratorio (quest’ultime connotate da un’impostazione analitica che
contrasta con l’innegabile sintesi del referto della prima) va
prendendo piede presso realtà istituzionali, come le regioni,
l’estensione anche all’anatomia patologica di programmi
per l’implementazione della qualità. Alcune regioni hanno
elencato, sulla base di indicazioni di gruppi di esperti, criteri
minimi per controlli di qualità interni. Altre realtà, come, ad
esempio, il Piemonte, la Lombardia, il Lazio, hanno esperimentato controlli di qualità esterni volti alla concordanza
diagnostica citoistologica o alla determinazione di fattori
prognostici. Altri programmi, previsti da regioni come la
Toscana e la Lombardia, possono comprendere variegate
tematiche e modalità di svolgimento: collaborazione paritetica tra gli esperti per la definizione di procedure, linee
guida ed ogni altra forma documentale, mediante incontri
115
organizzati su tematiche specifiche relative: alla definizione di protocolli comuni per il monitoraggio della qualità
dell’intero processo operativo (fasi preanalitica, analitica e
postanalitica), a linee guida per l’accreditamento professionale, anche mediante audit, alla costruzione o alla revisione
delle modalità di attuazione del controllo di qualità interno;
incontri di formazione professionale rivolti agli operatori;
effettuazione di visite ispettive da parte di esperti operanti
in ambiti territoriali diversi, sino alla valorizzazione delle
strutture regionali coinvolte nella gestione della valutazione
esterna di qualità. Alcune realtà istituzionali sono ben consce, attraverso il parere di esperti chiamati a costituire dei
veri e propri comitati di riferimento, della particolarità della
nostra disciplina, che rende difficoltoso istituire programmi
di verifica di qualità che accertino l’intero processo anatomopatologico: ad esempio la non ancora risolta questione
di poter riconoscere, anche in anatomia patologica, valori
critici (diagnosi critiche) che possano essere utilizzate come riferimento per definire la qualità di tutto il processo di
produzione di una diagnosi. E di conseguenza chiedono alla
società scientifica riferimenti tecnici e procedurali.
Nell’ambito del pianeta qualità, dal punto di vista istituzionale, la SIAPEC-IAP ha costituito nel 2002 un gruppo di
lavoro per definire le strategie dell’Associazione in merito
a linee guida che possano essere rilevanti per l’attività anatomopatologica, redatte secondo i seguenti principali criteri:
multidisciplinarietà del gruppo di lavoro autore delle linee
guida, esplicito processo di ricerca bibliografica e grado
dell’evidenza secondo manuale ASSR (ora AGENAS).
Nell’ambito della collaborazione con AIOM stati istituiti
gruppi di esperti che hanno redatto raccomandazioni per
specifici argomenti di carattere biomolecolare, mentre alcuni
gruppi di studio hanno pubblicato le modalità per una refertazione completa ed accurata di determinate patologie. Uno
dei compiti che attende ora, inderogabilmente, la società
scientifica è sicuramente la sistematizzazione e l’imprimatur
istituzionale e la successiva diffusione di quanto prodotto,
i primi attraverso uno stimolo ai vari gruppi di studio di
patologia ad individuare dei parametri minimi da osservare strettamente e con un sistema di convalida da parte del
gruppo di lavoro, che li formalizzi e li renda in un certo qual
senso vincolanti per tutti gli operatori, e la seconda con la
pubblicazione in uno spazio dedicato sul sito istituzionale
in cui sia possibile la discussione e l’aggiornamento degli
argomenti. Ben più difficile, ma ugualmente essenziale e
non più procrastinabile, lo stabilire le regole del gioco nel
campo della verifica di qualità: in questo campo le necessità
da soddisfare sono parecchie: definire, in primis, la qualità e
la riproducibilità di quanto deve essere controllato e l’estensione del controllo a tutte le fasi del processo diagnostico,
con criteri di valutazione trasparenti e riproducibili. Rientrano nell’ambito della verifica di qualità: la valutazione
di processo, di estrema rilevanza in considerazione della
relativamente scarsa automazione dei processi diagnostici;
la valutazione delle dotazioni e delle competenze logistiche
e strumentali; la valutazione delle competenze tecniche, che
comprende la valutazione delle tecniche immunocitochimiche e biomolecolari, anche attraverso sistemi di valutazione
internazionale; la valutazione delle competenze diagnostiche, singole o di squadra. Nel caso della valutazione esterna
di qualità risulta ancora più cruciale il ruolo delle segreterie
regionali della società scientifica, indispensabile tramite tra
le indicazioni tecniche della società e le esigenze di controllo
delle istituzioni.
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La responsabilità professionale in Anatomia
Patologica
V. Cirese
Roma
Gli errori dovuti alla “medical malpractice” suscitano ancora
grande preoccupazione. Nel 2010 in Italia si sono riscontrati
circa 32.000 casi di contenzioso tra medici e pazienti per
ipotesi di non corretta prestazione professionale. Il notevole
ampliamento del campo di azione dell’Anatomia Patologica,
ha fatto registrare un incremento di responsabilità anche in
questa specializzazione. Il medico, come ogni professionista,
è soggetto, nell’esercizio della propria attività, a responsabilità
disciplinare, civile e penale. Può essere chiamato a rispondere
in sede civile dei danni causati al paziente nell’erogazione
delle cure e trattamenti terapeutici; in sede penale per i reati
eventualmente connessi a prestazioni professionali censurabili
per negligenza, imperizia, imprudenza(ad es. lesioni, omicidio colposo) in presenza di un nesso causale tra condotta ed
evento.Volendo ovviare al preoccupante contenzioso medicolegale, è importante definire gli standard qualitativi auspicabili
che dovrebbero essere comuni a tutte le strutture di Anatomia
Patologica, nel rispetto delle linee guida inerenti le procedure,
dal campionamento alla refertazione, sia in citopatologia che
in istopatologia, come richiama il Piano Oncologico Nazionale
2010/2012. Grande attenzione, pertanto, deve essere posta
alle procedure, dovendo il Patologo garantire il paziente e se
stesso in merito alla validità del metodo, alla sua accuratezza
ed alla sua riproducibilità. Lo studio dei prevalenti indirizzi
della giurisprudenza e della dottrina rappresenta senza dubbio
uno strumento di grande interesse per fornire alcune delle
risposte che la classe medica attende. È agevole constatare
come la giurisprudenza di legittimità e di merito abbiano impresso al diritto vivente una continua evoluzione in materia
di responsabilità professionale medica civile e penale, che
giocoforza esercita la sua influenza anche nel campo dell’anatomopatologia. L’attività medica, nelle sue manifestazioni
(prevenzione, diagnosi, prognosi, intervento,trattamento terapeutico), ha guadagnato in efficienza.Dal panorama normativo
e giurisprudenziale, nonostante le oscillazioni e le contraddizioni purtroppo rinvenibili, emerge un dato costante, costituito
dall’esigenza di assicurare la piena ed effettiva tutela della
salute degli individui (art. 32 Cost.). Il dovere d’informazione
che grava sul sanitario è funzionale al consapevole esercizio,
da parte del paziente, del diritto di scelta se sottoporsi o meno
all’intervento terapeutico di elezione, che la stessa Carta costituzionale, agli art. 13 e 32, comma 2, a lui solo attribuisce.
Costituiscono un’ eccezione infatti i casi di trattamento sanitario obbligatorio per legge o emergenza-urgenza integrante
stato di necessità. Gli esiti infausti residuati ad un intervento di
chirurgia o trattamento medico eseguito in violazione del dovere di informazione da parte del sanitario, possono integrare
gli estremi dell’ “alterazione anatomo-patologica dell’organismo” e, conseguentemente, l’elemento oggettivo del reato di
lesioni colpose al cui accertamento il giudice è chiamato al
fine di pronunciarsi sulla risarcibilità del danno, allorquando
tali esiti non siano riferibili ad interventi in cui le possibilità
di simili conseguenze dannose erano già state preventivamente
ed esaurientemente rappresentate al paziente dall’operatore
sanitario (Cass. civ, sez. III, 6 ottobre 1997, n. 9705). La mancanza del consenso,(opportunamente “informato”) del malato
o la sua invalidità per altre ragioni determina l’arbitrarietà del
trattamento medico chirurgico e la sua rilevanza penale, in
quanto posto in violazione della sfera personale del soggetto e
del suo diritto di decidere se permettere interventi di estranei
sul proprio corpo. In materia di consenso informato, il dato
saliente da rilevare è relativo all’irrompere anche in campo
penale, della tesi civilistica, della “autolegittimazione” dell’attività medica, la quale rinviene il proprio fondamento non tanto
nella scriminante tipizzata del consenso dell’avente diritto ex
art. 50 c.p., quanto nella stessa finalità: la tutela della salute
ex art. 32 Costituzione, sul presupposto della abilitazione alla
professione. Infatti, in caso di lesioni in ambito terapeutico, le
Sezioni Unite della Cassazione, con sentenza del 18 dicembre
2008, n. 2437/2009, hanno affermato che: “non integra il reato
di lesione personale, né quello di violenza privata, la condotta
del medico che sottoponga il paziente ad un trattamento chirurgico diverso da quello in relazione al quale era stato prestato il
consenso informato, nel caso in cui l’intervento, eseguito nel
rispetto dei protocolli e delle leges artis, si sia concluso con
esito fausto...”.
Diverso è il caso dell’omessa o errata diagnosi. In proposito si
segnala una recentissima decisione della Cassazione (Cass. civile, Sez. III, 14/06/2011, n. 12961) in materia di responsabilità in Anatomia Patologica. La Corte di Cassazione chiamata
ad occuparsi di un caso di errore istopatologico ha escluso che
questo errore avesse determinato - sia pure in termini probabilistici - un danno alla paziente, nel senso che il successivo exitus non fu influenzato, neppure nella durata della residua vita
o nella qualità degradata della stessa, dalla mancata diagnosi
precoce della malattia tumorale.Resta il fatto che l’inadempimento del professionista, specie se consistente nell’errore o
omissione di diagnosi in relazione alla propria obbligazione, e
la conseguente responsabilità colposa per imperizia, è sempre
valutato alla stregua del dovere dell’agente modello, particolarmente qualificato in relazione allo svolgimento della sua
attività professionale specialistica.
Ai fini della causalità, la giurisprudenza (Cass. Sez. Unite,
11/01/2008, n. 581, 576 ed altre) e la dottrina prevalente, in applicazione dei principi penalistici di cui agli art.
40 e 41 c.p., hanno ribadito che un evento sia da considerare causato da un altro se, ferme restando le altre
condizioni, il primo non si sarebbe verificato in assenza del secondo (c.d. teoria della condicio sine qua non).
Il rigore del principio dell’equivalenza delle cause, posto
dall’art. 41 c.p., in base al quale, se la produzione di un
evento dannoso è riferibile a più azioni od omissioni, deve
riconoscersi ad ognuna di esse efficienza causale, trova il suo
temperamento nel principio di causalità efficiente, desumibile
dall’art. 41 c.p., comma 2, in base al quale l’evento dannoso
deve essere attribuito esclusivamente all’autore della condotta
sopravvenuta, solo se questa condotta risulti tale da rendere
irrilevanti le altre cause preesistenti, ponendosi al di fuori
delle normali linee di sviluppo della serie causale già in atto
(Cass. 19.12.2006, n. 27168; Cass. 8.9.2006, n. 19297; Cass.
10.3.2006, n. 5254; Cass. 15.1.1996, n. 268). Nel contempo
non è sufficiente tale relazione causale per determinare una
causalità giuridicamente rilevante, dovendosi, all’interno delle serie causali così determinate, dare rilievo a quelle soltanto
che, nel momento in cui si produce l’evento causante non
appaiano del tutto inverosimili, ma che si presentino come
effetto non del tutto imprevedibile, secondo il principio della
c.d. causalità adeguata o quella similare della c.d. regolarità
causale (Cass. 1.3.2007; n. 4791; Cass. 6.7.2006, n. 15384;
Cass. 27.9.2006, n. 21020; Cass. 3.12.2002, n. 17152; Cass.
10.5.2000 n. 5962).
Nel danno da inadempimento per condotta omissiva come
per esempio per omessa diagnosi, il giudizio causale assume
come termine iniziale la omessa condotta rispetto al compor-
relazioni
tamento dovuto (Cass. n. 20328 del 2006; Cass. n. 21894 del
2004; Cass. n. 6516 del 2004; Cass. 22/10/2003, n. 15789).
Non può riconoscersi la responsabilità per omissione quando
il comportamento omesso, ove anche fosse stato tenuto, non
avrebbe comunque impedito l’evento prospettato: in altre
parole, la responsabilità non sorge non perché non vi sia stato
un comportamento antigiuridico ma perché quell’omissione
non è causa del danno lamentato. Il Giudice pertanto è tenuto
ad accertare se l’evento sia ricollegabile all’omissione (causalità omissiva) nel senso che esso non si sarebbe verificato se
(causalità ipotetica) l’agente avesse posto in essere la condotta
doverosa impostagli, con esclusione di fattori alternativi.
L’accertamento del rapporto di causalità ipotetica passa attraverso l’enunciato “controfattuale”. Essendo questi i principi
che regolano il procedimento logico - giuridico ai fini della
ricostruzione del nesso causale, ciò che muta sostanzialmente
tra il processo penale e quello civile è la regola probatoria,
in quanto nel primo vige la regola della prova “oltre il ragionevole dubbio” (cfr. Cass. Pen. S.U. 11 settembre 2002,
n. 30328, Franzese), mentre nel secondo vige la regola della
preponderanza dell’evidenza o “del più probabile che non”,
(in questo senso Cass. 16.10.2007, n. 21619; Cass. 18.4.2007,
n. 9238; 11/05/2009, n. 10741; Cass. 22837 del 2010; Cass.
16123 del 2010). Dopo queste necessarie premesse, venendo
a trattare piu’ specificamente il tema della responsabilità in
anatomia-patologica può notarsi subito una particolarità rispetto al rapporto con il paziente.
Invero nel corso del suo iter diagnostico e terapeutico il paziente normalmente incontra: il medico di base, il radiologo,
il chirurgo senologo, l’anestesista nella valutazione preoperatoria e nel periodo perioperatorio, nel periodo postoperatorio
l’oncologo e il radioterapista. Nel corso della malattia incontrerà anche il patologo clinico per le analisi di laboratorio
prima del ricovero e nei controlli successivi e forse anche il
cardiologo per eventuali approfondimenti diagnostici cardiologici prima dell’intervento e il chirurgo plastico per eventuali
ricostruzioni a livello della mammella.
Di certo incontra numerosi altri medici per consulti e consulenze varie. Difficilmente il paziente incontrerà l’anatomopatologo, anzi sicuramente non si porrà il problema che possa
esistere uno specialista dedicato alla tipizzazione delle lesioni
tumorali e non tumorali, un laboratorio con medici specializzati e tecnici di laboratorio esperti nel trattamento dei tessuti
e delle cellule.
Medico assai diverso anche dagli stereotipi televisivi: l’antomo-patologo appare come un medico abbastanza statico,
seduto al suo microscopio ad analizzare tessuti, a valutare
la morfologia di un nucleo cellulare o l’espressione di una
qualche molecola da parte di una popolazione neoplastica, a
consultare libri, a riflettere su algoritmi diagnostici.
La medicina moderna si basa su gruppi integrati di medici
che con diverse specializzazioni, insieme, confrontandosi ed
integrandosi raggiungono la diagnosi più corretta, al fine di
fornire la terapia più idonea. Di questi gruppi deve necessariamente far parte anche l’anatomo-patologo e questo perché,
non è possibile trattare i pazienti senza analisi istopatologica.
Val la pena ricordare lo slogan creato alcuni anni fa dalla
SIAPEC che diceva: “come un albero vive grazie alle sue radici nascoste, così un ospedale non potrebbe esistere senza la
figura dell’anatomo-patologo, spesso invisibile ai pazienti”.
Nel suo laboratorio l’anatomo-patologo lavora per tipizzare
lesioni in quasi tutti i campi della medicina; l’anatomopatologo lavora a stretto contatto con tutti i chirurghi (toracici,
addominali, oncologici, plastici, urologi, ginecologi, neurochirurghi, ecc.) che sono i suoi interlocutori principali; ma la-
117
vora anche con molti altri colleghi come i dermatologi (tipizzazione di lesioni cutanee), con gastroenterologi (tipizzazione
di biopsie gastriche-coliche-duodenali e/o di lesioni asportate
endoscopicamente), gli ematologi (tipizzazione di linfonodi e
biopsie osteomidollari), gli oncologi e i radioterapisti. È infatti
indispensabile una corretta analisi cito/istologica di una lesione, per esempio per studiare le caratteristiche prognostiche e
predittive di un certo tumore.
I tumori esistenti ad oggi riconosciuti e classificati dalla
WHO (organizzazione mondiale della sanità- World Health
Organization) sono classificati in alcune migliaia di differenti
istotipi, alcune neoplasie si rivelano rapidamente fatali, altre
presentano un andamento clinico più indolente e a lenta evoluzione, altre ancora sono assolutamente benigne.
Definire la benignità o la malignità di una neoplasia è utile
per prevedere l’andamento clinico della malattia e prendere
di conseguenza delle decisioni per stabilire la terapia più
idonea.
Esistono lesioni facili da “catalogare” e lesioni molto difficili,
per le quali più anatomo-patologi devono confrontarsi tra loro
e discutere per giungere ad una conclusione.
L’anatomo-patologo deve quindi fornire un esame morfologico che sia prima di tutto corretto e attendibile. Spesso non è
agevole raggiungere il consenso su una data lesione attesa la
complessità dello studio morfologico.
La prudenza e la perizia, l’approfondimento diagnostico,
sono senz’altro doti indispensabili per l’anatomo-patologo
che voglia adempiere alle sue prestazioni professionali con
coscienziosità e successo e non incorrere in censure sotto il
profilo medico legale.
Non vi è dubbio che gli orientamenti dominanti, recentemente, si siano dimostrati piu’ rigorosi anche nella valutazione
della colpa medica in ambito anatomo-patologico, in considerazione del peso giuridico di un errore professionale in un
settore così specializzato e tuttavia così ampio, nonché per la
maggior severità di valutazione dell’errore commesso dallo
specialista (a fronte delle specifiche capacità tecniche che
l’acquisizione del titolo specialistico comporta, legittimanti
maggiori aspettative).
Fra i profili rilevanti della colpa medica, anche in ambito anatomo-patologico, meno ricorrente anche alla luce dell’esperienza giudiziaria, risulta quello collegato all’inosservanza
delle leggi e regolamenti, ordini o discipline, anche se a volte
ciò si rinviene e anzi l’inosservanza, fonte della colpa, può in
teoria riflettere, non solo norme consacrate in leggi o regolamenti, ma anche norme di servizio o di disciplina, contenute,
ad esempio nei regolamenti interni dell’ospedale o di altri
enti, o frutto di singole disposizioni impartite in forza di una
posizione di superiorità gerarchica o funzionale.
In tema di malpratica sanitaria, la casistica degli ultimi anni
ha evidenziato che, nell’ambito dell’istopatologia umana, il
rischio dell’errore diagnostico (per mancata o errata diagnosi)
ricorre con maggiore frequenza al momento della scelta della
metodica del taglio da eseguire o nell’esecuzione del taglio
stesso. È stato rilevato, inoltre, che la responsabilità professionale può conseguire anche alla mancata conservazione dei
vetrini.
Occorre premettere che la metodica del taglio, fase centrale e
altresì fondamentale dell’attività specialistica del settore, non
è attualmente regolata da specifici protocolli.
Tale mancanza, se da una parte garantisce al clinico che deve
procedere all’escissione del pezzo, maggiore libertà nella scelta tecnica, potendo egli applicare o meno una ben precisa metodica, dall’altra rischia di esasperare la diversità “di scuola”
nella scelta della metodica, che in alcuni casi è già ben eviden-
118
te. È stata riscontrata, ad esempio, una diversità d’esecuzione
(di sezione e di prelievo) per identiche patologie tumorali, a
seconda delle istituzioni ospedaliere ove s’interveniva. A titolo esemplificativo, basti citare che nell’asportazione radicale
della prostata, vi sono scuole che sezionano l’intera ghiandola
prostatica e altre invece che preferiscono inciderne solo un
pezzo. Come vi sono cliniche che sostengono che l’escissione
(del tumore) ideale è quella di spaccare a metà la sezione, lasciando al centro la parte tumorale e ben in evidenza i margini,
i cui contorni devono essere valutati con estrema precisione
per l’identificazione della patologia: ciò in particolare nel
caso di tumori cerebrali e del seno. Ed è proprio la diversità di
pratica media descritta la causa piu’ ricorrente nei contenziosi
civili e dei processi penali, che vedono come protagonisti
gli anatomi patologi, chiamati a rispondere, come prestatori
d’opera intellettuale, esclusivamente per inadempimento di
un’obbligazione di mezzi e non di risultato.
Tipici casi sui quali la giurisprudenza ha avuto modo di esprimere indirizzi precisi sono:
gli errori diagnostici di primari ospedalieri (di reparti di Anatomia e Istologia Patologica) che, dopo aver eseguito esami
di frammenti di cute provenienti dalle sale operatorie, hanno
diagnosticato erroneamente (ad esempio, l’esistenza di un
melanoma anziché di un angioma), inducendo il radiologo a
praticare al paziente terapie irradianti non indicate.
errate diagnosi istologiche di carcinoma, che hanno comportato interventi chirurgici di asportazione di mammella e utero
in donne in età feconda. Trattandosi di tipiche contestazioni di
“imperizia”, si rileva la distinzione tra diritto penale e civile
perchè solo in quest’ultimo ambito si esige per l’affermazione della responsabilità,la colpa grave, nell’accoglimento del
criterio, della non ricorrenza di speciale difficoltà tecnica. Va
osservato però che trattandosi di condotta omissiva, il metodo
di accertamento causale è di tipo probabilistico e scaturisce
dal fatto che il nesso tra condotta ed evento è in forza della
previsione dell’art.40 c.p. (non impedire un evento che si ha
l’obbligo giuridico di impedire, equivale a cagionarlo), costituito in termini ipotetici e non naturalistici com’è invece per
la causalità del reato commissivo.
A volte si è verificato un errore in presenza di due diagnosi
diverse fatte da due patologi; quando alla revisione superiore,
ad esempio, in un programma di controllo di qualità, viene
posta una diagnosi diversa dalla prima;ovvero il follow-up
clinico-laboratoristico, compreso l’eventuale controllo istologico successivo, contraddice la diagnosi primitiva.
Può verificarsi che la mancata rilevazione di un allarme, peraltro presente nel preparato (“il falso negativo”), non induca
gli esami successivi, compreso il controllo istologico, che
dimostrerebbero con certezza la presenza di una condizione
neoplastica o pre-neoplastica, così cagionando ommisione di
diagnosi o ritardo diagnostico che può avere delle gravi conseguenze per la salute del paziente. Meno grave, anche se spesso
molto ansiogeno e costoso, è il “falso positivo”. La mancata
conferma di una diagnosi citologica fa in genere terminare
il processo diagnostico-terapeutico. Allorquando un clinico
richiede un esame citologico, si attende una risposta certa:
negativa o positiva.
Se esiste un colloquio aperto tra clinico e patologo il primo,
anche alla luce di ulteriori notizie cliniche, in genere ignote al
patologo, potrà scegliere l’atteggiamento diagnostico-terapeutico più appropriato per il paziente; in caso contrario il quesito
diagnostico potrebbe non essere chiarito.
Con il “Quality Control” si deve essere in grado di monitorare,
ottimizzare e standardizzare ogni singolo passo del processo
diagnostico, affinchè, attraverso la “Quality Assurance” si sia
in grado di fornire un prodotto finale (la diagnosi) sempre più
preciso e utile per il cliente/paziente, anche se la via verso la
qualità ha comunque dei costi elevati.
Certamente attraverso programmi di “quality control” e “quality assurance” è possibile migliorare sensibilmente le prestazioni professionali, ma occorre prestare attenzione anche
alla comunicazione: essere certi che il soggetto intermedio
(il clinico e/o il medico di medicina generale) e quello finale
(il paziente) comprendano veramente il significato di una
diagnosi, peraltro formalmente esatta. Alcune diagnosi sono
assolutamente criptiche e sembrano fatte per diagnosticare
tutto e il suo contrario e far nascere problemi medico-legali.
Una chiara e precisa comunicazione tra patologo e clinico è la
chiave per prevenire censure di “malpratice”ed è particolarmente utile al paziente, seppur in alcuni casi non è possibile
essere chiari e si sia costretti ad esprimere un certo margine
di incertezza.
Non v’è dubbio che il professionista anatomopatologo debba
riconoscerci come parte di un sistema complesso, in cui giocano il loro ruolo anche il clinico e il paziente e con cui occorre
interagire e comprendersi. Nella specialistica di settore è necessario garantire standard procedurali e diagnostici elevati,
monitorati frequentemente, nella consapevolezza che l’errore
è grave, a volte perfino letale per il paziente. Le conseguenze
sono anche a carico del medico ed è interesse comune ridurne
l’incidenza.
La valutazione HTA
P. Dalla Palma
Anatomia Patologica Ospedale S. Chiara, Trento
L’Health Technology Assessment (HTA) è una branca multidisciplinare di “policy analysis” che studia le implicazioni
mediche, etiche, sociali ed economiche dello sviluppo, dello
sviluppo e dell’uso delle tecnologie sanitarie il cui scopo è
quello di fornire gli input necessari ai processi decisionali e
alla loro realizzazione specialmente in un contesto di risorse
limitate. Rappresenta perciò un ponte tra scienza (EBM) e
decisione e deve misurare gli effetti sulla salute della tecnologia in esame tenendo conto sia dei costi che dei valori.
Altre valutazioni secondarie della strumentazione riguardano
caratteristiche tecniche e sicurezza, efficacia teorica e pratica,
efficienza (costo-efficacia e costo-beneficio), impatto sull’organizzazione ed disponibilità ed accettabilità degli operatori al
cambiamento (aspetti etici e sociali).
La valutazione HTA si fa attraverso passi successivi: si parte
con stimare i bisogni e fissare le priorità per passare poi alla
vera valutazione e al trasferimento delle conoscenze attraverso lo studio delle varie banche dati e tenedo conto della
Evidence based medicine.
Tutto questo processo avviene attraverso il coinvolgimento di
varie strutture (ministero, Università, agenzie regionale, ecc).
Proprio in questo ambito è appena stato concluso il progetto
strategico “Strumenti e metodi per il governo dei processi di
innovazione tecnologica, clinica ed organizzativa nel Servizio
Sanitario Nazionale- Un sistema integrato di ricerca.” coordinato dall’ASSR e finanziato dal Ministero della Salute per la
ricerca finalizzata. In tale progetto, era previsto un sottoprogetto denominato “Valutazione dell’impatto delle diverse modalità di coinvolgimento del Servizio Sanitario nella produzione
delle evidenze sulla trasferibilità delle conoscenze” coordinato
da Lazio Sanità ASP cui ho avuto la fortuna di partecipare per
una valutazione sulla lettura computer assistita dei pap test.
119
relazioni
L’obiettivo finale di una valutazione HTA è a tre livelli: un
livello “macro” (ad esempio quando si voglia introdurre uno
screening nazionale) a livello “meso” (quando ad esempio si
debba acquisire una strumentazione di rilevante impatto economico come ad esempio era l’acquisizione di un sistema di
lettura computer assistito del valore superiore aa 1 milione di
euro) e infine a livello “micro” quando ad esempio si decida
di cambiare un farmaco sostituendone un altro o nel nostro
campo quando si voglia abbandonare una metodica ce sostituirla con un’altra).
Si tratta chiaramente di un processo molto complesso che
richiede l’apporto di molteplici attori di diversa estrazione.
Nella sperimentazione che ho condotto abbiamo seguito una
metodologia che prevedeva la costituzione di un Gruppo
di Lavoro (GL) costituito in modo preminente da esperti
del settore che riportando la loro esperienza e confrontando
i risultati con quelli derivanti dall’analisi della letteratura
“indipendente” che nel caso specifico era rappresentata per
lo più anche se non esclusivamente da documenti ufficiali
a livello Nazionale (Inghilterra, Canada, Nuova Zelanda,
Australia) hanno stilato una bozza di documento utilizzando
anche competenze extra-patologiche come economisti, epidemiologi, dirigenti di medicina pubblica. Tale documento è
poi stato fatto valutare ai vari “stakeholders” interessati come
Altri Patologi, gestori di Sanità pubblica, economisti sanitari,
direttori Generali, rappresentanti dell’Agenzia Sanitaria Regionale, rappresentanti del CCM, ma anche delle associazioni
volontarie come la lega per la lotta contro i Tumori (LILT) e
infine i rappresentati dell’industria interessata. Solo a questo
punto il documento è stato licenziato e messo a disposizione
dell’ASSR e del Ministero per la sua diffusione ad Assessorati
e Direttori Generali.
Emolinfopatologia
Moderatore: Vito Franco (Palermo)
Pathobiology of peripheral t-cell lymphomas
S.A. Pileri , G. Inghirami , B. Falini , E. Sabattini , F.
Bacci*, C. Agostinelli*, P.P. Piccaluga*
*
**
***
*
Chair of Pathology and Unit of Haematopathology, Department of
Haematology and Oncological Sciences “L. and A. Seràgnoli”, University of Bologna, Italy; **Department of Pathology and Centre for
Experimental Research and Medical Studies (CeRMS), University
of Torino, Italy; ***Institute of Haematology, University of Perugia,
Italy
*
Peripheral T-cell lymphomas (PTCLs) correspond approximately to 12% of lymphoid neoplasms, being definitely rarer
than B-cell tumours but slighter commoner than Hodgkin
lymphoma in Western Countries 1. They represent a heterogeneous group of diseases that can be roughly subdivided into
specified and not otherwise specified (NOS) forms. However,
three subtypes represent about 70% of all cases: PTCL/NOS,
angioimmunoblastic T-cell lymphoma (AITL) and anaplastic
large cell lymphoma (ALCL) with or without expression of
the ALK protein (ALK+ or ALK- respectively) as consequence of the t(2;5) translocation and variants.
PTCLs usually occur in the fifth-sixth decade of life, without sex predilection, more often in advanced clinical stage,
with both nodal and extra-nodal involvement 1. A haemophagocytic syndrome is at times encountered. On clinical
grounds, they are among the most aggressive non-Hodgkin
lymphomas (NHLs), their response to conventional chemotherapy being frustrating with 5-year-relapse free and overall
survival rates not exceeding 26% and 20%, respectively. In
particular, conventional chemotherapeutic regimens do not
appear as effective as in B-NHLs and the addition of anthracyclins does not provide significant benefits 2. Noteworthy,
resistance to anthracyclins as well as to other conventional
agents has been demonstrated in experimental models by
our group 3.
In the light of the above, the better knowledge of the pathobiology of PTCLs is warranted if one aims to positively modify
the outcome of these neoplasms. To this hand, our Group has
been intensively working during the last few years.
In 2006, we published an extensive report based on the construction of tissue micro-arrays from 193 PTCLs (148 NOS
and 45 AITL) that were tested by immunohistochemistry and
EBER in situ hybridization 4. Both tumors demonstrated frequent loss of CD5 and CD7, with CD3 being the conventional
marker most commonly expressed in NOS, and CD2 in AITL.
CD4 was detected in 46% of cases and CD8 in 15% of cases.
Interestingly, a huge number of PTCLs/NOS and AITLs
(55%) turned out to be either CD4/CD8 double-negative or,
more rarely, double-positive. Such profiles, which are usually
observed during intra-thymic T cell development, had previously been reported in isolated PTCL cases and a proportion
of cutaneous T-cell tumors. This study demonstrated that in
the setting of PTCLs there is no marker that – like the Ig light
chain restriction of B-cell lymphomas – can surrogate a clonality assay. Else, it is the aberrancy of the global profile that
supports the neoplastic nature of a given population. Interestingly, in this study high Ki-67 expression, EBV positivity
and CD15 staining were associated with the worst outcome
among PTCLs/NOS. No other phenotypic marker alone or in
combination was associated with a poor prognosis, although
patients with tumors expressing a CD57 or CD4+/CD8−
profile showed a tendency towards a more favorable course.
Based on such observations and previous publications in the
literature, a new score for PTCL/NOS was developed integrating patient- and tumor-specific characteristics (age ≥60 years,
high performance status, elevated LDH values, and Ki-67
marking >75%): this score identified three clear-cut groups of
patients with different prognosis and seems to be more effective than previous indices, including the IPI and PIT 4.
In 2007, we published the first comprehensive study illustrating the gene expression profile (GEP) of PTCL/NOS 3. It was
based on 28 lymph node biopsies containing an amount of
neoplastic cells exceeding the 70% value of the whole examined population. The messenger RNA extracted from these
120
cases was hybridized on the HG U133 2.0 Plus gene chip. The
results obtained were compared with those of six AITLs, six
ALCLs (two ALK+ and four ALK-) and 20 samples of normal
T-lymphocytes, which had been purified from the peripheral
blood and tonsil and corresponded to the main T-cell subsets
(CD4+, CD8+, resting and activated). Such study significantly
differed from the previous ones of Martinez-Delgado et al. 5
and Ballester et al. 6 who had evaluated very heterogeneous
cases, often containing a prominent reactive component that
had influenced the global signature. Notably, for the first
time, our study provided the rationale for possible targeted
therapies in PTCL/NOS by offering clear evidence of their
ex vivo effectiveness. In particular, GEP indicated that PTCL/
NOS are distinct from normal T- and B-lymphocytes and are
more closely related to activated rather than resting T-cells.
As in normal mature T-lymphocytes, it was possible to identify two main subgroups of PTCL/NOS, with GEPs related to
either CD4 or CD8 elements. Notably, this characteristic did
not reflect the expression of CD4 and CD8 molecules. More
importantly, two small subsets were identified provided with
cytotoxic and follicular T-helper (FTH) profile. The former
was shown to herald a very poor prognosis by Iqbal et al.
two years later 7. The latter will be further discussed in the
following.
In addition to histogenetic information, our analysis provided
several insights into the functional alterations of PTCL/NOS.
A careful comparison of PTCLs/NOS with the closest normal
counterparts revealed the systematic deregulation of 155 genes
controlling functions that are typically damaged in malignant
cells, such as matrix remodeling, cell adhesion, transcription,
proliferation and apoptosis. In particular, our findings might
explain the dissemination pattern of PTCL/NOS, with frequent extranodal and bone-marrow involvement and spread
to peripheral blood, by showing the up-regulation of genes
that promote local invasion and metastasis in different types
of human cancer. In addition, it revealed the deregulation of
genes involved in apoptosis (e.g., MOAP1, ING3, GADD45A
and GADD45B) and chemo-resistance (such as CYR61 and
NNMT). Immunohistochemistry provided in situ validation
of the genomic data by showing correspondence between
messenger RNA and protein expression, as seen, for example,
with PDGFRalpha and BCL10. In addition, by comparison
with normal tissues, immunohistochemistry allowed the identification of staining patterns corresponding to the synthesis
of ectopic or para-physiological products by neoplastic cells.
Finally, the phenotypic test highlighted the possibility that
some of the results obtained by GEP may depend on nonneoplastic components present in the analyzed sample, as seen
for Caldesmon. In the course of the same study, we found that
all ALCLs tended to cluster together – irrespective of their
ALK positivity or negativity – showing a signature distinct
from those of PTCL/NOS and AITL. Some of these findings
have been the object of further research activities and will be
detailed in the following.
In the same year and almost at the same time, our own and
De Leval’s GEP analyses revealed that AITL has a gene
signature that is indeed close to that of FTH cells, i.e. of Tlymphocyte taking part in the regulation of the germinal center B-cell life 8 9. This explains why AITL expresses CD10,
Bcl-6, CXCL13, PD1, ICOS, SAP, and CCR5 in variable
combinations. In fact, such molecules are physiologically
carried by FTH lymphocytes. This was regarded as a tool for
the straightforward differentiation of AITL from PTCL/NOS
and the staining for one of the above mentioned markers as
the diagnostic proof of AITL. Unfortunately, both concepts
turned out wrong. In fact, further studies carried out by our
Group have demonstrated on a large series of cases that the
FTH phenotype can also be found in tumours that lack the
hallmarks of AITL, i.e. hyperplasia of follicular dendritic
cells and high endothelium venules and are thus classified
as PTCLs/NOS 10. This suggests that a new histogenetically
homogeneous category of PTCLs can be envisaged that includes morphologically different pictures. In addition, on
immunohistochemistry the expression of one single FTH
marker is not enough to sustain the derivation of the process
from FTH-related cells: in fact, at least three of these markers must be simultaneously detected since they can be singly
expressed as the result of cell plasticity 11. In the course of the
GEP study on AITL, in agreement with De Leval et al. 9, we
observed up-regulation of the VEGF gene. However, by immunohistochemistry on tissue microarrays, we showed that
neoplastic cells strongly express both VEGF and its receptor
KDR. This fact suggests the possible existence of an autocrine loop and sensitivity to anti-angiogenetic drugs.
Three additional findings merit attention. Firstly, we found
that PTCL/NOS presents global down-regulation of NF-κB
genes in comparison with normal T- lymphocytes 3. This observation was corroborated by a subsequent SNP array study
carried out in cooperation with Martin Hansmann’s Group 12,
as well as by the consistent cytoplasmic localization of NFκB molecules, the latter moving to the nucleus in the case of
NF-κB pathway activation. Our data differ to some extent
from those reported by other Groups that displayed up- or
down-regulation of NF-κB molecules, with possible prognostic implications, not confirmed in our series 5 6. However,
such discrepancies might reflect the fact that other studies
included a limited number of PTCLs/NOS or anyway cases
with prominent non-neoplastic components. Secondly, since
silencing of certain genes (such as GADD45A and GADD45B)
can be regulated by epigenetic mechanisms including acetylation, we tested a histone deacetylase inhibitor against PTCL/
NOS primary cells. Notably, the compound induced dramatic
G0–G1 cell cycle arrest and apoptosis at therapeutic concentrations, suggesting a possible role for this class of drugs in
PTCL/NOS therapy. This issue has been developed by Owen
O’Connor and co-workers: their results that will be the object
of a presentation at the next ASH meeting, support the efficacy of histone deacetylase inhibitors in the treatment of
PTCLs (personal communication). Thirdly, the regular detection of PDGFRalpha over-expression at the messenger RNA
and protein levels, as well as its consistent phosphorylation
prompted us to design ex vivo experiments aimed testing the
sensitivity of PTCL/NOS cells to Imatinib, a well-known
PDGFRalpha inhibitor. The results obtained were of interest,
with about 50% cytotoxic effect seen at 48 h with a 1 μmol
concentration. Such an effect became even higher (75%) with
a 10 μmol dose. Notably, Imatinib exerted a limited effect on
the viability of normal lymphocytes.
PDGFRalpha has been one of the main objects of our most recent research activity. In fact, the same alteration has been found
in T-cell tumors other than the NOS and AITL ones, such as
extranodal NK/T-cell lymphoma nasal type, mycosis fungoides
and ALCL, thus suggesting that it can represent an important
pathogenetic mechanism. For this reason, we have extensively
evaluated the mechanisms that can sustain PDGFRalpha activation by coming to the conclusion that this is sustained by an
autocrine loop. In addition, we have obtained evidence that the
activation of PDGFRalpha pathway might vicariate the NF-κB
one – that is instead of pivotal importance in B-cell lymphomas
– by sustaining proliferation and rescue from apoptosis.
relazioni
All these pieces of information further support the original ex
vivo observation concerning the potential therapeutic efficacy
of TKIs. On this respect, in co-operation with Lukas Kenner’s
and Giorgio Inghirami’s Groups, it was found that ALCL
(both ALK+ and ALK-) is also highly sensitive to Imatinib 13.
This was shown in the mouse model and a limited number of
patients refractory to all therapies with either ALCL or PTCL/
NOS. Imatinib produced regression of the transplanted tumor
in mice and disease stabilization or even complete remission
in humans.
Combined GEP and TMA studies have provided additional
relevant contributions as to the in vivo administration of the
humanized monoclonal antibody Campath-1H (Alemtuzumab) targeted to CD52, repeatedly proposed for the treatment
of patients with PTCL. Although other factors can affect its
response in vivo, the lack of CD52 expression may play a major role in causing refractoriness to the compound 14. We studied the expression of CD52 on tissue microarrays containing
97 PTCLs/NOS 14. In addition, in 28 cases for which frozen
material was available, GEPs were generated and compared
with those of 20 samples of normal T-lymphocytes. We found
that 60% of PTCLs/NOS showed CD52 gene expression level
at least two log lower than the lowest one recorded in normal
T-cells. In addition, the gene product was detected by immunohistochemistry in 41% PTCLs. Based on these findings, we
think that the estimation of CD52 expression may provide a
rationale for the selection of patients with higher probability
of responding to Alemtuzumab, by avoiding the risk of unwanted toxicity.
In 2010, the same combined GEP and TMA approach was applied to a large series of ALK+ and ALK- ALCLs with frozen
material available 15. This study aimed to definitely clarify
whether or not ALK- ALCL should be lumped with PTCL/
NOS, as suggested at the time of the last WHO Classification
drafting in spite of the fact that the International PTCL Trial
had reveled that ALK- ALCL – although more aggressive
than the ALK+ form – has 5-year failure-free and overall survival rates that are significantly better than PTCL/NOS. The
profiles of ALK- ALCL were compared to other PTCLs, and
14 genes were discovered capable to distinguish ALK- ALCL
from PTCL-NOS and AILT samples. Unexpectedly, all 14
ALK predictors were similarly expressed by ALK+ ALCL,
suggesting the existence of a common ALCL signature. This
hypothesis was subsequently confirmed comparing all ALCL
samples to PTCLs. A class prediction analysis led to the identification of an overlapping list of genes which included 34
probes. The new classifier clearly separated ALCLs from PTCLs/NOS, AITLs and normal T-cells. The identified fingerprint was confirmed by Q-RT-PCR in independent cases using
4 targets (TNFRSF8, SNFT, NFATC2, and PERP), which resulted differentially regulated in ALCL patients. As predicted,
also the immunostaining revealed weak/rare expression of
NFATC2 in ALCL, while it was consistently expressed in the
neoplastic compartments of PTCL/NOS samples. Notably, by
massive parallel sequencing Feldman et al. have recently observed the occurrence of the t(6;7)(p25.3;q32.3) translocation
in about one third of ALK- ALCLs causing downstream the
same effects as t(2;5) in ALK+ ALCLs 16.
The most recent developments of our research activity deal
with gene expression and microRNA profiling and deep sequencing of PTCLs.
GEP studies have for a decade found a major limitation in the
need for cryopreserved material. This has obviously affected
the clinico-molecular correlations with special reference to rare
tumors as PTCLs actually are. In fact, it is indeed difficult to
121
collect series large enough to achieve high statistical power.
We have recently applied the novel DASL technology from
Illumina to 144 PTCLs. Such technique allows profiling of
formalin-fixed, paraffin-embedded tissue samples by using a
pool of probes spanning about 50 bases that make possible to
analyze partially degraded RNA. Interestingly, all the observations published in the Journal of Clinical Investigation in 2007 3
by frozen samples, were indeed confirmed. This – for instance –
regarded the malfunctioning of the NF-κB pathway. In addition,
by supervised analysis signatures could be constructed clearly
differentiating PTCLs/NOS, AITLs and ALK+ and ALK- ALCLs, which over all showed different regulation of relevant
cellular programs. Interestingly, by comparing the signature
of the different subsets of normal T-lymphocytes with that of
the neoplastic cells, PTCL/NOS clusters could be identified
corresponding to activated central memory T-cells, cytotoxic Tlymphocytes and FTH elements. In the same series of cases, the
microRNA profile was studied by using the Applied Biosystem
card A. While the unsupervised analysis did only distinguish
neoplastic cells from normal T-lymphocytes, the supervised one
revealed up-regulation of miR-146 and miR-222 in ALCL and
differential expression of 7 miRNAs between AITL and PTCL/
NOS and 26 miRNAs between the latter and ALK- ALCL.
Notably, by gene set enrichment analysis the microRNA profile
turned out to significantly impact the GEP. These preliminary
results demonstrate the feasibility of high-tech studies by using
archival material and the usefulness of the combined evaluation
of gene expression and microRNA profiles.
As to the next generation sequencing approach, at the moment we are enrolling patients who agree to provide both their
normal and pathological DNA according to the guidelines of
our Ethical Committee. The former is obtained from saliva,
peripheral blood or skin shave biopsy, depending on the clinical manifestations and disease spread in each single patient. By
the Illumina HiScan SQ platform different types of analysis
are ongoing: whole genome, whole exome and transcriptome
sequencing. The aim is to identify driving mutations which
can better explain the pathobiology of PTCLs and allow the
development of novel therapeutic options as already happened
for hairy cell leukemia.
References
1
Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of
Tumours of Haematopoietic and Lymphoid Tissues. 4th Edition. Lyon:
IARC Press 2008, pp. 157-166.
2
International Peripheral T-cell Lymphoma Project. International peripheral T-cell and natural killer/T-cell lymphoma study: pathology
findings and clinical outcomes. J Clin Oncol 2008;26:4124-30.
3
Piccaluga PP, Agostinelli C, Califano A, et al. Gene expression
analysis of peripheral T-cell lymphoma, unspecified, reveals distinct profiles and new potential therapeutic targets. J Clin Invest.
2007;117:823-34.
4
Went P, Agostinelli C, Gallamini A, et al. Marker expression in peripheral T-cell lymphoma: a proposed clinical-pathologic prognostic
score. J Clin Oncol 2006;24:2472-9.
5
Martinez-Delgado B, Melendez B, Cuadros M, et al. Expression
profiling of T-cell lymphomas differentiates peripheral and lymphoblastic lymphomas and defines survival related genes. Clin Cancer Res
2004;10:4971-82.
6
Ballester B, Ramuz O, Gisselbrecht C, et al. Gene expression profiling
identifies molecular subgroups among nodal peripheral T-cell lymphomas. Oncogene 2006;25:1560-70.
7
Iqbal J, Weisenburger DD, Greiner TC, et al. Molecular signatures to
improve diagnosis in peripheral T-cell lymphoma and prognostication
in angioimmunoblastic T-cell lymphoma. Blood 2010;115:1026-36.
8
de Leval L, Rickman DS, Thielen C, et al. The gene expression profile
of nodal peripheral T-cell lymphoma demonstrates a molecular link
between angioimmunoblastic T-cell lymphoma (AITL) and follicular
helper T (TFH) cells. Blood 2007;109:4952-63.
122
9
10
11
12
13
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Piccaluga PP, Agostinelli C, Califano A, et al. Gene expression
analysis of angioimmunoblastic lymphoma indicates derivation from T
follicular helper cells and vascular endothelial growth factor deregulation. Cancer Res 2007;67:10703-10.
Agostinelli C, Hartmann S, Klapper W, et al. Peripheral T-cell lymphomas with follicular T-helper phenotype: a new basket or a distinct
entity? Revising Karl Lennert’s personal archive. Histopathology
2011, in press.
Laurent C, Fazilleau N, Brousset P. A novel subset of T-helper
cells: follicular T-helper cells and their markers. Haematologica
2010;95:356-8.
Hartmann S, Gesk S, Sholtysik R, et al. High resolution SNP array
genomic profiling of peripheral T cell lymphomas, not otherwise specified, identifies a subgroup with gains and rearrangement of REL. Br J
Haematol 2010;148:402-12.
Laimer D, Dolznig H, Vesely PW, et al. The novel AP-1 target gene
PDGFRB represents an effective target for imatinib in NPM-ALK
positive Anaplastic large cell lymphoma. Nature 2011, submitted.
Piccaluga PP, Agostinelli C, Righi S, et al. Expression of CD52
in Peripheral T-Cell Lymphoma Unspecified. Haematologica
2007;92:566-7.
Piva R, Pellegrino E, Agnelli L, et al. Gene expression profiling uncovers molecular classifiers for the recognition of Anaplastic Large
Cell Lymphoma within Peripheral T-cell neoplasm. J Clin Oncol
2010;28:1583-90.
Feldman AL, Dogan A, Smith DI, et al. Discovery of recurrent
t(6;7)(p25.3;q32.3) translocation in ALK-nagative anaplastic large
cell lymphoma by massively parallel genomic sequencing. Blood
2011;117:915-9.
Linfomi cutanei: aspetti classificativi
e problematiche diagnostiche
M. Lucioni, G. Fiandrino, M. Nicola, R. Riboni, S. Molo, C.
Franco, M. Paulli
Sezione Anatomia Patologica, Dipartimento di Scienze Pediatriche e
di Patologia Umana ed Ereditaria, Università di Pavia e Fondazione
IRCCS Policlinico “S. Matteo”, Pavia
Non-Hodgkin’s lymphomas arising from tissues other than
lymph nodes are defined as primary extranodal lymphomas,
and constitute at least 25-35% of newly-diagnosed lymphoma
cases 1.
Beyond its role as outer physical barrier, the skin represents
a complex and active immune organ, equipped with its own
microenvironment and hosting both residing and migrating
specialized cells. Therefore, it should not surprise that the cutaneous district often represents the site of onset or localization
of lymphoproliferative disorders. Notably, primary cutaneous
lymphomas (PCL) account for around 20% of cases of extranodal lymphomas. The estimated incidence of PCL is around
10.7/1.000.000 person-years in the United States with an increasing trend, being second only to primary lymphomas of the
gastrointestinal tract (SEER program data) 2. On the contrary,
secondary skin involvement in the course of a systemic lymphoma constitutes an entirely separated disease: beyond obvious and often challenging morphologic analogies with PCL,
it carries distinct implications both for diagnosis and therapy,
and will not be the focus of this short review. Since the seminal
description of mycosis fungoides (pian fongoïde) in 1806 by
the French dermatologist J.M.L. Alibert 3, followed by a morein-depth coverage by E. Bazin 4, many leaps forward have
been accomplished both in the classification and treatment of
cutaneous lymphomas. As of today, they appear to represent
a family of acquired neoplasms with multifactorial and mostly
unidentified etiologies. In addition, there is a lack of accurate
epidemiological studies in the different geographic areas. Data
on genetic susceptibility are also limited; sporadic associations
have been identified in the rare familial cases, though in the
absence of specific genotype polymorphisms 5. Basic research
is today focused on the processes of lymphocytic mitosis, apopotosis, and skin-homing, together with the study of the immunobiologic properties of the cutaneous microenvironment
and microbiologic agents. This is likely to pave the way for
tailored and more efficient immuno-molecular therapies.
In the past, PCL have been classified according to the Kiel
classification and the Working Formulation; however, these
lacked adequate correlations with prognosis. Currently,
PCL are subdivided along the schemes elaborated by 2005
World Health Organization (WHO)-European Organization
for Research and Treatment of Cancer (EORTC) joint classification 6, with successive and notable adjustments in the
2008 WHO Classification of Tumors of Haematopoietic and
Lymphoid Tissues 7. The two main diagnostic branches of
PCL are represented by T-cell (PCL-T) and B-cell (PCL-B)
lymphomas. Exceptions are represented by plasmacytoid
dendritic cell neoplasm, now considered a different disease
altogether, and by cutaneous pseudolymphomas, a broad and
poorly defined category of reactive, benign and often selfhealing lymphoprolipherations, with either B- or T-cell predominance. Clinical staging is currently a standard of care
for PCL-T, with the Tumor-Node-Metastasis (TNM) and/
or the Tumor Burden Index (TBI) systems; most recently,
a Cutaneous Lymphoma Prognostic Index (CLPI) was also
proposed 8. In fact, PCL-T make up for the vast majority
of PCL cases (70-80%), usually affect adults, and are more
commonly reported in males than in females. Incidence
increases with age, with a peak at about 80 years. Multiple
skin biopsies, complete blood count, flow cytometry and
peripheral blood smear are required for the initial evaluation.
Extracutaneous disease must be excluded by lymph node
biopsy if palpable nodes are present; bone marrow biopsy is
only advised for advanced stages.
Histologically, PCL-T include mycosis fungoides (MF),
with its variants and subtypes, Sézary syndrome, adult T-cell
leukaemia/lymphoma, primary cutaneous CD30+ lymphoproliferative disorders, subcutaneous panniculitis-like T-cell
lymphoma, extranodal NK/T-cell lymphoma of nasal type,
and primary cutaneous peripheral T-cell lymphomas of rare
subtypes. Overall 5-year survival rate for CTL-T is rather
high, ranging from 91% for patients with mycosis fungoides
to 40% for Sézary syndrome 2.
MF, a neoplasm of memory T-cell phenotype, is by far the
most common entity of PCL-T (>50%) with an estimated
incidence rate of 4.1/1.000.000 person-years in the United
States 2, and representing a major burden in health care
costs and diagnostic efforts. Its course is generally indolent,
and affects more often male than female individuals (ratio
2:1), in the 5th-6th decade. Clinically, it often presents with
confined, scaly patches on the trunk and buttocks that may
go misdiagnosed as trivial inflammatory dermatoses, possibly with spontaneous remission. It often takes years or even
decades before the patches progress to the plaque stage or
the tumor stage. In spite of recent advances in the definition of MF pathogenesis, the diagnosis of the early stages
of the disease still remains challenging for dermatologists
and pathologists alike, often requiring ancillary studies and
repeated biopsies 9. T-cell receptor (TCR) b or g chains are
clonally rearranged in the majority of cases, and a T-helper 2
phenotype is usually observed, with expression of peripheral
T-cell antigens such as CD2, CD3, CD4, and of CD45RO,
notably in the absence of CD8 and, often, with loss of
CD7 and/or CD5. However CD8+ cases are sometimes
observed, especially in younger patients. The MF family
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includes variants and subtypes, such as pagetoid reticulosis,
follicular MF, syringotropic cutaneous T-cell lymphoma,
granulomatous slack skin, bullous MF, granulomatous MF,
and poikilodermic MF, among others. Sézary syndrome
(SS), often assimilated to MF both for epidemiological,
clinical and therapeutic purposes, is a distinct, uncommon
PCL-T. It is classically defined by the triad of generalised
erythroderma, lymphadenopathy, and the presence of 5%
or more malignant T-cells with cerebriform nuclei (known
as Sézary/Lutzner cells) in peripheral blood lymphocytes.
The International Society for Cutaneous Lymphoma (ISCL)
has proposed that the diagnosis of this disease be based on
molecular and flow cytometric evidence in the blood of a
large clonal population of abnormal T cells in addition to
erythroderma, with lymphadenopathy becoming an optional
criterion 10.
The group made up by MF and SS is followed, for incidence, by primary cutaneous CD30+ lymphoproliferative
disorders [primary cutaneous anaplastic large cell lymphoma
(C-ALCL), lymphomatoid papulosis (LyP) and borderline
cases](~30%). C-ALCL is characterized by either anaplastic,
pleomorphic or immunoblastic morphology and expression of
CD30 antigen; its clinical behaviour is strikingly more favourable than its systemic anaplastic-lymphoma kinase(ALK)-negative counterpart; however, gene expression profiling (GEP)
has failed to highlight major differences between the two
groups, suggesting a role for the microenvironment and posttranscriptional events 11. LyP often affects the trunk, the buttocks and the arms of young adults, with crops of nodules and
papules regressing in a few weeks with scar formation. Three
histological subtypes (A, B, and C) are recognized. CD30 is
uniformely expressed, except in type B; TCR rearrangement
is detected in around 90% of cases with laser microdissection
of cells (personal data). Most recently, the existence of a type
D, simulating CD8-positive aggressive epidermotropic cytotoxic T-cell lymphoma, has also been claimed 12. Borderline
cases are represented by those instances in which, despite
careful clinicopathological correlation, a definitive distinction
between C-ALCL and LyP cannot be made. PCL-T of rare
subtypes (gamma-delta T-cell lymphoma, CD8-positive aggressive epidermotropic cytotoxic T-cell lymphoma or Berti’s
lymphoma, CD4-positive small/medium-sized pleomorphic
T-cell lymphoma) account for the remaining 15%, and often
pose difficulties in terms of differential diagnosis.
PCL-B are much rarer, accounting for about one fourth of
PCLs. Patients are usually old adults, of older patients, and is
very aggressive, with a survival of less than 50% at 5 years.
Even though studies based on GEP and array comparative genomic hybridization seem to provide a biological background
to the existence of a distinct PCDLBCL “leg-related” 18, much
is still to be investigated regarding both biological features and
identification/prognostication markers of this subset of PCL-B.
References
1
Newton R, Ferlay J, Beral V, et al. The epidemiology of non-Hodgkin’s
lymphoma: comparison of nodal and extra-nodal sites. Int J Cancer
1997;72:923-30.
2
Bradford PT, Devesa SS, Anderson WF, et al. Cutaneous lymphoma
incidence patterns in the United States: a population-based study of
3884 cases. Blood 2009;113:5064-73.
3
Alibert JL. Description des Maladies de la Peau: Observées a I’Hôpital St. Lousis et Exposition des Meilleurs Méthodes Suivies pour leur
Traitment 1806. Barrois l’Ainé et Fils, Paris, p 157.
4
Bazin E. Leçons sur le Traitement des Maladies Chroniques en Général Affections de la peau en Particulier l’Emploi Comparé des Eaux
Minérales de l’Hydrothérapie et des Moyens Pharmaceutiques 1870.
Adrien Delahaye, Paris, p 425.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Hodak K, Akerman L, David M, et al. Cytokine gene polymorphisms in
patch-stage mycosis fungoides. Acta Derm Venereol 2005;85:109-12.
Willemze R, Jaffe ES, Burg G, et al. WHO/EORTC classification for
cutaneous lymphomas. Blood 2005;105:3768-85.
Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of tumours of haematopoietic and lymphoid tissues. 4th Ed. Lyon, France:
IARC Press 2008.
Agar NS, Wedgeworth E, Crichton S, et al. Survival outcomes and
prognostic factors in mycosis fungoides/Sézary syndrome: validation
of the revised International Society for Cutaneous Lymphomas/European Organisation for Research and Treatment of Cancer staging
proposal. J Clin Oncol 2010;28:4730-9.
Pimpinelli N, Olsen EA, Santucci M, et al. Defining early mycosis
fungoides. J Am Acad Dermatol 2005;53:1053-63.
Hwang ST, Janik JE, Jaffe ES et al. Mycosis fungoides and Sézary
syndrome. Lancet 2008;371:945-57.
Eckerle S, Brune V, Döring C, et al. Gene expression profiling of isolated tumour cells from anaplastic large cell lymphomas: insights into
its cellular origin, pathogenesis and relation to Hodgkin lymphoma.
Leukemia 2009;23:2129-38.
Saggini A, Gulia A, Argenyi Z, et al. A variant of lymphomatoid papulosis simulating primary cutaneous aggressive epidermotropic CD8+
cytotoxic T-cell lymphoma. Description of 9 cases. Am J Surg Pathol
2010;34:1168-75.
Takino H, Li C, Hu S, et al. Primary cutaneous marginal zone B-cell
lymphoma: a molecular and clinicopathological study of cases from
Asia, Germany, and the United States. Mod Pathol 2008;21:1517-26.
Streubel B, Simonitsch-Klupp I, Mullauer L, et al. Variable frequencies of MALT lymphoma-associated genetic aberrations in MALT
lymphomas of different sites. Leukemia 2004;18:1722-6.
Van Maldegem F, van Dijk R, Wormhoudt TA, et al. The majority of
cutaneous marginal zone B-cell lymphomas expresses class-switched
immunoglobulins and develops in a T-helper type 2 inflammatory
environment. Blood 2008;112:3355-61.
Dalle S, Thomas L, Balme B, et al. Primary cutaneous marginal zone
lymphoma. Crit Rev Oncol Hematol 2010;74:156-62.
Paulli M, Arcaini L, Lucioni M, et al. Subcutaneous ‘lipoma-like’
B-cell lymphoma associated with HCV infection: a new presentation
of primary extranodal marginal zone B-cell lymphoma of MALT. Ann
Oncol 2010;21:1189-95.
Hoefnagel JJ, Dijkman R, Basso K, et al. Distinct types of primary
cutaneous large B-cell lymphoma identified by gene expression profiling. Blood 2005;105:3671-8.
Sarcoma a cellule follicolari dendritiche
F. Facchetti, W. Vermi
Anatomia Patologica 1, Spedali Civili, Università di Brescia
Le cellule follicolari dendritiche (FDC) sono cellule a morfologia dendritica localizzate nei follicoli linfoidi B primari e
secondari presenti sia in sedi nodali che extranodali; si distinguono dalle cellule dendritiche professionali e con funzione
di “antigen presenting cells” in quanto, incapaci di catturare
l’antigene per presentarlo a cellule T naïve, sono al contrario
“antigen-carrying cells” poiché recano sulla loro superficie
complessi antigene-anticorpo pre-formati che entrano in contatto con cellule immunocompetenti adiacenti. La loro origine
non è stata ancora definita con certezza, ma si ritiene non
derivino da precursori emopoietici come le cellule dendritiche
“classiche”, ma da precursori mesenchimali. Anche la precisa
funzione svolta dalle FDC nella reazione centro-follicolare
non è stata definitivamente chiarita, ma la loro presenza è determinante per il normale sviluppo dello switch isotipico.
Il sarcoma originato dalle FDC (FDCS) è una neoplasia rara,
insorge ad un’età media di 44 anni, egualmente distribuito
nei due sessi tranne che nella variante cosiddetta “similpseudotumore infiammatorio” (FDCS-IPT-like) che è più
frequente nelle donne; nei due terzi dei casi ha primitività
nodale, extranodale nel resto (FDCS-IPT-like tipicamente
insorge in sede addominale). Si è osservata un’associazione
124
con la malattia di Castleman specie di tipo jalino-vascolare
(10%-20 % dei casi), tanto da far considerare tale condizione
un precursore del FDCS. La malattia è asintomatica, tranne
che nella variante FDC-IPT-like che si manifesta con febbre
e sintomi sistemici. Considerata un tempo malattia indolente,
in realtà FDCS recidiva nel 50% dei casi e metastatizza nel
25%, con una mortalità del 17% a 5 anni. Sono fattori prognostici negativi la sede addominale (tranne che nella variante
IPT-like!), le dimensioni (>6 cm) e la presenza di anaplasia e
proliferazione elevate.
Morfologicamente il FDCS si presenta come una massa del
diametro medio di 7 cm (1-20 cm); si caratterizza per una
estrema variabilità istologica, sia per la morfologia cellulare
(fusata, ovoidale, epitelioide), che per il pattern di crescita
(storiforme, il più comune, trabecolare, simil- meningioma,
simil- timoma, simil-GIST), che per la cellularità di accompagnamento (tipica è la presenza di piccoli linfociti sparsi
fra le cellule tumorali, ma in alcuni casi si osserva una ricca
componente infiammatoria eterogenea, tipica della forma
FDCS-IPT-like). Una variante particolarmente inusuale è
stata descritta recentemente (Lorenzi L, et al. Hum Pathol,
in press) e consiste nella proliferazione di FDC nel contesto
di macronoduli linfoidi composti da cellule B mantellari
(simulante le varianti nodulari di linfoma di Hodgkin a predominanza linfocitica o classico ricco in linfociti), oppure in
aggregati coesivi di cellule atipiche circondate da un mantello
di linfociti B (simiulante un linfomaB centrofollicolare ad
alto grado).
Le cellule tumorali hanno bordi mal definiti e distintive caratteristiche nucleari, che ricordano molto quelle delle FDC
normali: i nuclei hanno fine cromatina e delicata membrana
nucleare, con nucleolo evidente eosinofilo; spesso duplici o
multipli, entrano in stretto con fenomeno di “molding”.
La diagnosi si fonda sulla dimostrazione di marcatori antigenici tipici delle FDC, fra i quali, in ordine di utilità e specificità si annoverano CD21, CD23, clusterina, CD35, CXCL13,
podoplanina, desmoplachina, quest’ultima a conferma della
presenza di desmosomi. Considerata la facilità di delezione
antigenica in questi tumori, è raccomandato l’uso di almeno due o tre marcatori. FDCS possono inoltre esprimere la
proteina S100, CD68, ma sono negativi per CD1a e CD117.
Occasionalmente si è riscontrata espressione di CD45RB e
CD20, ma il fenomeno potrebbe dipendere da un adsorbimento passivo di antigene da cellule contigue. Recentemente
è stata infine riportata espressione di TdT, fenomeno che in
ogni caso merita verifica in altri casi.
Nella variante IPT-like è dimostrabile la presenza di EBV con
tecnica di ibridazione in situ.
In conseguenza della estrema variabilità morfologica del
FDCS, la diagnosi differenziale, che si base essenzialmente
sullo studio immunofenotipico, include un’ampia gamma di
neoplasie epiteliali e stromali, nodali ed extranodali. In alcuni
casi di Malattia di Castleman jalino-vascolare l’incremento
di FDC nelle aree interfollicoalri può essere marcato e non
agevole da definire se reattivo o già espressione di crescita
tumorale; similmente, si possono avere quadri di espansione
di FDC simil-tumorali in casi di Linfoma T angioimmunoblastico, che tipicamente si caratterizza per iperplasia delle FDC
nelle aree di proliferazione linfoma tosa.
Non esistono dati consistenti genetico-molecolari sul FDCS.
La neoplasia esprime intensamente il fattore di crescita EGFR. In uno studio eseguito su 20 casi di FDCS, tutti EGFR
positivi, è stata riscontrata attivazione della pathway EGFR
dipendente, con fosforilazione di STAT3, ERK e AKT. Tuttavia, in nessun caso si sono documentate mediante FISH o
sequenziamento diretto amplificazione genica o mutazioni
transattivanti di EGFR (esoni 18-19-20). Inoltre, da una delle
neoplasie è stato possibile generare una linea cellulare (Dr. E.
Giurisato, Siena) che è risultata EGFR-dipendente per proliferazione e sopravvivenza, che venivano inibite dall’anticorpo
anti-EGFR Cetuximab® in forma dose-dipendente. Questa
osservazione apre una potenziale via al trattamento delle forme di FDCS ad evoluzione aggressiva, per le quali non è stata
a tutt’oggi identificata una terapia efficace.
Domande
Non è un marcatore distintivo del Sarcoma a cellule follicolari
dendritiche:
CD21
CD23
Clusterina
CD117
Le cellule follicolari dendritiche si espandono in forma patologica nel:
Linfoma follicolare
Linfoma mantellare
Linfoma a cellule marginali
Linfoma T angioimmunoblastico
Cellule follicolari dendritiche con aspetti “displastici” sono
osservabili tipicamente in:
Timoma
Malattia di Castleman, jalino-vascolare
Linfoma T angioimmunoblastico
Linfoma follicolare
Il Sarcoma a cellule follicolari dendritiche può simulare:
Un GIST
Un timoma
Un Linfoma di Hodgkin a predominanza linfocitica
Tutte le neoplasie indicate
Nessuna delle neoplasie indicate
Carrying out research projects in developing
countries: the contribution of APOF
L. Leoncini, M.R. Ambrosio, B.J. Rocca, K. Schurfeld, S.
Lazzi
Department of Human Pathology and Oncology, Anatomic Pathology
Section - University of Siena, Italy
Introduction
In the past 25 years revelations on the genesis of human
cancer have come at an increasing pace. Research on oncogenic infectious agents, especially viruses, has helped us to
understand the process of malignant transformations of cells
because the cellular events in viral-driven transformation mirror, often brilliantly, basic cellular processes that culminate
in cancer, even those not associated with viruses. Infectious
agents, especially viruses, account for several of the most
common malignancies-up to 20% of all cancers. Some of
these cancers are endemic, with a high incidence in certain
geographic location but sporadic/lower incidence in other
parts of the world. Lymphomas arise frequently in association with infectious agents such as Epstein-Barr virus (EBV),
human immunodeficiency virus (HIV), human herpes virus
8 (HHV8), Helicobacter pylori (HP), and hepatitis C virus
(HCV). Although accurate estimates are difficult given the
paucity of information, it is likely that approximately 30.000
non-Hodgkin lymphomas (NHL) occur in the equatorial belt
of Africa each year and these tumors are among the top 10
causes of cancer in this geographic region. The experience
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of APOF in establishing pathology services in many African
laboratories has allowed the collection of many lymphoma
cases and contribute to the research for understanding the role
played by infectious agents in pathogenesis of lymphomas.
Role of infectious agents in lymphomagenesis
In recent years insights into the roles played by pathogens
such as the EBV, the malaria-causing agent Plasmodium falciparum, HHV8, HIV and HP in lymphoid malignancies have
been gained. The infective agents may play a direct role in
lymphomagenesis or act indirectly as a cofactor, deregulating
immune cells. In addition, viruses may also encode products,
which may mimic cellular proteins (ie. v-cyclins) thus dis-
turbing mechanisms of cell cycle regulation. Recent literature
indicates that such interaction may involve, in particular, a
class of small noncoding RNAs, the microRNAs (miRNAs),
whose imbalance may alter and dysregulate gene expression
in infected cells.
Conclusions
The consistent association of lymphomas with various pathogens suggests that these factors may have a role in the malignant transformation or further evolution of lymphomas
and could even account for the differences in the pattern of
lymphomas observed between, for example, African and European countries.
Sabato, 29 ottobre, 2011
Patologia molecolare: come standardizzare le procedure
Moderatore: Generoso Bevilacqua (Pisa)
Identifying EGFR mutations
G. Pelosi
Dipartimento di Patologia Diagnostica e Laboratori, Fondazione
IRCCS Istituto Nazionale dei Tumori e Università degli Studi di Milano, Milano
The identification of EGFR mutation in exon 18 to 21 is the best
way to forecast the response to EGFR-tyrosine kinase inhibitors
in lung adenocarcinoma samples, either biopsies/cellblocks or
surgical specimens. Therefore, sensitive assays for mutation
detection using routine pathological specimens are demanded in
the clinical practice. Different methods are currently available,
either customer-designed or commercially available, either based
on DNA-based molecular assays or immunohistochemistry, but
several technical implications should be taken into account as
much carefully as possible in order to obtain the best results,
in terms of both reproducibility of procedures and diagnostic
accuracy of results. The detection of EGFR mutations, independently of the methods under assessment, always requires a precise
and rigorous stepwise procedure, which starts from an accurate
enrichment in tumor cells of tumor samples being analyzed and
then includes PCR amplification and eventual obtaining and
evaluation of the results. Molecular assays should also comprise
negative and positive controls to ensure the highest diagnostic accuracy. In this presentation, the technical implications of different
EGFR-based molecular assays in lung adenocarcinoma will be
taken into consideration, as well as the diverse implications that
different molecular assays may have in the clinical practice.
Mutazioni rare di EGFR: interpretazione
e significato clinico
M. Barberis
have been proved to predict activity of the EGFR-tyrosine kinase inhibitors (EGFR-TKI), gefitinib and erlotinib. While the
“common” EGFR mutations, like the L858R point mutation in
exon 21 and the in-frame deletional mutation in exon 19 have
been definitively associated with response to EGFR-TKIs, for
many others only occasionally detected, data on their correlation with response are still unclear.
We report here the experience of our Institution in the treatment with EGFR-TKIs of patients with advanced NSCLC
harbouring rare EGFR mutations.
Methods
The frequency of rare mutations has been investigated in 681
cases of NSCLC screened between 2006 and 2010. Mutations
in exons 18 and 20, uncommon mutations in exons 19 and 21,
and/or the presence of different mutations in a single tumour
(complex mutations) were considered rare.
Results: EGFR mutations were detected in 99 tumours (14.5%).
Eighteen cases carried rare mutations and ten out of these
patients were treated with erlotinib or gefitinib. The clinical
outcome was described case by case with references to the
literature. Of note, we found two EGFR mutations never identified before and one of unknown response to EGFR TKIs.
Conclusions
Gefitinib and erlotinib have different anti-tumour activity
according to the type of the EGFR mutation borne. Report
of cases harbouring rare mutations can support the decisionmaking process in this subset of patients.
Standardizing reports in molecular pathology
G. Troncone*, A. Fassina**
Milano
*
Department of Scienze Biomorfologiche e Funzionali, University of Naples
Federico II; **Department of Diagnostic Medical Sciences & Special Therapies, Surgical Pathology & Cytopathology Unit, University of Padova.
Introduction
Mutations of the Epidermal Growth Factor Receptor (EGFR)
Consistent rules should be followed in reporting molecular
pathology tests. This apply to both format and content. How-
126
ever, molecular pathology reports also need to be modulated,
reflecting different settings. In some instances, they can be
stand-alone reports; in other occasions, they may be part of
larger cytological or histological reports. In both settings
the reports should meaningful convey relevant information,
relative to patient/sample/laboratory identifiers, performed
test, obtained result, analytical and clinical interpretation and
controls. This should be done in a manner that is most understandable by clinicians. Thus, technical information are only a
part of molecular report. It is also relevant convey information
on what the result means for the patient and how clinicians
may integrate the obtained information with pertinent clinical
data.
International recommendations for reporting are well developed. Reports should be based on the International Organization for Standardization (ISO) 15189:2007 requirements for
medical laboratories and on a guideline document developed
by the College of American Pathologists regarding reporting
of molecular results. Some recommendations are general and
apply to most laboratory reports, whereas others are specific
to given molecular tests 1. As far as mutation nomenclature
for gene mutations is concerned, this has to comply with
the guidelines from the Human Genome Variation Society
(HGVS, http://www.hgvs.org) to stimulate uniform and unequivocal description of sequence changes. These state that
nucleotide number 1 should correspond to the A of the ATG
translation initiation codon. The description of all variants is
preceded by a letter indicating the type of reference sequence
used; “c.” relates to a coding DNA sequence and “p.” related
to a protein sequence 2.
A good example on how the above mentioned guidelines
can be adapted to a given molecular test can be derived from
the recent recommendation on KRAS testing 3. The external
quality assessment (EQA) scheme of the European Society
of Pathology applied selected 17 items to be covered for a
meaningful KRAS test report:
1) Sampling/arrival date; 2) Sample number; 3) Date of report; 4) Signature, 5) Unique identifier on each page; 6) Total
pages; 7) Name/address referral person; 8) Nature of the sample (Paraffin section, biopsies, formalin-fixed paraffin-embedded section, …); 9) Percentage of tumor cells; 10) Reason
for testing (KRAS testing, presence of KRAS mutation, …);
11) Genotype; 12) Use of correct nomenclature; 13) Interpretation of the data (Comments/results and conclusion/discussion); 14) List of mutations tested: 15) Method used (Name
of commercial kit or noncommercial assay); 16) Report title:
Refers to KRAS testing or KRAS molecular diagnosis and
clearly distinguished from the rest of the report; 17) Refers
to therapy: Positive test for KRAS indicates low response to
anti–epidermal growth factor receptor therapy (cetuximab,
panitumumab). A note statiting the rport issued according
to the International Organization for Standardization (ISO)
15189:2007 standard should be added.
References
1
Gulley ML, Braziel RM, Halling KC, et al. Clinical laboratory reports
in molecular pathology. Arch Pathol Lab Med 2007;131:852-63.
2
Ogino S, Gulley ML, den Dunnen JT, et al. Standard mutation nomenclature in molecular diagnostics: practical and educational challenges. J Mol Diagn 2007;9:1-6.
3
Bellon E, Ligtenberg MJ, Tejpar S, et al. External quality assessment
for KRAS testing is needed: setup of a european program and report
of the first joined regional quality assessment rounds. Oncologist
2011;16:467-78.
Il sequenziamento di seconda generazione per
la diagnosi molecolare su campioni citologici
F. Buttitta
Ospedale Clinicizzato-Anatomia Patologica, Chieti
Lung cancer has the highest rate of cancer-related mortality
worldwide, and non-small cell lung cancer (NSCLC) accounts
for approximately 80% of all lung cancer subtypes. Recent
advances in cancer treatment have been achieved with agents
that are designed to target specific molecules that are crucial
to growth or survival avoiding the side effects of conventional
chemotherapy. In lung tumors therapies targeting the epidermal growth factor receptor (EGFR) have shown significant
improvements in tumor response and survival, albeit in a
select group (10%) of NSCLC patients.
EGFR (ErbB1, HER1), is a transmembrane receptor protein
with an extracellular binding domain and a cytoplasmic tyrosine kinase domain. Ligand binding leads to activation of the
tyrosine kinase domain with subsequent cell cycle progression
and inhibition of apoptosis. EGFR kinase domain is a suitable
target for pharmacologic inhibition. Specific EGFR tyrosine
kinase inhibitors occupy the tyrosine kinase ATP binding
site, preventing activation and downstream effects responses
in 10% to 20% of patients, who were subsequently shown to
have specific somatic mutations in the EGFR tyrosine kinase
domain.
Since almost 70% of patients present with locally advanced
or metastatic disease at the time of diagnosis, and only a
small proportion of NSCLC patients are eligible for surgical
resection, great attention has been focused on cytology specimens and small biopsies, often the only available material for
molecular testing. Over the last decades, the ‘‘gold standard’’
method for research and clinical testing for gene mutations
has been Sanger sequencing. This technique is not very
sensitive and is limited by interference from non-malignant
cells in a heterogenous samples. Cytology specimens have
not been widely used for sequence analysis due primarily to
heterogeneity within samples and/or a very low abundance
of neoplastic cells. The development of a much more sensitive technology, the massive parallel sequencing (MPS), also
known as “next generation sequencing” (NGS), offers new
diagnostic opportunities. Briefly, single DNA strands with
adaptor sequences are attached to beads and then clonally amplified by PCR in an oil–water emulsion. The beads are mixed
with DNA polymerase and deposited in plates containing over
1 million wells, with one bead per well. Nucleotides then flow
sequentially over the wells and as each nucleotide is added to
form complementary DNA strands, pyrophosphate is released
and detected in a chemiluminescent flash (pyrosequencing chemistry). In this study, we test the feasibility of NGS
analysis for the detection of EGFR mutations in cytological
specimens with a limited amount of neoplastic cells (less than
of 10%). Results indicate that NGS analysis is by far superior
to Sanger sequencing for the detection of genetic mutations in
cytological samples.
127
relazioni
Diagnosi e procedure in tema di biopsie prostatiche
Moderatori: Maurizio Colecchia (Milano), Filippo Fraggetta (Catania)
Prostate biopsy: idea of saturation biopsy
P. Pepe
Cannizzaro Hospital, Urology Unit, Catania
The adoption of extended transrectal-ultrasound (TRUS) guided biopsy schemes (12-18 cores) with an increased number of
cores on the peripheral portion of the gland constitutes, nowadays, the only suitable method to increase the detection rate of
prostate cancer (PCa) considering the absence of a suspicious
TRUS pattern in case of early diagnosis. Saturation prostate
biopsy (SPBx) (> 20 cores) 1 has been introduced in clinical
practice to increase cancer detection rate but a greater number
of cores combined with PCa screening protocols leads to an
increasing incidence of low volume PCa with the consequent
risk of overdiagnosis of clinically insignificant PCa (IPCa)
defined as < 0.5 cc3 in volume without Gleason grade 4 or 5
disease 2. SPBx is recommended in PCa diagnosis and staging; moreover, it is useful in the revaluation of microfocus
of cancer and in patients enrolled in active surveillance (AS)
programs.
SPBx and PCa diagnosis. SPBx should be performed in case
of repeat biopsy for persistent suspicious of PCa 3; in fact, the
SPBx does not afford any advantage compared with extended
schemes in case of primary biopsy, but increases PCa detection rate at second and third biopsy in comparison with 18
cores scheme (22.6 vs 10.9% and 6.2 vs 0%, respectively) 4.
Moreover, SPBx increases microfocus of PCa and IPCa detection rate resulting equal, in our experience, to 41% and 20%
5
, respectively. A transition and anterior zone sampling is recommended at repeat biopsy 1 3, although the cancer detection
rate in this region ranges from 1.8 to 41.8% 6.
Saturation biopsy and prostate cancer staging. To improve the
diagnostic accuracy, multiple biopsy variables, all included
in the term “quantitative histology” (i.e., total percentage of
cancer, greatest percentage of cancer etc), were found to be
predictive of advanced cancer. The need to consider multiple
biopsy findings is suggested by the occurrence of a nonorganconfined (OC) PCa even in patients who were supposed
to be at low risk (i.e.,clinical stage T1c with PSA ≤ 10 ng/
mL and biopsy GS ≤ 6) 7. The predictive value of quantitative
histology is emphasized by the comparison with the results
obtained using the Partin’s tables: according to them, in our
experience on 69 patients submitted to radical prostatectomy
after SPBx 8, the risk of non-OC PCa should have been equal
to 24%, an incidence lower than expected from quantitative
histology (40.6%) and even lower than the definitive pathological stage (46.4%).
SPBx and microfocus of PCa and/or AS programs. SPBx has
been proposed as a staging tool for revaluating patients with
microfocal PCa, characterized by a single microfocus (5%
or less) of Gleason score (GS) 6 cancer on primary biopsy.
Boccon-Gibod 9 reported on the clinical use of repeat SPBx
in patients with a diagnosis of microfocal PCa on a 10-core
biopsy set: SPBx was helpful to distinguish the 30% of men
who probably have minimal disease, on the basis of negative
SPBx, from the 70% with multifocal disease or grade 4 PCa
that were undetected in the primary biopsy set. This result
could be explained considering that, despite with SPBx more
of 20 cores are taken, the amount of tissue available represents a minimal percentage of peripheral zone volume (about
2-3% of the entire peripheral zone) 10. In our experience 4, 48
(87.3%) out of 55 patients with a single microfocus of GS 6
PCa diagnosed by SPBx had significant disease in the radical
prostatectomy specimen; in conclusion, patients with GS 6
microfocal cancer in a single core found at SPBx should be
advised that they may harbour more aggressive disease with a
risk of non-OC PCa equal, in our series, to 27.3% of the cases.
Some authors suggest the use of SPBx in the follow up of patients in expectant management, but the rate of misclassification is equal to 39-56% when the patients are enrolled in AS
programs 11; Duffield 12 reported that the highest percentage
of cancer progression at SPBx is detected after first (44% of
the cases) and second year (75% of the cases) of surveillance
underlining the power accuracy of SPBx.
Conclusions. SPBx is mandatory in patients submitted to
repeat biopsy; moreover SPBx is useful in PCa staging especially in “low risk” patients.
References
1
Chun FK, Epstein JI, Ficarra V, et al. Optimizing performance and
interpretation of prostate biopsy: a critical analysis of the literature.
Eur Urol 2010;58:851-64.
2
Epstein J, Walsh P, Carmichael M. Pathological and clinical findings
to predict tumour extent of non palpable (stage T1c) prostate cancer.
JAMA 1994;271:368-74.
3
Heindenreich A, Bolla M, Joniau S, et al. Guidelines on Prostate Cancer. European Association of Urology 2011.
4
Pepe P, Aragona F. Saturation prostate needle biopsy and prostate cancer detection at initial and repeat evaluation. Urology
2007;70:1131-5.
5
Pepe P, Candiano G, Fraggetta F, et al. Is a single focus of low grade
prostate cancer, diagnosed on saturation biopsy, predictive of clinically insignificant cancer? Urol Int 2010;84:440-4.
6
Pepe P, Candiano G, Fraggetta F, et al. Is still useful transition zone
sampling at repeated saturation biopsy? Urol Int 2010;85:324-7.
7
D’Amico AV, Renshaw AA, Cote K, et al. Impact of the percentage
of positive prostate cores on prostate cancer-specific mortality for
patients with low or favourable intermediate-risk disease. J Clin Oncol
2004;22:3726-32.
8
Pepe P, Fraggetta F, Galia A, et al. Is quantitative histology useful to
predict non-organ confined prostate cancer when saturation biopsy is
performed? Urology 2008;72:1198-2002.
9
Boccon-Gibod LM, Barry de Longchamps N, Toublanc M, et al. Prostate saturation biopsy in the revaluation of microfocal prostate cancer.
J Urol 2006;176:961-4.
10
Pepe P, Panella P, D’Arrigo L, et al. Should men with serum prostatespecific antigen < 4 ng/ml and normal digital rectal examination
undergo a prostate biopsy? Oncology 2006;70:81-9.
11
Suardi N, Capitanio U, Chun FK, et al. Currently used criteria for active surveillance in men with low-risk prostate cancer: an analysis of
pathologic features. Cancer 2008;113:2068-72.
12
Duffield AS, Lee TK, Miyamoto H. Radical prostatectomy findings
in patients in whom active surveillance of prostate cancer fails. J Urol
2009;182:2274-8.
128
Atrophyc lesions as pitfalls of prostate cancer
diagnosis
E. Bollito1, M. Fiorentino2, J. Rider-Stark3, F. Giunchi2, A.
Fornari1, R. Montironi4, M. Papotti1, M. Loda5
Pathological Anatomy, University of Turin at San Luigi Gonzaga
Hospital, Orbassano, Turin (Italy); 2Pathological Anatomy, University of Bologna at Policlico Sant’Orsola-Malpighi, Bologna (Italy);
3
Department of Epidemiology, Harvard School of Public Health,
Boston, MA, (USA); 4Pathological Anatomy, Polytechnic University
of Marche Region, Torrette, Ancona (Italy); 5Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, (USA)
1
Focal atrophy is an extremely frequent occurrence in prostate
specimens. Atrophic prostate lesions are a group of benign
histological findings that was considered as minor, trivial and
negligible changes until a few years ago. The interest in such
prostate lesions has recently increased because some Authors
reported that some types of atrophic schanges may mimic
prostate cancer. The problem of a correct differential diagnosis between prostatic cancer and cancer-mimicking atrophic
lesions is only one of the multiple recognised reasons to improve studies on atrophic prostate changes. Indeed De Marzo
suggested that chronic prostatitis and atrophy may play a role
in the origin of prostate cancer 3, and also proposed a classification of the atrophic prostate lesions 5 including a punctual
description and adequate pictures for each atrophic subtype.
Despite being since 2006 and and its worldwide recognition
by uro-pathologists, this classification has been poorly applied
4
and the sub-types of atrophic changes are exceptionally reported. However, the couple “prostatitis – atrophy” has been
suggested as a prostate intra-epithelial neoplasia (PIN) and/or
prostate cancer precursor in some works other than those 5-7,
of De Marzo.
However, in the daily diagnostic practice, the main problem
is the possible role of some atrophic findings as cancer mimickers. Simple atrophy and post-atrophic hyperplasia may
appear as small glands showing a pseudo-infiltrative pattern
that occasionally may simulate prostate cancer. The most
insidious sub-type of prostate atrophy in mimicking prostate cancer is the partial atrophy: this uncommon pattern is
characterized by small or medium size glands including cells
with variable amount of clear cytoplasm sometimes similar
to prostate cancer mostly with Gleason’s pattern 3. Partial
atrophy is nowadays the most common benign mimicker of
prostate cancer and reaches 4.3% (170/3916 prior diagnoses
as atypical glands) in a series of consultation cases reported by
Epstein and coworkers who showed that the similarities were
not only morphological but an overlap in the immunoprofile
was also possible using p63-HMWCK-racemase cocktail 1. In
addition, Worschech and coworkers recently described an aberrant expression of alpha-methylacyl-coenzymeA racemase
in 47/143 (32.9%) cases of their partial atrophy series 2, a finding particularly dangerous for prostate cancer misdiagnosis. In
conclusion prostate atrophy, mostly partial atrophy, is actually
the most frequent mimicker of prostate adenocarcinoma and
should be well known and recognized among pathologists
attending prostate specimens. Further investigations are also
needed to clarify the role of prostate atrophy, particularly postatrophic hyperplasia, in the development of prostate cancer.
References
1
Wang W, Sun X, Epstein JI. Partial atrophy on prostate needle biopsy
cores: a morphologic and immunohistochemical study. Am J Surg
Path 2008;32:851-7.
2
Worschech A, Meirelles L, Billis A. Expression of alpha-methylacyl
coenzyme A racemase in partial and complete focal atrophy on pro-
3
4
5
6
7
8
state needle biopsies. Anal Quant Cytol Histol 2009;31:424-31.
De Marzo AM, Meeker AK, Zha S, et al. Human prostate cancer
precursors and pathobiology. Urology 2003;62(5-suppl 1):55-62
De Marzo AM, Marchi VL, Epstein JI, et al. Proliferative inflammatory atrophy of the prostate: implications for prostatic carcinogenesis.
Am J Surg Path 1999;155:1985-92.
De Marzo AM, Platz EA, Epstein JI, et. al. A working group classification of focal prostate atrophy lesions. Am J Surg Path 2006;30:128191.
Montironi R, Mazzucchelli R, Lopez-Beltra A, et al. Mechanisms
of disease: high-grade prostatic intraepithelial neoplasia and other
proposed preneoplastic lesions in the prostate. Nat Clin Pract Urol
2007;4:321-32.
Mikuz G, Algaba F, Beltran AL, et al. Prostate carcinoma: atrophy or
not atrophy that is the question. Eur Urol 2007;52:1293-6.
Wang W, Bergh A, Damber JE. Morphological transition of proliferative inflammatory atrophy to high-grade intraepithelial neoplasia and
cancer in human prostate. Prostate 2009;69:1378-86.
Standardizzazione delle pocedure
G. Mikuz
Institute of Pathology, Medical University, Innsbruck
Prostate needle biopsy can give important clinical information
on tumor extension and grading, useful prognostic parameters
for the therapeutic choice and prognostic definition. The best
method to perform a prostate biopsy includes the use of 18gauges needles, single specimen identification and subsequent
orientation of every bioptic fragment by inking its proximal
end. The labelling of the single biopsies according to their has
advantages for pathologists and urologists. Laterality of cancer can be important for the planning of nerve sparing surgery.
Basal or apical cancer localization may modify the surgical
technique. A focus of a suspect lesion can help to
redirect part of the repeat biopsies. The biopsies are stretched
between two nylon meshes enclosed in a tissue cassette in
formalin (preembedding technique). The full length of the
biopsy is within the section plane. This „sandwich“ technique
has furnished evident advantages for the pathologist, optimizing the visible area for section plane in comparison to that
obtainable from free floating core biopsies. From each biopsy
serial histological section are performed. Alternatively one
slide is HE stained and the next one remained unstained and
can be used for immunohistochemistry or further HE staining.
The primary goal of the pathologists is to detect the cancer, to
classify it according its morphology (histological type) and to
grad it using the modified (2005) Gleason system. Gleason’s
original only records the most common grading pattern (primary pattern) and the next most prevalent pattern (secondary
pattern), adding them together to arrive at a Gleason
score. The typical scenario, however, with tertiary patterns
on biopsy includes tumors with patterns 3, 4, and 5 in various
proportions. Urological pathologists (ISUP) agreed that tumors should be classified overall as high grade (Gleason score
8–10) given the presence patterns 4 and 5 on needle biopsy. It
was the consensus that these tumors, on needle biopsy, should
not be graded by listing the primary, secondary, and tertiary
patterns.
129
relazioni
Scienze Biomediche
Good Practice Guidelines
La valorizzazione delle responsabilità
e dell’autonomia professionale…
un’occasione da non perdere
A. Esposito
Responsabile del Miglioramento dei Processi Assistenziali P.O. Clinicizzato “SS. Annunziata” Chieti, ASL 02 Abruzzo, Italy
Introduzione
Gli anni dal 1990 al 2006 sono stati caratterizzati da grandi
innovazioni e conquiste per le Professioni Sanitarie. Si sono
evidenziati cambiamenti sul percorso formativo e di esercizio
professionale. L’attuale assetto normativo riconosce al Tecnico di Laboratorio Biomedico (TSLB) da un lato un ambito di
competenze ben definite, ricche di autonomia e responsabilità,
dall’altro un ruolo strategico nel Sistema organizzativo della
Medicina di Laboratorio, delle Strutture Sanitarie, nei Percorsi
Formativi Universitari, di Aggiornamento e nella Ricerca.
Metodi
Sono stati analizzati i riferimenti legislativi nel periodo in
considerazione e la relativa evoluzione nell’ambito professionale con particolare riferimento alla Disciplina dell’Anatomia
Patologica e della Citodiagnostica. Scopo del lavoro è delineare il superamento del concetto di “Professione Ausiliaria”
ri-definendo il TSLB come un Professionista proiettato nel
futuro scientifico, tecnologico ed organizzativo.
Risultati e conclusioni
L’evoluzione del TSLB operante nella Disciplina non è
determinata solamente dal percorso normativo ma da un
“necessario” cambiamento culturale dello stesso; in contesti multiprofessionali ad alto tasso di specializzazione ed
innovazione tecnologica deve essere sviluppata, quindi, la
capacità di verificare, quotidianamente, il sapere individuale proiettando, il proprio agire, verso la Ricerca applicata.
Il mutamento di mentalità e la consapevolezza del Core
Competence Professionale in continua evoluzione, infatti,
saranno elementi fondamentali che favoriranno il consolidamento dell’Anatomia Patologica e Citodiagnostica a totale
vantaggio dell’Utente.
Bibliografia
1
Chiari P, Mosci D, Naldi E. Evidence-Based Clinical Practice-La
pratica clinico-assistenziale basata su prove di efficacia. II ed. Milano: McGraw-Hill 2011.
2
Zangrandi A (a cura di). Economia e Management per le professioni
sanitarie. Milano: McGraw-Hill 2010.
3
Baraldi S. Il Balanced Scorecard nelle aziende sanitarie. Milano:
McGraw-Hill 2005.
4
Benci L. Le Professioni Sanitarie (non mediche). Milano: McGrawHill 2002.
Innovazione e laboratorio: necessità o moda
F. Caruso
Pavia
L’Anatomia patologica nasce come disciplina scientifica nel
XVII secolo ad opera del medico italiano Gianbattista Morgagni (1682-1771).
Nel XIX secolo assistiamo ad ulteriori progressi grazie ai
perfezionamenti ottenuti nel campo della microscopia ottica
e delle metodiche di fissazione e colorazione delle cellule e
dei tessuti.
Vengono migliorate le procedure di microtomia che consentono il taglio dei tessuti in sezioni estremamente sottili.
Nel XX secolo si osserva un ulteriore grande sviluppo
legato,soprattutto, ad un continuo aggiornamento tecnologico
che riguarda tutte le fasi dell’attività istopatologica.
L’obiettivo di questa relazione è quello di mettere in evidenza
i progressi fatti in materia d’igiene e sicurezza dell’ambiente
e delle persone ed, ancora, di sottolineare l’evoluzione nel
tempo dei reagenti e degli strumenti di lavoro nel laboratorio
di anatomia patologica.
Per quanto concerne la sicurezza del personale e la tutela
dell’ambiente sono state emanate tutte una serie di leggi e
disposizioni.
Ad esempio il T.U. 81/08 impone al datore di lavoro di organizzare un Servizio di Prevenzione e protezione.
I dispositivi atti alla tutela dell’ambiente e del personale vanno dall’utilizzo di cappe chimiche, banchi e armadi aspiranti
ai dispositivi di protezione individuale quali guanti, occhiali,
mascherine ed altro: inoltre tutti i reagenti, dopo l’uso vengono raccolti in appositi contenitori ed inviati a centri che
provvedono al loro smaltimento.
Si è infine cercato di eliminare o sostituire le sostanze particolarmente tossiche, ove possibile, con altre che lo sono meno
e non lo sono affatto.
A riguardo dei reagenti si può affermare che oggi, in commercio, è possibile trovare qualsiasi colorante già pronto all’uso
mentre un tempo era necessario prepararli in laboratorio.
Parlando infine degli strumenti di lavoro possiamo distinguere
i materiali di consumo dagli apparecchi.
Tra i primi interessante è l’evoluzione nel mondo d’includere
i campioni istologici si è passati dall’utilizzo delle forme di
Leuckhart alle attuale formelle metalliche. Come supporto al
blocchetto di paraffina si è passati dal pezzetto di legno all’anello ring ed infine alle cassettine d’inclusione su cui è possibile
scrivere il numero che identifica il campione bioptico.
Per quanto concerne gli apparecchi si è passati da una
processazione manuale dei campioni ai primi processatori
automatici le «Istochinette» sino ad arrivare agli attuali processatori gestiti da un sistema computerizzato. La colorazione
delle sezioni e le reazioni di immunoistochimica venivano
fatte manualmente, oggi ci si avvale dell’ausilio di strumenti
automatici.Sono in commercio stazioni robotizzate dove
viene inserito il vetrino, eseguono la colorazione, la reazione
immuno-istochimica o metodiche biomolecolari e lo restituiscono montato.
Anche la biologia molecolare, se pur di più recente applica-
130
zione, ha subito una notevole innovazione tecnologica nel giro
di pochi anni.
Altri apparecchi che hanno avuto uno sviluppo tecnologico
sono stati i microtomi: è possibile ottenere sezioni dello spessore di pochi micron, secondo nuovi standard di sicurezza e
operativi.
Infine per ottenere sezioni da tessuti freschi si è passati dai
primi microtomi congelatori agli attuali criostati impiegati
nella pratica quotidiana degli esami intraoperatori.
Il laboratorio d’istopatologia, nonostante i progressi tecnologici e l’automazione, rimane una struttura in cui l’operatore
riveste un ruolo di primaria importanza.
Da un lato vi è il tecnico che deve allestire i preparati istologici in maniera ottimale, dall’altro l’anatomopatologo che deve
formulare una diagnosi dalla lettura dei preparati.
Le varie apparecchiature del laboratorio di istopatologia non
possono sostituire alla manualità che è caratteristica essenziale del tecnico istopatologico, ma costituiscono sicuramente un
supporto per il miglioramento della qualità, la standardizzazione dei preparati ottenuti e le riduzioni dei tempi di refertazione, nonché un aiuto per migliorare la qualità della vita del
personale che vi opera, rispondendo ai criteri di sicurezza che
impone la legislazione vigente.
Necessità o moda? Diventa necessità se risponde ai requisiti richiesti ai fini di una accuratezza diagnostica, migliora
le condizioni lavorative degli operatori, garantendo più
sicurezza interne al laboratorio e un minor impatto ambientale.
È moda quando si acquisiscono tecnologie che non rispondono alle necessità generando inefficienze operative e spreco di
risorse.
L’organizzazione secondo i principi di “Lean
Economy”
T. Ragazzini*, A. Bondi**
Anatomia ed Istologia Patologica Osp. Bellaria Università degli Studi di Bologna; **Anatomia Patologica Osp. Maggiore AUSL Bologna
*
I volumi di attività di un’unità operativa di Anatomia Patologica possono variare molto non solo in funzione delle
dimensioni della struttura ospedaliera, in termini di numeri di
posti letto, ma anche in relazione al rapporto che al suo interno esiste tra discipline chirurgiche e mediche. Poiché spesso
i servizi dell’U.O. sono accessibili anche all’utenza esterna,
un’altra variabile è costituita dall’ampiezza del territorio di
riferimento, dalla sua popolazione e dalla densità di presidi
sanitari pubblici e privati.
Gli spazi fisici funzionali in cui si svolgono le attività in genere sono organizzati logisticamente secondo il percorso che
il campione oggetto dell’indagine segue dal momento in cui
viene consegnato al laboratorio.
La produttività dipende dal tipo di laboratorio, dal carico di
lavoro, dallo staff e dalle sue competenze, dalla presenza di
strumentazione adeguata e dal livello di automazione; fondamentalmente però dipende da come è organizzato il lavoro.
Questa è la ragione per cui i laboratori (soprattutto quelli con
almeno 20.000 casi all’anno) beneficiano dell’analisi del flusso di lavoro tramite le tecniche di management.
Da alcuni anni si stanno utilizzando, soprattutto negli Stati
Uniti, metodologie di management finalizzate ad incrementare la produttività in Anatomia Patologica. La più efficace
risulta la teoria della “Lean Production” (termine coniato da
Womak e Jones nel libro “La macchina che ha cambiato il
mondo”) in cui i due studiosi hanno analizzato in dettaglio
e confrontato le performance del sistema di produzione dei
principali produttori mondiali di automobili con la giapponese
Toyota, rivelando le ragioni della netta superiorità di quest’ultima rispetto ai suoi concorrenti.
La Lean Production è dunque una generalizzazione e divulgazione in occidente del Toyota Production System che ha superato il sistema di produzione di massa sviluppato da Henry
Ford nel 1908 ed applicato tutt’oggi alla quasi totalità delle
aziende occidentali.
Il primo obiettivo dei pionieri della Lean Production in laboratorio è stato quello di progettare un ambiente razionale ed
efficiente, superare gli ostacoli stratificati dalle consuetudini,
ridefinire i singoli passaggi del flusso operativo e creare il
concetto di “celle di lavoro”, settorializzare alcune mansioni,
applicare tecniche di governo ed analisi dei flussi per determinare dove viene peso tempo e come si muove lo staff, riorganizzare il personale e, budget permettendo, portare maggiore
automazione. In alcuni laboratori la scelta è stata quella di
snellire il flusso di lavoro e di aumentare la produttività degli
strumenti in uso. Altri hanno introdotto la telepatologia e
sistemi di riconoscimento vocale come ausili informatici utili
alla riduzione del TAT.
Di impatto significativo è l’esperienza del 2000 dell’Henry
Ford Hospital di Detroit, dove lo sforzo teso al miglioramento
della qualità, applicando i principi Lean al laboratorio di Anatomia Patologica, in particolare ha provocato una trasformazione culturale del ruolo manageriale e dell’approccio degli
operatori al lavoro.
Nella stesura di una tesi di laurea magistrale in Scienze delle
Professioni Sanitarie ci si è posti l’obiettivo di disegnare un
progetto di laboratorio secondo i principi dell’efficienza, della
riduzione degli sprechi e dell’uso razionale delle risorse, mutuando tecniche ed esperienze dalla grande industria di produzione. Il tutto per una ottimizzazione delle attività di Anatomia Patologica, un controllo della qualità delle prestazioni,
un’organizzazione coordinata del lavoro medico e tecnico e
che sfrutti le nuove potenzialità della tecnologia e rafforzi il
ruolo di riferimento culturale diagnostico della disciplina.
Per poter strutturare un progetto non solo teorico si è utilizzato
l’area metropolitana bolognese come ipotesi applicativa e si è
partiti dall’analisi del contesto (popolazione di 985.000 abitanti circa, tre Aziende Sanitarie Pubbliche, un’Azienda Ospedaliera Universitaria, un IRCCS) e dall’analisi in dettaglio
delle realtà di Anatomia Patologica presenti (n. di prestazioni
istologiche, citologiche e di riscontro autoptico, personale
medico, biologo, tecnico, amministrativo e di altra qualifica,
strutture ed ambienti dedicati, strumentazioni avanzate, attività specialistiche peculiari).
Una prestazione di Anatomia Patologica si compone schematicamente di due diversi momenti relizzativi: una prima fase
con presa in carico, esame macroscopico ed allestimenti di
laboratorio; una seconda fase di osservazione, interpretazione
e report diagnostico. Tradizionalmente questi due momenti
sono realizzati nello stesso edificio.
La seconda fase è realizzata da dirigenti medici o biologi e
si configura sostanzialmente nello studio al microscopio e
nell’inquadramento diagnostico del caso sulla base dei reperti
osservati, delle notizie disponibili, della discussione con i
responsabili clinici e talvolta con la visita diretta del paziente
o l’osservazione di reperti di imaging o di altre metodiche
diagnostiche. È un’attività più affine alla pratica clinica che a
quella di laboratorio, che anche fisicamente è più appropriato
immaginare vicino al tavolo operatorio che non al processatore di tessuti e che comunque può essere realizzata indipendentemente dalla vicinanza fisica del laboratorio.
131
relazioni
I passi successivi sono: la riorganizzazione delle automazioni
consolidate e la gestione di quelle “avanzate”; la scelta del
sistema informativo che deve essere un sistema di collegamento, controllo e gestione delle prestazioni unico, condiviso
e distribuito, con specifiche caratteristiche; lo studio della
logistica e l’organizzazione di una efficace rete di collegamenti (compresa la tecnologia del vetrino digitale); la cura dei
sistemi di tracciabilità e degli incarichi di responsabilità (risk
management); il calcolo del personale, la sua distribuzione
sulle attività e la definizione di orari e turni di lavoro, in particolare per il personale tecnico.
Una tale organizzazione vede un laboratorio unico centralizzato sfruttato su un’ampia area territoriale (sei ospedali),
mentre parte delle unità di interpretazione diagnostica rimane
in periferia. La stima fatta sugli operatori indica che è possibile una riduzione del personale rispetto all’attuale e lo staff,
divenuto numeroso in seguito all’accorpamento, facilita le
reciproche sostituzioni e specializzazioni. Si raggiunge il massimo della produttività avendo l’opportunità di trattare almeno
50.000 casi istologici all’anno.
Si tratta della teorizzazione della possibilità di istituire un laboratorio metropolitano di Anatomia Patologica nell’area provinciale bolognese al fine di ottimizzare le risorse ed adeguare gli
standard quali-quantitativi rispetto a quei paesi che hanno già
introdotto sistemi di “Lean Production” nelle loro istituzioni.
La medicina moderna, ad elevato grado di specializzazione ed
alti costi, richiede strutture di dimensioni adeguate, con ampio
bacino di utenza e produttività conseguentemente elevata.
In un’organizzazione sanitaria centrata sul servizio pubblico
l’appropriatezza della spesa e la razionalizzazione degli investimenti sono un segno di civiltà ed un dovere etico. L’Anatomia Patologica non sfugge a questo principio generale.
Problematiche tecnico-organizzative della
fissazione: quale evoluzione?
M. Cadei, P.G. Grigolato
Cattedra di Anatomia Patologica II, Università di Brescia
Il laboratorio di Anatomia Patologica storicamente non ha subito sostanziali trasformazioni metodologiche nelle procedure
di allestimento dei campioni istologici.
Dalla fissazione del campione alla processazione, la principale
implementazione è stata più legata alla innovazione strumentale (processatori, coloratori ecc.) che ai reagenti utilizzati.
Da qualche tempo, anche in conseguenza all’introduzione della legislazione sulla sicurezza è stata posta attenzione anche
riguardo all’utilizzo di sostanze che, da sempre considerate
indiscutibili (formalina, xilolo, alcool), hanno invece cominciato a trovare sul mercato validi concorrenti.
Indubbiamente questo merito è da riconoscere anche alle
Aziende del settore che, in anni più recenti, hanno fortemente
sviluppato la ricerca di possibili prodotti non tossici in grado
di competere con i reagenti tradizionali.
Così è stato per i sostituti della processazione istologica
che hanno ormai trovato applicazioni routinarie in molti laboratori ospedalieri, mentre maggiori difficoltà sussistono,
ancora oggi, per sostanze in grado di sostituire validamente
la formalina.
La nostra esperienza, iniziata qualche anno fa con l’utilizzo
di “prodotti alternativi” della fissazione dei tessuti, si è perfezionata nel tempo con la sperimentazione su tessuti umani
di routine giunti presso il nostro Servizio per la diagnosi istopatologica. Oggetto del nostro studio sono stati la valutazione
morfologica del preparato istologico, le tecniche istochimiche
ed immunoistochimiche e le indagini bio-molecolari, che ad
oggi sempre più frequentemente vengono richieste ad integrazione della diagnostica.
L’obiettivo è finalizzato a dimostrare l’esistenza sul mercato
di un fissativo adatto alle attuali esigenze normative, di buona
qualità morfologica ed adeguato alle necessità metodologiche
più evolute (biologia molecolare).
Ruolo della fissazione e problemi organizzativi
connessi
Mara Dal Santo
Anatomia Patologica, Ospedale S. Chiara, Trento
In Anatomia Patologica la fissazione riveste un ruolo primario
nella preservazione dei tessuti istologici, in quanto attraverso
l’inibizione dei processi autolitici e l’inibizione della crescita
batterica permette la conservazione di tutti i componenti cellulari. Nel processo di fissazione dei tessuti la reazione cruciale
è la formazione di legami crociati che stabilizzino le proteine,
formando un componente che mantenga i rapporti fra proteine
solubili e proteine strutturali.
Il fissativo ideale deve:
prevenire il danno osmotico e il restringimento tissutale
mantenere in situ i componenti tissutali
preservare completamente le originali reattività chimiche e le
caratteristiche antigeniche delle molecole che costituiscono i
tessuti
creare un legame indissolubile fra i costituenti cellulari mantenendo l’originale struttura e rapporti relativi
conferire resistenza ai costituenti tissutali a trattamenti fisicochimici bruschi (marcati sbalzi di tenmperatura, forno a microonde) o con effetti marcatamente deleteri sui tessuti come
acidi, chelanti, enzimi.
Il metodo standard per la fissazione in istologia è per immersione nel fissativo, subito dopo la sua escissione, in rapporto
tra volume del pezzo e quantità di fissativo di 1:10 – 1:20. Il
contenitore deve avere una capacità idonea a contenere sia il
pezzo che il fissativo, in modo da non deformare il materiale
inserito.
Il fissativo finora comunemente utilizzato, che corrisponde
a questi requisiti è la formaldeide o aldeide formica, commercializzata in soluzione acquosa al 4%, tamponata con
fosfati sodici mono e bibasici a ph 7,2–7,4. La velocità di
penetrazione nel campione è di circa 1 mm/ora. Su questa
influiscono: il rapporto ottimale (1:10 – 20) fra volume del
campione e fissativo, lo spessore e composizione chimica del
campione, la temperatura e l’uso di mezzi fisici (vuoto, onde
elettromagnetiche, ultrasuoni). Il meccanismo di fissazione
della formaldeide avviene mediante la formazione di ponti
metilenici da parte della forma attiva, il glicole metilenico.
Induce cross-links tra residui adiacenti di lysine (K), arginine
(R), tyrosine (Y), asparagine (N), histidine (H), glutamine
(Q), serine (S), zuccheri.
L’effetto fissativo della formaldeide è di stabilizzare le strutture secondarie, legando peptidi adiacenti (della stessa o di
proteine contigue) con legami covalenti.
Pregi e difetti della formaldeide
Vantaggi: previene l’indurimento dei campioni da parte degli
alcoli usati durante la successiva processazione; tessuti fissati
con formalina possono essere post-fissati con numerosi altri
fissativi (a seconda delle successive indagini), possono essere
successivamente congelati (es ricerca istochimica dei lipidi);
elevata sensibilità nel tempo, che rende possibile la conservazione prolungata di pezzi anatomici in formalina, infine con-
132
sente di eseguire indagini FISH e permette la conservazione
del DNA per indagini molecolari.
Svantaggi: in assenza di tamponi neutralizzanti e di stabilizzanti (metanolo), la formaldeide in soluz acquosa si ossida in
acido formico che produce precipitati neri nei campioni; lo
stesso se utilizzata in soluzioni contenenti cromati.
Ma lo svantaggio più importante nell’utilizzo della formaldeide è legato alla sua tossicità acuta e cronica nei confronti
dell’uomo.
Le proprietà pericolose delle soluzioni di formaldeide sono
funzione della percentuale di formaldeide presente e di quella
di eventuali additivi o stabilizzanti.
In base alla normativa europea relativa alla classificazione
imballaggio ed etichettatura delle sostanze pericolose, ovvero
la normativa che definisce i criteri per identificare le proprietà
pericolose dei prodotti chimici e gli elementi (simboli, Frasi
di Rischio, …) di comunicazione dei pericoli, le soluzioni
acquose più concentrate di formaldeide sono classificate attualmente come tossiche per inalazione, contatto cutaneo e
per ingestione; corrosive e sensibilizzanti per contatto con la
pelle, cancerogene di categoria 3 (possibilità di effetti cancerogeni – prove insufficienti).
La classificazione delle soluzioni di formalina utilizzata per
la fissazione è identificabile dai simboli di pericolo, frasi
di rischio (frasi R), consigli di prudenza (frasi S), presenti
sull’etichetta delle confezioni e dalle informazioni contenute
nella Scheda dei Dati di Sicurezza.
Da segnalare che nel 2004 l’Agenzia Internazionale di Ricerca sul Cancro (IARC), ha classificato la formaldeide come
cancerogeno del gruppo 1, ovvero cancerogeno “certo” per
l’uomo. Gli esperti della IARC hanno valutato che si dispongono di indicazioni sufficienti per ritenere che la formaldeide
provochi tumori naso-faringei nell’uomo e che l’esposizione
inalatoria a formaldeide può costituire un fattore di rischio di
leucemie, di cancro alle fosse nasali e dei seni paranasali.
Per il momento, in Europa, la classificazione regolamentare
della formaldeide rimane “cancerogeno di categoria 3” ovvero
cancerogeno “possibile”. Il gruppo di lavoro che opera per
definire le classificazioni e le etichettature armonizzate in seno
alla Comunità Europea sta però discutendo una proposta più
severa di revisione dell’attuale classificazione delle proprietà
cancerogene della formaldeide. Si ricordi che la formaldeide è
utilizzata anche nella manifattura di resine usate come adesivi
e leganti ad esempio nei prodotti del legno e della carta. Un
altro uso massiccio della formaldeide riguarda la produzione
di materie plastiche e rivestimenti. Viene anche impiegata
nel fissaggio dei tessuti e pellami, come intermedio chimico
e come disinfettante. Inoltre viene impiegata nell’industria
alimentare (E240) come conservante contro i funghi per disinfettare contenitori, tuature, recipienti vari e nella produzione
di vaccini ed altri prodotti farmaceutici
L’utilizzo professionale della formaldeide comporta per il
datore di lavoro la valutazione dei rischi prevista per gli
agenti chimici pericolosi, conformemente a quanto previsto
dal D.Lgs. 81/2008 e s.m.e i (che sostituisce il D.Lgs 626/94).
L’articolato quadro normativo richiede di organizzare i processi lavorativi con presenza, impiego o sviluppo di agenti
chimici in modo tale da eliminare i rischi per la salute e la
sicurezza dei lavoratori o, se questo non è possibile, di ridurli
massimamente e di classificare il rischio per gli operatori in
base alla valutazione della loro esposizione. Il percorso di valutazione dei rischi deve quindi basarsi su una metodologia di
analisi che permetta di individuare gli agenti chimici pericolosi, di caratterizzare e valutare le condizione di esposizione,
stimare le possibilità che si possano verificare dei danni alla
salute degli operatori e di definire le misure di tutela più idonee, da integrare nell’organizzazione del lavoro, per eliminare
o ridurre massimamente tale evenienza.
Prioritariamente, tra le misure di tutela, se tecnicamente possibile, la normativa impone la sostituzione dell’agente chimico
pericoloso con uno non pericoloso o meno pericoloso. In
subordine, se l’utilizzo dell’agente chimico deve essere mantenuto, le misure tecniche da adottare devono privilegiare la
protezione collettiva: utilizzo in apparecchiature funzionanti a
ciclo chiuso, utilizzo di cappe o di aspirazioni localizzate in
grado di avvolgere la sorgente inquinante, la meccanizzazione
di certe attività. L’utilizzo di dispositivi di protezione individuale (dpi) può risultare indispensabile ma non deve affatto
sostituirsi all’adozione delle misure di protezione collettiva.
Tutto il personale interessato alla manipolazione di agenti
chimici pericolosi deve essere informato sui rischi collegati
alla loro presenza e impiego e formati alle modalità per un
utilizzo sicuro.
L’efficacia delle misure adottate deve essere successivamente verificata attraverso la quantificazione dell’esposizione
personale attraverso misurazioni (in zona respiratoria) delle
sostanze aerodisperse nei luoghi di lavoro e misurazioni delle
sostanze utilizzate (o di loro metaboliti) presenti nei liquidi
biologici degli operatori (monitoraggio biologico).
Per quanto riguarda la formalina, più sostituti sono attualmente disponibili sul mercato ma tutti presentano delle difficoltà
di impiego che, a diverso titolo, rendono la sua sostituzione
non tecnicamente possibile.
Per il controllo dell’esposizione alla formaldeide devono
quindi prima essere identificate ed analizzate le attività che
implicano la manipolazione delle soluzioni: fissazione dei
tessuti, trasporto dei prelievi, ricevimento e registrazione,
macroscopia, campionamento dei pezzi, utilizzo delle apparecchiature automatiche di inclusione, stoccaggio ed eliminazione dei rifiuti, al fine di evidenziare gli eventuali aspetti
critici presenti che possono configurare condizioni di rischio.
Conseguentemente dovranno essere
In relazione alla volatilità delle soluzioni di formaldeide, per
controllare l’esposizione inalatoria e ridurre il numero di operatori esposti, le attività con utilizzo di formalina e di prelievo
e campionamento dei pezzi fissati, devono essere organizzate
sotto cappa o su banchi di lavoro provvisti di aspirazione che
avvolgano il più possibile la sorgente inquinante, confinati
all’interno di locali separati dagli altri e dotati di un impianto
di ventilazione meccanica mantenuto in leggera depressione (la
quantità d’aria immessa nel locale deve essere inferiore a quella
emessa all’esterno), in modo da confinare all’interno le eventuali aerodispersioni non catturate dall’aspirazione localizzata.
Nella formazione dei lavoratori dovranno essere prese in
considerazioni anche eventuali modifiche nelle modalità operative dei lavoratori, tali da evitare o ridurre massimamente
le esposizioni e quindi le condizioni di rischio. Gli operatori
dovranno conoscere l’importanza di assumere determinati
accorgimenti per interfacciarsi al meglio con i dispositivi di
protezione collettiva presenti e l’importanza dell’utilizzo dei
dpi nella protezione individuale.
Bibliografia
1
www.inrs.fr Istitut National de Recherche et de Securitè (INRS) “ED
5032 – Le formaldehyde”.
2
www.cramif.fr “Prévention des cancers professionnels - Les Fiches
d’Information et de Prévention: Exposition au formol au poste de
macroscopie dans les laboratoires d’anatomopathologie”.
3
http://www.afaqap.org/page.php3?id_rubrique=140&lang=fr#bilan
Association Francaise d’Assurance Qualité en Anatomie et Cytologie
Pathologiques.
relazioni
4
5
http://monographs.iarc.fr/ENG/Monographs/vol88/mono88.pdf
D.Lgs 81/2008 e s.m.e.i http://www.iss.it/dbsp/
Tecniche di analisi mutazionale nell’era
delle Target Therapies
C. Lupo
Anatomia Patologica e Patologia Molecolare Oncologica, Casa di
Cura di Alta Specialità “La Maddalena”, Palermo
La diagnostica molecolare in Anatomia Patologica, mediante
l’utilizzo di tecniche molecolari, oggi permette la determinazione di biomarcatori oncologici. Oltre alle caratteristiche
cliniche ed istologiche del tumore, è fondamentale identificare
le mutazioni associate alla risposta o alla resistenza della terapia mirata. Nel carcinoma del colon-retto, si valuta lo “status”
del gene K-RAS che può essere “wild type” o mutato. È un
fattore che permette di predire l’efficacia dell’azione dei farmaci biologici in questo tipo di tumori, distiunguendo pazienti
“responders” con il gene non mutato “K-RAS wild type”, e
pazienti “non responders”con il gene “K-RAS mutato”.
Nel carcinoma del polmone “non a piccole cellule” (Non Small
Cell Lung Cancer, NSCLC) si valuta lo “status” del gene EGFR. I pazienti che presentano mutazioni attivanti TK del gene
EGFR-M+, hanno un’elevata probabilità di risposta al farmaco
biologico, superiore alla terapia standard. Il medico oncologo
seleziona il paziente per il quale verrà richiesta l’analisi dello
stato mutazionale dei geni K-RAS ed EGFR, in base alla diagnosi anatomo-patologica. La successiva esecuzione del test
è di pertinenza del Servizio di Anatomia Patologica, poiché
questi test sono eseguiti sui campioni istologici e citologici
del paziente. L’anatomopatologo determina l’idoneità alla
successiva analisi molecolare, per il gene di riferimento, valutando la percentuale di cellule neoplastiche evidenziata nella
sezione tissutale analizzata. Il professionista abilitato all’esecuzione di tecniche molecolari, sia esso un medico, biologo,
biotecnologo o tecnico di laboratorio, dovrà avere un’adeguata
esperienza e formazione nel campo specifico. Il protocollo di
lavoro prevede l’utilizzo di tecniche tra le quali l’estrazione
del DNA genomico (da cellule e/o tessuto tumorale), l’amplificazione del DNA genomico e lo screening mutazionale
dei geni (K-RAS, EGFR) mediante primers specifici, che può
avvenire già nella fase di PCR, mediante PCR “Real-Time”o
successivamente attraverso metodiche di sequenziamento, pirosequenziamento, strip-assay e biochip array.
Il ruolo del Dipartimento
T. Zanin
E.O. “Ospedali Galliera”, S.C. Anatomia Patologica”
A partire dall’ultimo decennio il Sistema Sanitario Italiano
è stato caratterizzato dall’introduzione di concetti e tecniche
manageriali.
In accordo con il paradigma del New Public Management,
durante gli anni ’90 il SSN è stato oggetto di un profondo
ammodernamento volto a promuovere l’efficienza produttiva,
l’efficacia e l’appropriatezza dei servizi sanitari attraverso
l’introduzione di principi e strumenti di managerialità.
Alcune delle riforme più recenti hanno agito soprattutto sul
fronte degli assetti organizzativi delle aziende sanitarie, promuovendo l’introduzione di modelli di tipo dipartimentale.
L’organizzazione dipartimentale quindi è il modello ordinario
di gestione operativa delle attività a cui fare riferimento in
ogni ambito del Servizio Sanitario Nazionale (SSN) con la
133
finalità di assicurare la buona gestione amministrativa e finanziaria ed il governo clinico.
L’evoluzione legislativa dei dipartimenti ospedalieri attraversa cinque fasi fondamentali.
Il primo step è rappresentato dalla legge 132 del 1968 che per
lungo tempo regolamenta l’organizzazione interna degli ospedali stabilendo una precisa distinzione tra le divisioni ospedaliere e le unità operative. La divisione e l’organizzazione del
lavoro all’interno degli ospedali secondo tale prospettiva si
basa sulla specialità clinica, e la grandezza dell’organizzazione è collegata con il numero di posti letto senza tenere in considerazione le risorse impiegate. Successivamente il Decreto
legislativo del ministero della salute del 1976 introduce il concetto di dipartimentalizzazione basato sul modello dei Clinical
Directorates inglesi. In seguito numerosi interventi normativi
rinnovano profondamente tale sistema attraverso l’introduzione del modello divisionale (Achard, 1999): in particolare
il D.lgs 502/92, il D.P.R. del 27 marzo 1992, il D.lgs 229/99,
oltre che numerose normative a livello regionale completano
il quadro istituzionale del nuovo modello divisionale.
Nello specifico il D.lgs 502/92 stabilisce che le nuove aziende
ospedaliere debbano essere organizzate in base al modello
dipartimentale.
La riforma introdotta dal D.lgs 229/99 invece modifica l’organizzazione interna delle strutture sanitarie creando un contesto
organizzativo più integrato per la fornitura dei servizi, in
particolare tale decreto spiega come i dipartimenti ospedalieri
debbano essere strutturati e organizzati.
Infine le più recenti normative regionali riconoscono il dipartimento ospedaliero come il miglior contesto in cui possono
essere sviluppati gli strumenti di governo clinico.
I dipartimenti ospedalieri presentano inoltre un’origine internazionale, nel contesto sanitario durante gli anni ’60 viene
infatti introdotta l’organizzazione dipartimentale all’interno
dei policlinici universitari americani al fine di perseguire una
migliore forma di gestione.
Il Ministero della Salute in collaborazione con l’Università
Cattolica del Sacro Cuore di Roma ha realizzato nel corso
2005 una survey con il proposito di mappare la operatività
dipartimentale nelle aziende sanitarie in Italia. La ricerca è
stata condotta mediante un questionario strutturato, articolato
in 3 sezioni relative a: (i) informazioni anagrafiche dell’organizzazione, anno di istituzione dei dipartimenti, processo di
cambiamento della struttura organizzativa; (ii) informazioni
sull’assetto organizzativo del singolo dipartimento quali, ad
esempio, numero di unità organizzative accorpate e criterio di
aggregazione (strutturale, funzionale, aziendale, interaziendale); (iii) informazioni relative ai meccanismi operativi adottati
nel dipartimento, con particolare attenzione all’attivazione
degli strumenti per il governo clinico. Il questionario, inviato
a tutta la popolazione di aziende sanitarie presenti sul territorio nazionale (Aziende Ospedaliere, ASL, Policlinici Universitari, IRCCS), è attualmente disponibile sul sito web del
Ministero della Salute (www.ministerosalute.it/imgs/C_17_
pagineAree_233_listaFile_itemName_1_file.doc).
I dati raccolti sono stati analizzati attraverso un’analisi statistica descrittiva con il supporto del software SPSS. Prima di
procedere all’elaborazione dei dati, un panel di esperti si è
occupato di riclassificare i dipartimenti in relazione alla natura
delle unità organizzative accorpate.
Bibliografia di riferimento
Achard PO. Economia e organizzazione delle imprese sanitarie. Milano:
Franco Angeli 1999.
Anessi Pessina E, Cantù E. L’aziendalizzazione della sanità in Italia.
Milano: Egea 2001.
134
Anessi Pessina E, Cantù E.. L’aziendalizzazione della sanità in Italia.
Milano: Egea 2002.
Anessi Pessina E, Cicchetti A, Cifalinò, et al. Il punto di vista delle
aziende. In Baraldi S (a cura di). L’organizzazione dipartimentale
nelle aziende sanitarie. Accademia Nazionale di Medicina. 2003
Brailer DJ. Management of knowledge in the modern health care delivery
system. Journal of Quality improvement 1999;25(1): 6-19.
Cicchetti A. L’organizzazione dell’ospedale. Fra tradizione e strategie
per il futuro. Vita & Pensiero Editore 2002.
Cicchetti A. La progettazione organizzativa. Milano: Franco Angeli
2004.
Cicchetti A, Baraldi S. La diffusione del modello dipartimentale nel Ssn:
solo un fatto formale? Organizzazione Sanitaria 2001;1:71-81.
L’aggiornamento delle professioni sanitarie
ed il valore della formazione
A. Esposito*, S. Mennilli**
*
Responsabile del Miglioramento dei Processi Assistenziali; **Coordinatore Infermieristico UOC di Nefrologia e Dialisi P.O. Clinicizzato “SS. Annunziata” Chieti, ASL 02 Abruzzo, Italy
Introduzione
La nuova fase dell’Educazione Continua in Medicina (ECM)
contiene molte novità e si presenta quale strumento per progettare un moderno approccio allo sviluppo ed al monitoraggio
delle competenze individuali. Sono state introdotte, inoltre,
nuove tipologie formative, non ci sarà più solo il congresso/
convegno, ma altre forme di aggiornamento:
la formazione sul campo, che deve rispondere ai criteri di appropriatezza tra l’esercizio della professione e l’aggiornamento;
la formazione a distanza che riesce a raccogliere un numero
elevato di partecipanti abbattendo i costi e coniugando strategie formative universali, che arrivano agli operatori in modo
omogeneo. (http://www.salute.gov.it/ecm/)
Metodi
Viene analizzato lo stato dell’arte della realtà nazionale ed
illustrato il modello organizzativo nato dall’esperienza formativa nel Policlinico di Chieti.
Risultati e conclusioni
Oggi i tempi dell’apprendere e del lavorare non possono più
restare divisi. Il tempo “del sapere e saper fare” deve servire
soprattutto al sapere e saper fare professionale (Corsi di Laurea di I° livello); il tempo del lavoro deve diventare sempre
più anche un tempo d’apprendimento delle conoscenze e delle
capacità nuove e diverse, contribuendo sia al proprio sviluppo
individuale sia alla competitività dell’Organizzazione di appartenenza (ECM). L’apprendimento deve essere continuo,
quindi, e di conseguenza nel Sistema Sanità, la Formazione
ECM, specialmente quella Aziendale, non può essere assegnata a luoghi e tempi separati rispetto ai luoghi e tempi di
lavoro. La Formazione ECM, nelle classiche e nuove forme,
deve diventare realmente continua, distribuita capillarmente
nel tempo di lavoro, funzionale alla generazione di nuova
conoscenza e nuovo sapere ed in prospettiva dinamica, sviluppando, di fatto, una nuova cultura professionale al servizio
del Cittadino.
Bibliografia di riferimento
Benci L. Le Professioni Sanitarie (non mediche). Milano: McGraw-Hill
2002.
http://www.salute.gov.it/ecm/
http://cplps.altervista.org/blog/
Santianello M, Negrisolo A (a cura di). Quando ogni passione è spenta La sindrome del burnout nelle professioni sanitarie. Milano: McGrawHill 2010.
Zangrandi A (a cura di). Economia e Management per le professioni
sanitarie. Milano: McGraw-Hill 2010.
COMUNICAZIONI ORALI
Pathologica 2011;103:135-174
Patologia fetoplacentare
Acrania and anencephaly: a case report
L. Sollima*, G. Calvisi**, I. Cicchinelli*, G. Crisman*, P. Leocata*
*
Anatomia Patologica, Dipartimento di Scienze della Salute /Università
degli Studi dell’Aquila, L’Aquila, Italia; **Unità Operativa di Anatomia
Patologica/ Ospedale Civile “San Salvatore”, L’Aquila, Italia
A failure of fusion of the neural tube causes the so-called Neural
Tube Defects (NTDs), which include spina bifida, anencephaly
and encephalocele.
It has been estimated that NTDs represent the second most common birth defects with a worldwide reported incidence ranging
from 1.0 to 10.0 per 1,500 births. According to the literature,
the Italian incidence is 0.4-1/1,000. Maternal diabetes, obesity,
genetic gains or losses, race/ethnicity, hyperthermia, drugs, a previous history of NTD-affected pregnancy have been identified as
risk factors for NTDs, even though folic acid insufficiency seems
to play a pivotal role still.
An absence of the flat skull bones covering the brain is defined
by the term acrania, and represents a lethal malformation, which
could be diagnosed sonographically even in the first trimester if
a large mass of disorganized brain tissue covered only by a thin
membrane is detected. The sonographic differential diagnosis of
acrania should include anencephaly, cephalocele, osteogenesis
imperfecta, and hypophosphatasia.
We report on a case of a 41-years-old woman, presented with
good health’s conditions over all the pregnancy period, in absence
of any history of dysmetabolic disorders, or hyperthermia, or folic
acid insufficiency. At the 20th week of gestation, the ultrasound
revealed that her female fetus presented with anencephaly and
acrania. An interruption of the pregnancy has been induced thus,
placenta and fetus have been analyzed by the Pathology Unit of
the “San Salvatore Hospital” of L’Aquila (L’Aquila, Italy).
The gross analysis of the placenta revealed a twisting of the umbilical cord and a marginal hematoma of 3,5 cm in-diameter. The fetus
presented without any flat skull bone covering the brain, which was
totally absent. Interestingly, the spinal cords ends around the fifth
cervical vertebra thus, the fetus does not present the encephalic trunk
as well. No other malformation has been detected.
Acrania and anencephaly are two rare and lethal conditions and
the real aetiopathogenetic mechanisms are still poorly understood. The rarity of this entity and the peculiar findings of the
postmortem examination lead us to report on this case.
References
1
Weissman A, Diukman R, Auslender R. Fetal acrania: five new cases
and review of the literature. J Clin Ultrasound. 1997;25:511-4.
2
Dhaulakhandi DB, Rohilla S, Rattan KN. Neural tube defects: review
of experimental evidence on stem cell therapy and newer treatment
options. Fetal Diagn Ther 2010;28:72-8.
Raison d’etre, clinical relevance and legal
questions in early spontaneous abortion diagnosis
G. Lomazzo, V.R.L. Beltrami, G. Botta, M. Ribotta
Struttura Complessa di Anatomia Patologica e Citodiagnostica, Azienda
Ospedaliera “OIRM-Sant Anna” Torino, Italy
Early spontaneous abortion (ESA) is defined a miscarriage by 12
weeks gestation. It is a frequent event: 10-15% of pregnancy ends
with a spontaneous abortion and 85% of spontaneous abortion
occures by 12 weeks.
The abortive tissue sample by uterine evacuation includes villi,
decidua and embryo tissue. Associate to traditional histological
exam in selected case it is possible a cytogenetic exam or bacteriologic o virologic investigation.
The object was to determine whether histological examination of
ESA would be helpful.
From 1-1- 2007 to 30-06-2011 all surgical uterine evacuation for
ESA (3599 cases) were esamined histologically and in 966 cases
we associated also cytogenetic investigation.
In 29% of cases we observed morphological anomalies suggesting Karyotipe abnormalities (in 1/3 of case the citogenetic
investigation was available). Il 22,1% of cases a deficent villi
vascularization was present. In 18,1% of cases a detachment of
gestational sac, in 3,4% of cases an acute or chronic infection,
in 3,5% a hydropic degeneration was observed. In 4,6% of case
a molar pregnangy was diagnosed (3,8% partial mole and 0,8%
complete mole). In 0.7% of cases a trophoblastic non molar disease was present. In 14% of cases surgical uterine evacuation not
include material appropriate to hystological diagnosis (absence or
paucity or bad preservation of villi).
Histological examination of ESA allows in 70-80% of cases an
aetiological diagnosis. The cause of ESA sometimes are sure (for
example in case of kariotipe abnormality) or it specifies the conclusive event (for example in case of sac detachment) that include
a lot of maternal morbility. Furthermore histological examination of ESA is the more precise method of molar disease and
trophoblastic disease screening. Only to identify villi or embryo
tissue in a surgical uterine evacuation rules out an extrauterine
pregnancy.
In conclusion uterine evacuation for ESA should be routinely
histologically esamined to understand abortion aetiology, to
screen molar disease and to rule out ectopic pregnancy. A missed
examination may cause a professional responsibility.
Patologia cardiaca
PTHrP and PTHR1 myocardial expression in 66
explanted hearts
V. Arena*, I. Pennacchia*, E. Di Stasio**, R. Fiaccavento***, P. Di
Nardo***, G. Monego****
*
Institute of Pathology, Catholic University of Sacred Heart, Rome; **Institute of Biochemistry, Catholic University of Sacred Heart, Rome; ***
Laboratory of Cellular and Molecular Cardiology, ‘‘Tor Vergata’’ University, Rome; ****Institute of Human Anatomy and cell biology, Catholic
University of Sacred Heart, Rome
In a previous paper we reported the expression of Parathyroid
Hormone-related Protein (PTHrP) and Parathyroid Hormone Receptor type 1(PTH1R) in human myocardium. We showed for the
first time the expression of PTHrP by ventricular cardiomyocytes
in a set of autoptic hearts. The PTHrP/PTH1R signalling system
resulted upregulated in association with ischemic damage 1. This
paper prompted to wonder if PTHrP system should be considered
as “a friend or a foe” in myocardial ischemia 2. We try to answer
to this question starting from the association between the expression of PTHrP system and the heart ischemia.
In order to rule out PTHrP induction by stretching of myofibers due to mechanical overload, we chosen samples from hearts
136
showing similar levels of contractile function. 66 samples of ventricular myocardium from patients undergoing transplantation,
were collected on the basis of an ejection fraction < 40%. (9 cases
of ischemic cardiopathy, 28 cases of dilative cardiopathy, 8 cases
of valve pathology, 12 cases of hypertrophic cardiopathy, 8 cases
of non-compaction). The immunohistochemical expression of
both PTHrP and PTH1R was categorized using a score based on
the intensity of immunostaining. The population studied showed
a high number of cases with increased expression of PTHrP
@80% ranging from 100% of the ischemic (9/9) and non-compaction (9/9) groups, to @37,5% of valve pathology group (3/8).
The percentage of high PTHrP expression cases was @83,3% in
the dilative group (22/28) and @78,5% in the hypertrophic group
(10/12%). PTH1R resulted overexpressed in @33% of cases belonging to hypertrophic (4/12) and non-compaction (3/9) groups,
decreasing to @25% in the dilative (7/28) group. However, this
percentage increased to 50% (4/8) in the valve pathology group,
and reached 100% (9/9) in the ischemic group. These data are
suggestive for an upregulation of PTHrP as a response to heart
failure independently of the etiology of the underlying cardiac
pathology. This adaptive overexpression of the peptide resulted
associated with a low prevalence of the overexpression of the
cognate receptor, suggesting a sort of negative feed-back regulation mechanism, as reported in literature 3. In contrast with this
trend, the ischemic cases were characterized by upregulation of
both the components of the PTHrP/PTH1R signalling system,
showing increased expression of peptide and receptor.
Basing on these observations, we can assume that myocardium
responds to heart failing by upregulating cardiomyocytes expression of PTHrP, probably due to mechanical overload. Postischemic heart failure is significantly associated (p=0,01) with
increased expression of PTH1R by ventricular cardiomyocytes,
probably triggered by ischemic injury. Although the cases studied
are few in number for statistical analysis, we can outline a trend
of PTH1R overexpression under ischemic conditions, suggesting
the hypothesis that myocardial adaptation to ischemia could be
mediated by G protein coupled receptors such as PTH1R.
References
1
Monego G, Arena V, Pasquini S, et al. Ischemic injury activates
PTHrP/PTH1R expression in human ventricular cardiomyocytes. Bas
Res Cardiol 2009;104:427-34.
2
Schlüter KD, Schreckenberg R. Ischemic injury and the parathyroid
hormone-related protein system: friend or foe? Bas Res Cardiol.
2009;104:424-6.
3
Clemens TL, Cormier S, Eichinger A, et al. Parathyroid hormonerelated protein and its receptors: nuclear functions and roles in the
renal and cardiovascular systems, the placental trophoblasts and the
pancreatic islets. Br J Pharmacol 2001;134:1113-36.
Epatopatologia
High mobility group A1 (HMGA1): a potential role in
hepatocarcinogenesis
F. Trapani*, S. Piscuoglio* **, L. Tornillo*, M. Matter*, L. Terracciano*
Institute of Pathology, Molecular Pathology Division, University of Basel,
Basel, Switzerland; ** Research Group Human Genetics, Department of
Biomedicine, University of Basel, Basel, Switzerland
*
Background. Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide and incidence rates are
increasing, it is a primary malignancy of the hepatocyte, generally
leading to death within 6-20 months. HCC frequently arises in the
setting of cirrhosis, appearing 20-30 years following the initial
insult to the liver. High mobility group A (HMGA) proteins play
an important role in the regulation of transcription, differen-
tiation, and neoplastic transformation. The HMGA gene family
includes HMGA1, which encodes the HMGA1a and HMGA1b
protein isoforms. These chromatin-binding proteins function in
transcriptional regulation and recent studies also suggest a role in
cellular senescence. HMGA1 proteins also appear to participate
in cell cycle regulation and malignant transformation.
Aims. In this study, the expression of HMGA1 in 341 liver tissues of mainly HCC type has been studied by immunohistochemistry in order to evaluate its feasibility as HCC marker.
Materials and methods. We investigated the expression of
the HMGA1 gene in a tissue microarray platform consisting of
normal, cirrhotic and HCC tissues by immunohistochemistry.
HMGA1 was detectable in 341 patients (72 normal, 87 cirrhotic
and 182 HCC). Immunoreactivity was scored semi-quantitatively
by evaluating the number of positive tumor cells over the total
number of tumor cells. Scores were assigned using 5% intervals
and ranged from 0% to 100%, and statistical correlations between
numerical variables were tested using a T test (unpaired).
Results. Our results demonstrated that HMGA1 are over-expressed in HCC tissues compared to normal (p<0,001) and cirrhotic liver (p=0.0015). It is tempting to speculate that HMGA1
could be implicated in HCC carcinogenesis.
Conclusions. This study demonstrates that HMGA1 over-expression seems to play an important role in liver carcinogenesis.
Additional studies are needed to demonstrate that HMGA1 expression could be used as prognostic marker of HCC.
Neuropatologia
CD90 expression in meningiomas
E. La Mantia*, G. Scognamiglio*, V. Relli*, G. Rossi**, L. Sparano***, A. Mirabella***, R. Franco*, G. Rocco****, G. Botti*
*
Pathology Unit, National Cancer Institute, Pascale Hospital, Naples,
Italy; **Pathology Unit,Modena and Reggio Emilia University, Modena,
Italy; ***Pathology Unit,M. ScarlatoHospital, Scafati (SA), Italy; ****Thoracic Surgery Unit, National Cancer Institute, Pascale Hospital, Naples,
Italy
Introduction. CD90 (Thy-1) is a 25–37 kDa glycosylphosphatidylinositol (GPI) -anchored glycoprotein and it is the smallest
member of the immunoglobulin superfamily.
This is expressed in many cell types, including T cells, thymocytes, neurons, endothelial cells, and fibroblastic, but its role has
not yet been fully elucidated.
Several studies have shown that CD90 has a number of immunological effects, modulating also immunologicl response to cancer.
In fact it was shown that CD90 is a tumor suppressor in ovarian
cancer, nasopharyngeal carcinoma and hepatocellular carcinoma.
In Nervous Central System (CNS), Thy-1 is highly expressed
in normal adult neurons and in in astrocytic and neural tumors.
Tumors affecting CNS rarely develop extraneural metastases,
probably because of inherent biologic charateristics of these types
of tumors and intrinsic anatomic characteristics of brain. In particular extraxial metastasis have been described in meningiomas.
Aim. The aim of our study was evaluation of CD90 expression in
primary tumors of CNS and correlation with distant metastases.
Methods: A TMA of primitive CNS tumors has been built. TMA
included 35 tumors, 10 meningiomas, 9 astrocitomas, 13 glioblastomas. Morover in our archive 4 meningioma metastasis have
been collected, 3 lung metastasis and 1 liver metastasis. 1 out of
three lung metastasis corresponded to primitve meningioma of
our series.
Immunohistochemical expression analaysis of CD90 has been
performed. Moreover in the case of meningioma lung metastasis
with corresponding primitive lesion cytoflurietric analysis of
CD90 expression has been performed.
137
comunicazioni orali
Results. CD90 high expression has been observed in astrocytic
tumors, while 8/10 meningiomas were negative. 2 cases of meningiomas were positive. All meningiomas metastasis showed
CD90 high expression. Interestingly both primitive and relative
metastasis were CD90 immunostained. Cytofluorometry of metastasis cases parallel immunohistochemical results, showing a
significant high expression.
Conclusion. CD90 is generally negative in meningiomas. The
expression in metastasis could suggest a relative more aggressive behavior of meningiomas. CD90 identification in primitive
tumors could help to better stratify meningioma patients.
Collateral trigone choroid plexus papilloma
with extreme stromal sclerosis
A. Cimmino*, G. Ingravallo*, R. Rossi*, P.I. D’Urso**, S.V.
Scarcella*, L. Resta*
* Dipartimento di Anatomia e Istologia Patologica, Università di Bari, **
Clinica Neurochirurgica, Università di Bari, Italia
Choroid plexus papillomas (CPPs) are relatively rare and usually
benign neoplasms. CPPs account for 0,4 to 0,6% of all intracranial neoplasms. In rare instances they may be congenital and more
exceptionally bilateral. Stromal changes and unusual histological
features in choroid plexus papilloma, such as oncocytic changes,
mucinous degeneration, melanization and tubular glandular architecture may occur in choroids plexus papilloma, but massive
fibrovascular stroma collagenization of a choroid plexus papilloma has not previously reported.
A 60-year-olf female was referred with MRI evidence of a left
intraventricular collateral trigone lesion, manifested with symptoms of increased intracranial pressure. A smaller lesion was also
demonstrated in the contralateral ventricle. Patient underwent to
surgical removal.
Microscopic examination revealed a fibrosclerotic mass containing scattered islands of epithelial papillary fronds, covered
by a single layer of uniform columnar/cuboidal epithelial cells.
The tumor was almost entirely effaced by collagenized stroma.
Fibrous sclerosis extensively interested the lesion, reducing the
papillomatous component to nodular scars containing sparse
benign glandular elements. Neoplastic stroma was constituted
by fibroblasts, a conspicuous amount of collagen fibers, inflammatory cells and macrophages. There was no evidence of malignancy. Immunohistochemical analysis showed immunoreactivity
for S-100 protein and CK-pool, patchy positivity for CK7, and
absence of reactivity for GFAP and CK20 in the epithelial cells.
In the epithelial cells, there was not immunoreactivity for the
specific markers of more frequent metastatic carcinomas with
papillary architecture (TTF-1, estrogen receptor and CDX-2).
Electron microscopy showed a papillary structure lined by low
columnar cells with numerous short microvilli on the luminal
surface. The basal surface was followed by a well defined basal
lamina. The lateral surfaces had typical junctional complexes
near the luminal end and numerous interdigitations of the cell
membranes. The nuclei were rounded, contained finely granular
chromatin and small inconspicuous nucleoli. A moderate amount
of rough endoplasmic reticulum and mitochondria were uniformly distributed throughout the cytoplasm. Cytoplasmic filamentous
inclusions of variable morphology were present.
A diagnosis of CPP with extreme stromal sclerosis was made.
Interestingly, massive fibrovascular stroma collagenization of
human CPP has not been previously reported. Similar event is
common in breast papilloma, a benign tumor of the epithelium
of mammary duct. In the current case, the massive collagenization may be due to an ischemic injury, as consequence of an
imbalance between tumor growth and inadequate angiogenesis
or blood flow.
Paleopatologia
Genetic susceptibility to rheumatoid arthritis
in Cardinal Carlo de’ Medici
G. Fontecchio1, L. Ventura2, V. Giuffra3, A. Vitiello3, S. Giusiani3, A. Fornaciari4, D. Caramella5, N. Villari6, G. Fornaciari3
Centro Regionale di Immunoematologia e Tipizzazione Tissutale, Ospedale San Salvatore, L’Aquila, Italia; 2U. O. C. di Anatomia Patologica,
Ospedale San Salvatore, L’Aquila, Italia; 3Dipartimento di Oncologia, dei
Trapianti e delle Nuove Tecnologie in Medicina, Divisione di Paleopatologia, Storia della Medicina e Bioetica, Università, Pisa, Italia; 4Dipartimento di Archeologia e Storia dell’Arte, Sezione di Archeologia Medievale, Università, Siena, Italia; 5Dipartimento di Oncologia, dei Trapianti e
delle Nuove Tecnologie in Medicina, Divisione di Radiologia Diagnostica
ed Interventistica,Università, Pisa, Italia; 6Dipartimento di Fisiopatologia Clinica, Sezione di Radiologia Clinica, Università, Firenze, Italia
1
A paleopathological study was carried out on the skeletal remains
of Cardinal Carlo de’ Medici (1595-1666), son of the Grand Duke
Ferdinando I, to investigate the articular pathology described in
the archival sources. The skeletal remains, buried in the Basilica
of San Lorenzo in Florence, have been exhumed and submitted to
macroscopic and radiologic examination, revealing a concentration of different severe pathologies. These include Klippel-Feil
syndrome, cervical spine tuberculosis (Pott’s disease), from
which Carlo suffered in his infancy, and a post-cranial ankylosing
disease, symmetrical and extremely severe, involving the large
and small articulations, and characterized by massive joint fusion,
that totally disabled the Cardinal in his last years of life. The final
diagnosis of this latter polyarthritis suggests an advanced, ankylosing stage of rheumatoid arthritis (RA), rather than a psoriasic
arthritis (PsA).
Molecular analysis was performed to seek “risk factor” genes
HLA-related to RA or PsA.
Genetic susceptibility to RA and PsA is linked to some genes
belonging to HLA system, a wide polymorphic region containing
an high number of alleles related to the manifestation of hundreds
of autoimmune diseases (AIDs). The “risk genes” include the
HLA-DRB1*01 (phenotype DR1) and HLA-DRB1*04 (DR4)
predisposing to RA and widely represented in Italian population,
while HLA-Cw*06 (Cw6) and HLA-DRB1*07 (DR7) are associated with both PsA and psoriasis.
Genotyping tests were performed starting from a rib fragment of
Carlo. After the extraction of aDNA from bone, this was purified with a method based on sodium acetate/2-propanol. In all
aDNA processing steps all precautions to avoid contamination by
exogenous DNA were taken. The HLA assays for the DRB and C
loci were undertaken by means of PCR-Sequence Specific Primers low resolution technique. The typing test assigned to Carlo
the genotype DRB1*04/DRB1*11 for DRB locus and Cw*04/
Cw*12 for the locus C. These results confirm the presence of RA
susceptibility gene DR4 and the presence of Cw4, but the lack of
Cw6. The concomitant absence of Cw6, as well as DR7, excludes
the predisposition for psoriasis and PsA.
The exact RA molecular basis has not completely defined yet and
several theories have been proposed. The “molecular mimicry” is
the pathogenetic mechanism which can better explain the pathogenesis of RA. It consists in a cross-reactivity between some amino acid sequences (i.e. EQK/RRAA), common to DR1 and DR4,
named “shared epitopes” and identical or similar peptide motifs
of host-determinants and infection agents, including Proteus
mirabilis, Cytomegalovirus, Escherichia coli and Mycobacterium
tuberculosis. Thereby, autoreactive T-cells elicit a strong autoimmune response with production of antibodies raised against both
microbial and self-components. This sequence homology has
been demonstrated between M. tuberculosis 65-kDa heat shock
protein and DR4. Some studies report a sequence similarity be-
138
tween M. tuberculosis protein and cartilage components as proteoglycans, while others demonstrated antibodies against Type
A synoviocytes and Type II collagen in DR4 patients. All these
data, together with the features that Cardinal Carlo (i) undoubtly
suffered from tuberculosis, (ii) was bearing the genetic risk factor
DR4 and (iii) was negative for Cw6 or DR7, are more in favour
to the onset of RA rather than psoriasis or PsA. In addition, since
Cw6 is the major genetic risk factor for psoriasis, it is likely that
Carlo was not even affected by this disease. We underline that
HLA-typing has not to be considered an assay with diagnostic
significance but it can give an important support to paleopathological examination of ancient remains in case of doubtful diagnosis, as it often happens with this kind of specimens.
Paleopathology of the “Queen of the Moors”, a XIX
century natural mummy from Scicli (south-eastern
Sicily)
L. Ventura1, V. Pensiero2, C. Caruso3, G. Romeo4, B. Grimaldi 4,
F. Marampon5, G.L.Gravina5, G. Fornaciari2
U. O. C. di Anatomia Patologica, Ospedale San Salvatore, L’Aquila, Italia; 2Divisione di Paleopatologia, Storia della Medicina e Bioetica, Dipartimento di Oncologia, Trapianti e delle Nuove Tecnologie in Medicina,
Università, Pisa, Italia; 3R. S. A., Ospedale Busacca, Scicli (RG), Italia;
4
U. O. C. di Diagnostica per Immagini e Radiologia, Ospedale Maggiore,
Modica (RG), Italia; 5Divisione di Radioterapia e Radiobiologia, Dipartimento di Medicina Sperimentale, Università, L’Aquila, Italia
1
The church of Santa Maria della Consolazione in Scicli (Ragusa
province, south-eastern Sicily) was started to build in the XVI
century on the site of a pre-existing chapel. After surviving
undamaged to a major earthquake in 1693, it was expanded in
a Baroque style and finished in the beginning of XIX century.
The funerary character of the church is suggested by the name
itself (consolation for the dead) and witnessed by the several discoveries of human remains, mortuary chapels and crypts within
the building through the years. During the Second World War,
beneath the frontal staircase of the building, a mummy (named
by local people the “Queen of the Moors”) was recovered and
moved to the church of the Carmine, where it is still preserved
and displayed in a glass/wooden case. Despite the absence of
clothes, objects and documents related to the subject, it could be
postulated that the mummy dated back to the second half of the
XIX century.
At visual inspection the body was almost complete (except the
feet) and appeared in a very good state of preservation, without
external signs of anthropogenic manipulation and belonging to a
female subject. The age at death, according to the dental wear and
the preservation conditions of the body, was 45-55 years. Without
the feet, the mummy measured 141 cm in length and the extimation of her stature could be 152-155 cm.
Direct radiograms in different projections were obtained with
the digital system GMM Opera T, whereas CT scanning was
performed by using a General Electric LightSpeed Pro 32 scanner
with 1 mm thick sections, obtained at reconstruction intervals of
1,25 mm, generating a total of 1269 scans. Tomodensitometric
evaluations were made according to the Hounsfield scale, and 3D
reconstructions were carried out with a Vitrea 2.1 workstation.
Amorphous material (remnants of encefalic tissues) was highlighted in the posterior cranial fossa, along with portions of the
meningeal wrappings, which were also visible within the entire
vertebral column. Tissue remnants were also present inside the
orbits. Thoracic and abdomino-pelvic organs appeared extremely
well-preserved and readily recognizable. All these findings confirmed the natural mummification process, due to rapid dehydration, possibly related to hot dry climate.
Radiography and 3D reconstructions of CT scans were also extremely useful to determine accurately the dental status. All but
eight superior and all the inferior elements were present, with
two molars displaced postmortem into the right cheek and the
larynx. Focal deposits of tartar were observed on the anterior
teeth, which also displayed mild periodontitis and severe dental
wear. Transverse lines on the anterior teeth were reconducted to
enamel hypoplasia.
Diffuse right pleural adhesions were observed, along with subpleural tiny calcifications of the lung and a paratracheal calcified
nodule measuring 21 x 16 x 12 mm. Such findings were consistent with primary pulmonary tuberculosis. Left lung appeared
normally collapsed.
Areas of calcification were also noted in the wall of the abdominal aorta and both iliac arteries, as a sign of atherosclerosis;
small (2-3 mm), round calcifications (reactive lymph nodes?)
were present in the pelvis. Pseudocystic areas in sacroiliac joints
could be related to sacroileitis or previous pregnancies. The
rectosigmoid appeared distended with endoluminal fecal material, whereas a relative hyperdensity zone could be noted in the
rectovaginal space.
Neither growth arrest (Harris) lines, nor fractures could be noted
in the standard radiograms of the long bones. Further histological
and molecular studies are planned to obtain additional information about this subject and her diseases.
The “Queen of the Moors” represents a rare example of natural
mummification in the scenario of Sicily, characterized by huge
numbers of artificial mummies. Her uneviscerated mummified
body allowed to obtain abundant data about her life and health.
Patologia renale
The role of t regulatory lymphocytes in lupus
nephritis
K. Giannakakis*, A. Gigante**, A. Afeltra***, R. Cianci**, A.
Amoroso**, D. Margiotta***, F. Pugliese**, T. Faraggiana*, A.
Onetti-Muda****
*
Anatomia Patologica, “Sapienza”Università di Roma; **Medicina Clinica “Sapienza” Università Roma; ***Medicina Interna, Università Campus
Bio-Medico di Roma; ****Anatomia Patologica, Università Campus BioMedico di Roma
Background. Regulatory T cells (Tregs) play a key role in the
maintenance of immune tolerance and in the development of autoimmune diseases. Expression of Foxp3, a member of forkheadbox family of transcription factors, is specific for Treg cells and
can be used for the identification of these cells. Systemic Lupus
Erythematosus (SLE) is a prototype autoimmune disease characterized by dysregulated activation of T and B lymphocytes, causing multiple organ damage. There is a high incidence of renal involvement during the course of the disease with varied pathologic
and clinical features. Several studies describe a quantitative and/
or qualitative abnormalities of peripheral Tregs in SLE. However,
the role of Tregs in lupus nephritis (LN) is still unclear.
Aim of the study. The study aims to investigate the variations of
Tregs Foxp3+ in the kidney biopsies inflammatory infiltrate of
different LN classes (according to ISN/RPS 2003 criteria) compared to that of ANCA associated crescentic glomerulonephritis
(ANCA- CrGN), acute tubulointerstitial nephritis (ATIN) and
nephroangiosclerosis (NAS).
Materials and methods. Investigation was carried out on renal
biopsy samples of 27 patients with histologically proven LN
classified according to the ISN/RPS 2003 criteria (class III: 3
patients, class IV: 17 patients, class V: 7 patients), 3 patients
with ANCA-CrGN, 6 patients with ATIN, and 2 patients with
NAS. Sections of paraffin embedded tissue have been stained by
immunohistochemistry with anti-CD3 and anti-FoxP3 antibodies, performed separately on consecutive sections. The number
139
comunicazioni orali
of FoxP3 positive cells and CD3 positive per mm2 was counted
after digitalization of slides and application of a dedicated image
analysis software.
Results. Amount of CD3+ cells was higher in ATIN (5713/mm2)
and in ANCA-CrGN (5121/ mm2) than in LN-IV (3558/ mm2),
LN-III (2491/ mm2), NAS (2379/ mm2) and LN-V (2220/ mm2).
Instead, we found that the ratio of FoxP3+/ CD3+ cells was significantly lower in patients with LN-IV (1,6) and, although less
significantly, in patients with CrGN (3) than in course of NAS
(3,9), ATN (4), and LN-V (4,5).
Conclusion. The data presented herein, demostrate a decrease
of Foxp3+ Treg cells in the inflammatory infiltrate of lupus
nephritis. These results, although preliminary, suggest an important role of Tregs in the pathogenesis of autoimmune diseases,
particularly during the most active phases of LN, as observed in
LN-IV class.
Loss of 9p and 14q predict the risk of progression
after nephrectomy in patients with nonmetastatic clear cell renal cell carcinoma
D. Segala*, V. Ficarra**, M. Brunelli*, G. Novara**, S. Gobbo*, C.
Cannizzaro*, A. Mosca***, C. Porta****, G. Martignoni*
Dipartimento di Patologia e Diagnostica, Università di Verona, Verona,
Italia; **Dipartimento di scienze oncologiche e chirurgiche, Università di
Padova, Padova, Italia; ***S.C.D.U. Oncologia Medica, AOU Maggiore
della Carità di Novara, Novara, Italia; ****Istituto di Medicina Interna e
Oncologia Medica, IRCCS Policlinico San Matteo, Pavia, Italia
*
The identification of predictors of progression-free survival
(PFS) in patients with clear cell renal cell carcinoma (CCRCC)
is an important task to improve the quality of the post-operative
counseling, to plan adequately the follow-up schedule, to facilitate the interpretation of the results of ongoing RCTs and to
evaluate the efficacy of targeted therapies as adjuvant treatment
after nephrectomy.
In the past decade, pathologic variables were integrated to generate predictive models with an higher predictive accuracy in
comparison with the single variable. Leibovich score is one of the
most important prognostic tools calculating the risk of recurrence
in CCRCC and the UCLA Integrated Staging System (UISS) can
be used to predict the PFS of patients surgically treated for RCC
regardless the histologic subtype. Today, ideal candidate for adjuvant trials were identified using both this models.
Several studies evaluated the impact of the molecular and cytogenetic markers to predict the oncologic outcomes in patients
with CCRCC. Nevertheless, the impact of cytogenetic alteration
on the risk of progression was not tested in the literature. Only
two reports highlighted the independent role of loss 9p to predict
cancer-specific survival.
The aim of the study was to evaluated the potential role of loss 9p
and 14q to predict the risk of progression in a cohort of patients
with non-metastatic CCRCC. Then, we tested if the simultaneous
presence of loss 9p and 14q was associated with a different risk
of progression in the subgroup of patients candidate for adjuvant
therapy with targeted therapies.
Using the interphase cytogenetic Fluorescence In Situ Hybridization (FISH) analysis on tissue microarrays, we evaluated the loss
of 9p and 14q in 196 patients who underwent partial or radical
nephrectomy between 1990 to 2000 for CCRCC. Statistical
analyses were performed using the SPSS software package, 16.0
version with p<0.05 considered statistically significant.
Twenty-one (10.7%) patients with distant metastases at diagnosis
were excluded from the analysis. According to Leibovich score,
70 (40%) patients were classified as low; 68 (39%) as intermediate and 37 (21%) as high-risk group. According to UISS, 54
(30.9%) patients were classified as low-risk; 110 (62.9%) as
intermediate risk and 11 (6.3%) as high risk.
At follow-up (mean 55.1 ± 44.4 months), 136 (77.7%) patients
were alive and disease-free; 6 (3.4%) alive but in progression; 7
(4%) had died of other causes and 26 had died of RCC.
Loss of 9p was present in 31 (17.7%) cases, loss of 14q was detected
in 26 (14.9%) cases and the contemporary presence of both cytogenetic alterations was reported in 17 (9.7%) cases. Only loss of 9p
resulted significantly correlated with the Leibovich score (p=0.01)
and UISS (p=0.05). Loss of 9p (p=0.002), loss of 14q (p=0.02) and
the contemporary presence of both cytogenetic alterations (p=0.005)
resulted able to predict PFS at univariable analysis.
The simultaneous presence of loss 9p and 14q turned out an
independent predictor of PFS once adjusted for the effects of
pathological factors combined in the Leibovich score or for the
effects of clinical and pathological variables included in the
UISS. Conversely, the presence of these cytogenetic abnormalities were able to further stratify the intermediate risk group in two
categories with different progression-free survival.
In conclusion, this study demonstrated for the first time that
simultaneous loss of 9p and 14q is an independent predictor of
PFS in patients who underwent partial or radical nephrectomy for
non-metastatic CCRCC once adjusted for the effects of Leibovich
score and UISS. Moreover, the data showed that the simultaneous
presence of loss 9p and 14q is able to stratify ideal candidates for
adjuvant treatment in two subgroups with a significant different
risk of progression.
Renal and lung features in Wegener
Granulomatosis
R. Passantino*, G. Li Cavoli**, A. Ferrantelli**, L. Bono**, C. Tortorici**, C. Giammaresi**, C. Zagarrigo**, U. Rotolo**
Unità Operativa di Anatomia Patlogica/Ospedale ARNAS Civico Di Cristina Benfratelli di Palermo, Palermo, Italia; **Unità Operativa di Anatomia Patologica/ Ospedale ARNAS Civico Di Cristina Benfratelli di Palermo, Palermo, Italia
*
Wegener Granulomatosis (WG) is the most common of the ANCAassociated vasculitides. The complete clinical triad (upper airway
disease, lower respiratory tract disease and glomerulonephritis) is
often not present at initial presentation. In this case we observed
the initial simultaneous involvement of larynx, lung and kidney.
A Caucasian 55-year-old man, bricklayer and heavy smoker, was
admitted for acute renal failure. From 3 months he was suffering
from dysphonic voice, loss of weight, anaemia and malaise. Lung
CT scan showed multiple bilateral nodules, like septic or metastatic
dissemination, without other typical features. Bronchoscopy and
bronchoalveolar lavage were negative except for a larynx polyp;
p-ANCA test was positive; microbiological and other immunological tests were unremarkable. Renal biopsy disclosed a crescentic
glomerulonephritis with wide destruction of Bowman’s capsule,
large semicircunferential cellular crescents with moderate infiltration by neutrophils and predominant mononuclear leukocytes,
segmental glomerular sclerosis and loss of many capillary loops.
Lung histology displayed a diffuse severe necrotizing chronic
granulomatosis inflammation with multinucleated giant cells and a
leukocytolitic angiitis. Larynx histology showed a laryngeal nodule
ulcerated with angiomatoid features of the stroma, severe necrotizing chronic active aspecific phlogosis without multinucleated giant
cells or hemorrhage. We performed diagnosis of WG. In nephrological reports of renal injury in WG, there are few histological findings
of lung involvement. We made lung biopsy because of lung CT scan
picture and the important malignancy risk. The exclusion of neoplastic disease was indispensable for following immunosuppressant
therapy. Although c-ANCA is the marker more frequent associated
in WG, in this case we detected a p-ANCA positivity.
References
Falk RJ, Gross WL, Guillevin L, et al. Granulomatosis with polyangiitis
(Wegener’s): an alternative name for Wegener’s granulomatosis. Arthritis Rheum 2011;63:863-4.
Kamali S, Erer B, Artim-Esen B, et al. Predictors of damage and survival
140
in patients with Wegener’s granulomatosis: analysis of 50 patients. J
Rheumatol 2010;37:374-8.
Xiao H, Heeringa P, Hu P, et al. Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice. J Clin Invest 2002;110:955-63.
Risk management
Patients’s perspective in disease oriented
biobanking activity: the Trentino Biobank project
experience
S. Fasanella*, S. Giuliani*, C. Cantaloni*, M. Macilotti**, P. Dalla
Palma*, M. Barbareschi*
*
Trentino Biobank, Unit of Surgical Pathology, S. Chiara Hospital, Trento;
Faculty of Law, Department of legal sciences, University of Trento;
**
Trust between donors, biobank managers and researchers is
essential in biobanking. Informed consent (IC) is a standard re-
quirement and its formulation in biobanking activity is a matter of
debate. We describe our experience in daily contact with patients
focusing on how they answer the questions of the IC.
Since 2009 we run a disease-oriented public biobank, and submitted our broad IC to 620 potential donors of left-over tissues in
a general hospital. Our IC includes a description of the project,
donor rights and 8 questions (see: www.tissuebank.it).
All patients agreed to donate their biomaterials; 0.3% denied to
provide biomaterials to private companies for industrial research,
27.5 % patients did not want to be recontacted by the biobank in
case of unexpected findings; 19% patients didn’t allow to share
clinical and genetical data with relatives. Most patients felt that
the complex consent module was unnecessary.
All patients understood the importance of biobanking and were
highly compliant, underscoring the importance of trust as the
fundamental element in biobanking. Broad IC was not a constraining element in their decision to donate biospecimens. Helping patients make this decision requires transparent terminology
together with the ability to listen and respecting the patient’s right
to self-determination.
Dermatopatologia
Aberrant expression of posterior locus HOX C gene
in metastatic melanoma
M. Farina*, A. Anniciello*, M. Cantile*, G. Scognamiglio*, A.
Manna*, N. Chicchinelli*, P. Ferraiuolo*, P. Ascierto**, F. Fulciniti*, R. Franco*, G. Botti*
Pathology Department, National Cancer Institute “Fondazione G. Pascale”, Naples, Italy; **Melanoma Department, National Cancer Institute
“Fondazione G. Pascale”, Naples, Italy
*
Introduction. The process of malignant transformation, progression and metastasis development of melanoma is not completely
understood. Recently, the microarray technology has been used to
survey transcriptional differences that might provide insight into
the metastatic process, but variation of gene expression during
metastatic progression is poorly investigated.
The role of the HOX gene network in tumour evolution, has been
widely investigated in several types of human cancers, suggesting its involvement in tumor development and progression. Recently we have identified the prognostic role of HOX D13 gene
in pancreatic cancer. Moreover, deregulated paralogous group
13 HOX genes expression has also been detected in melanoma,
cervical cancer and odonthogenic tumors. Among these, Hox C13
is recently identified as a member of human DNA replication
complexes.
Aim. In this study, to investigate HOX C13 role in melanoma
progression, we have compared its expression pattern between
juctional, compound and dermic naevus, as a representative
of non-malignant melanocytes, primary and metastatic human
melanoma both hystologic and cytological, derived from our Institutional BioBank, and commercially melanoma cell lines.
Methods. The TMA was constructed employing 5 benign nevi
and 15 primary cutaneous melanomas, 15 matching metastases.
20 samples lymph node cytological metastases were examined,
all working to the same score (15% = 1 + cell population, 15%
-50% = 2 +, 50 +% = 3 +). A melanoma progression TMA has
been built comprehending to perform immunohistochemistry
analysis of HOX C13 expression. Moreover, used the same
normal controls, tumor samples and cell lines have been used to
analyze gene expression profile by Real Time PCR analysis. The
human cell lines MEL-Juso and WM115 cell lines are melanoma
malignant primitive lines; Human SK-MEL lines-2, SK-MEL-3,
SK-MEL-5, HBL, NA-8 and A-375 are cell lines of metastatic
melanoma.
Results and conclusion. The 20 cytology samples all showed
a nuclear immunoreactivity (1+ 1 case; 2 + 8cases, 3+11cases).
Among the histological samples do not have results immunoreactive nevi, the melanoma early I showed a positive 1 +/ 2 +
metastatic melanoma compared to the results of 2+/ 3+, with
proportional increase of all examined case The results of the cell
lines confirmed the data obtained with low expression in primary
cell lines that increases in metastatic cell lines. Our results show
the strong and progressive over-expression of HOX C13 in metastatic melanoma tissues and cytological samples when compared
to nevi and primary melanoma tissues, suggesting the HOX C13
role in metastatic melanoma switch.
MHC class I molecules delocalization and
deregulation in melanoma metastases
G. Scognamiglio, A.M. Anniciello, A. Barbato, M. Farina, V.
Relli, M. Cerrone, R. Franco, G. Botti
Pathology Unit, National Cancer Institute, Pascale Hospital, Naples,
Italy
Introduction. The process of malignant transformation, progression and metastasis of melanoma is not completely understood.
MHC class I antigens play a crucial role in the interaction of
tumor cells with the host immune system, in particular, in the
presentation of peptides as tumor-associated antigens to cytotoxic
lymphocytes (CTLs) and in the regulation of cytolytic activity of
natural killer (NK) cells.
Nevertheless, the meccanism with regulate MHC class I expression on tumor cells are not clear.
Aim. In the present study we have investigated the expression
comunicazioni orali
and protein localization and the of MHC class I molecules in human melanoma tissue samples.
Methods. We have built a Tissue Micro Array (TMA) containing primary human melanomas and corresponding metastases
samples from the same patient.
The expression analysis was conducted by immunohistochemistry with mouse monoclonal antibody for HLA class I (HC-A2)
Results and conclusion. Preliminary results show a strong cell
surface expression of HLA class I in primitive melanoma cells,
while metastatic cells of the same samples present a prevalent
cytoplasmatic expression of this molecule.
Our data suggest that during tumoral progression of this neoplasia
can be realized an alteration in the receptor assembly with its
ligand which determines different localizations of the molecule in
primary lesions compared to corresponding metastases.
We will propose to analyze by immunohistochemistry and molecular investigations the complete pathway of processing (proteasome and immunoproteasome), transport (TAP1 and TAP2)
and assembly (chaperone molecule) of MHC class I antigens in
melanoma progression.
Superficial atypical melanocytic proliferations
of uncertain significate (SAMPUS) and
melanocytic tumours of uncertain malignant
potential (MELTUMP): theory and practice in the
consultation of experienced dermatopathologist
D. Morichetti, T. Pusiol, M.G. Zorzi, F. Piscioli
Institute of Anatomic Pathology, Rovereto Hospital, Italy
Introduction. Various definitions of superficial atypical melanocytic proliferations of uncertain significate (SAMPUS) and
melanocytic tumours of uncertain malignant potential (MELTUMP), has been reported. These lesions evoke a distressing divergence of histological interpretation. Second opinion consultations
on difficulties cases are an important part of pathology practice
1
. We report our experience regarding the opinion consultations
of SAMPUS and MELTUMP performed by Prof. David E. Elder
and Prof. George Murphy.
Material and methods. Prof. Murphy has diagnosed four cases
of MELTUMP, while Prof. Elder. two cases of SAMPUS and one
case of MELTUMP, using only Hematoxilin-Eosin (H&E). Prof.
Murphy has used in one case HMB45 and MIB-1 that resulted
negative.
Results. In the diagnostic report of the Prof. Murphy, the lesions
where diagnosed as: “lentiginous compound dysplastic nevus with
severe atypia of both the intraepidermal and dermal components”,
“atypical epithelioid cell compound melanocytic proliferation,
most consistent with epithelioid cell/Schwannian transformation
in a lentiginous compound nevus”, “Severely atypical compound
epithelioid and spindle cell melanocytic proliferation”, “Severely
atypical combined melanocytic proliferation with Spitzoid features (MELTUMP)” and refered in 3 cases in the comment/recommendation as MELTUMP. In the diagnostic report of the Prof.
Elder, the lesions where diagnosed as: “SAMPUS, cannot rule out
a superficial invasive melanoma”, “SAMPUS” and “MELTUMP,
favour an atypical deep penetrating nevus but cannot rule out an
unusual spindle cell melanoma”.
Discussion. Various atypical, ambiguous melanocytic lesions
have been defined by Barnhill et al.1: Spitz tumor with atypical
features (atypical Spitz tumor), Spitz nevus/tumor with atypical
features and indeterminate biologic potential, Blue nevi with
atypical features, Blue nevus-like melanocytic neoplasms with
indeterminate biologic potential. The pathologist is not able to
predict with certainty
the disease outcome, because the histological characteristics are
“atypical” or “ambiguous” and not let the categorization of the
lesion as benignant or malignant. In the realm of melanocytic
141
neoplasia, Ackerman accepted only 3 diagnoses: melanoma,
nevus and “I don’t know”. With (H&E) and immunohistochemistry, the pathologist makes only a morphological diagnosis and
can not formulate an opinion about the biological outcome of the
lesion. Fluorescence in situ hybridization (FISH) has emerged as
preferred molecular technique to interrogate chromosomal abnormalities. Gerami and Zembowicz 2 believe that FISH is not a standalone test and must be interpreted in conjunction with evaluation
of routine sections. Various Authors 1 3 believe that the difficulty
in correctly classifying these cases as “benign” or “malignant”
reflects an inherent biologic problem, namely, the fact that they
probably represent a spectrum or group of one or more low-grade
melanocytic tumors with potential for lymph node involvement
and rarely for distant metastases. These Authors believe that
problems in diagnosis did not result from insufficient discriminatory power, from inadequate criteria for histopathologic diagnosis, but that their diagnosis in dubious cases reflected exactly the
biologic nature of the neoplasms in question. It is evident that
the diagnosis of MELTUMP or SAMPUS may be performed
only after molecular studies. In our experience the diagnosis of
MELTUMP or SAMPUS of Prof. Elder and Prof. Murphy were
performed only with histologic criteria used routinely for the
distinction of benign melanocytic from melanoma. Molecular
studies were not made and these pathologist have included in the
MELTUMP or SAMPUS atypical or ambiguous melanocytic lesions and melanomas.
References
1
Barnhill RL, Cerroni L, Cook M, et al. State of the art, nomenclature,
and points of consensus and controversy concerning benign melanocytic lesions: outcome of an international workshop. Adv Anat Pathol
2010;17:73-90.
2
Gerami P, Zembowicz A. Update on fluorescence in situ hybridization
in melanoma: state of the art. Arch Pathol Lab Med 2011;135:830-7.
3
Cerroni L, Barnhill R, Elder D, et al. Melanocytic tumors of uncertain
malignant potential: results of a tutorial held at the XXIX Symposium
of the International Society of Dermatopathology in Graz, October
2008. Am J Surg Pathol 2010;34:314-26.
Analysis of the staminal phenotype in cutaneous
melanomas
M. Siano, G. Ilardi, M. Mascolo, M.L. Vecchione M., P. Cascone,
G. De Rosa, S. Staibano
Department of Biomorphological and Functional Sciences, Pathology
Section, University “Federico II”, Naples
Cutaneous melanoma (CM) is the most lethal skin malignancy,
with a constant increase in incidence. These tumors show a biological behavior unpredictable by the classical parameters Moreover, conventional therapies are ineffective to treat advanced
stage case. Therefore, the identification of molecular alterations
involved in their biological aggressiveness represents a major
challenge for researchers. Accumulating evidence supports the
involvement of cancer stem cells (CSC) in initiation, progression,
chemoresistance and therapeutic failure of malignant melanoma,
and the aggressive subsets of melanoma cells have been frequently associated with molecular markers shared by stem cells.
The aim of this study was to evaluate, in a selected series of primary CM, with corresponding metastases, the expression of stem
cell markers, correlating the results to clinical and follow-up data.
Results were also correlated with the expression of CAF-1/p60
and PARP-1, on the same CM cases.
Were selected formalin-fixed, paraffinized blocks of 89 primary
CM, of which 20 with brain metastases, from the archive files of
the Department of Biomorphological and Functional Sciences,
Pathology Section, University Federico II of Naples, relative to
cases examined between January 1985 and December 2009. All
the cases were tested for anti-CD166, anti-nestin, anti-CD133,
anti-CD44 and anti-CD44v6, by conventional immunohisto-
142
chemical technique. Our results suggested a link between the expression of stem markers and the outcome of melanoma patients,
being expressed strongly in the subgroup of patients with a worse
prognosis and brain metastases. The aggressive cases of CM coexpressed also CAF1/p60 and PARP1 proteins, this suggesting
a potential role of all these molecules for the prognostic evaluation of melanoma as well as possible targets of new molecular
therapy.
In conclusion, we demonstrated the suitability of ScreenCell_
filtration devices for the identification of CTCs and for recovery
of nucleic acids from the isolated tumor cells, with a higher detection rate in comparison with PCR based techniques. Present
results indicate the need for larger prospective studies with a
number of purposes, including whether individual CTCs may
predict the behavior of the entire pool of occult tumor cells and
drug sensitivity of the corresponding tumor tissue.
Evaluation of screen-cell devices for the
detection of circulating tumour cells in metastatic
melanoma patients
Variant “Pyogenic Granuloma-Like” of Kaposi’s
Sarcoma in ACRAL location: our experience
C. Scatena*, F. Salvianti **, P. Pinzani**, V. De Giorgi***, M. Paglierani*, M. Pazzagli**, D. Massi*
*
Dipartimento di Scienze per la Tutela della Salute “G. D’Alessandro”,
Sezione di Anatomia Patologica “Paolo Craxi”, Università degli Studi di
Palermo, Italia
Circulating Tumor Cell (CTC) analysis is a promising new diagnostic field for cancer patients and can be used as a liquid biopsy
approach for prognostic and predictive purposes. Development
and optimization of new technologies to identify and characterize CTCs and to establish their potential clinical significance are
highly relevant. In melanoma patients, clinical applications have
mostly focused on the identification of CTCs by cytometric and
PCR based indirect techniques. PCR is commonly used to amplify trascriptors specific for melanocytes, including tyrosinase,
MART-1 and GP-100, in multimarker assays. We recently validated the use of a direct method for CTC detection in melanoma
patients, called Isolation by Size of Epithelial Tumor cells (ISET).
ISET directly isolates CTCs by filtration of peripheral blood
through polycarbonate membranes with 8 µm pores. ScreenCell_
filtration devices are new methods of CTC isolation based on
blood filtration which enable easy, rapid (filtration of 3 ml of
peripheral blood is usually completed within approximately 2
minutes) and open access to CTCs avoiding the use of any dedicated instrument.
To the best of our knowledge, CTC detection rate by ScreenCell_
filtration devices in melanoma patients is presently unknown.
We applied ScreenCell_ filtration devices (ScreenCell® CY kit
and ScreenCell® Molecular Biology Kit) to isolate and characterize CTCs in peripheral blood of metastatic melanoma patients.
The results were compared to those obtained by ISET and by a
reverse transcription polymerase chain reaction (RT-PCR) assay for the detection of specific melanoma molecular markers.
Moreover DNA, extracted from Screen Cell_ positive filters was
investigated for the presence of BRAFV600E mutation.
Peripheral blood from 10 metastatic melanoma patients was filtered according to the manufacturer’s instructions. Hematoxylin
and eosin stain of isolated CTCs showed that cell morphology
is retained and that CTCs are characterized by large size, high
nucleus/cytoplasmic ratio and irregular nuclear shape. The
melanocytic nature of CTCs in selected cases was confirmed
by immunocytological staining with S100 protein, HMB45 and
CD45. For RT-PCR assay, we proceeded with cell isolation,
whereby tumor cells were enriched by an antibody-mix linked to
magnetic particles and mRNA isolated from the selected tumor
cells. Secondly, molecular biological detection and analysis was
performed whereby the isolated mRNA was transcribed into cDNA and a multiplex PCR was carried out for the analysis of tumor
associated gene expression. A complete concordance in terms of
CTC detection rate was found between the two filtration methods
and CTCs were isolated in 8/10 (80%) patients. RT-PCR assay
showed a lower sensitivity in CTC detection. By ScreenCell®
Molecular Biology Kit, we demonstrated the ability of the system
to recover the nucleic acids from the isolated tumor cells for subsequent BRAFV600E evaluation by a real-time PCR method.
A new entity showing both features of pyogenic granuloma (PG)
and of Kaposi’s sarcoma (KS) was initially named ‘Kaposi’s
sarcoma–like pyogenic granuloma’ (KS-l PG) and considered
benign 1. Due to the clinical course and the presence of human
herpesvirus-8 (HHV-8) DNA it is now considered a true KS and
therefore renamed ‘pyogenic granuloma–like Kaposi’s sarcoma’
(PG-like KS) 2 3. Like KS, it has been mostly reported on the
lower extremities. We reviewed our casuistry (2005-2011) of 50
acral nodular lesions (10 on the foot and 40 on the hand) clinically and histologically more suggestive of PG and found 6 cases of
PG-like KS (10%), 3 on the foot (3/10= 30%) and 3 on the hand
(2/40= 5%). These 6 cases represent 17% of all acral KS (29)
coming to our observation in the same period, 26 of wich located
on the foot, and, only three located on the hand, noteworthy, the
last ones, all were of the variant PG-like KS. All the patients were
over sixty years of age, human immunodeficiency virus (HIV)
negative men, and presented solitary skin red nodules (diameter
ranging from 0,4 to 2,4 cm).
The morphological features were consistent with a pyogenic
granuloma: exophytic, polipoid-like silhouette, covered by thickened and ulcerated epidermis forming a collarette at the base.
A well circumscribed, lobular proliferation of well-formed
capillaries with a feeder vessel and fibrous septae between the
lobules were clearly evident. Solid areas of spindle cells, typical
of KS, were scanty, obscured by the severe inflammation, edema
and hemorrhage and difficult to detect at low magnification on
haematoxylin–eosin stain. These areas lacked a true sarcomatous
appearance, showing very mild atypia and less than 1 mitosis
×10HPF.
Immunohistochemistry is useful in the differential diagnosis
between PG and KS, because in PG it highlights the presence of
a dual cell population, consisting of perycites, positive for SMA
and the mature endothelial cells forming vessels, positive for
FVIII, CD34 and CD31. On the other hand, in the KS, the solid
spindle cells areas are positive for CD31 and CD34 and typically
negative for FVIII and SMA, due to the absence of both mature
endothelial cells and pericytes. In our cases, we found the scanty
spindle cells areas expressing SMA and FVIII, like PG, and
CD31 and CD34, like KS, probably because they consisting of
immature endothelial cells not forming vessels.
Immunostaining with anti-latent nuclear antigen 1 (LNA-1) for
HHV-8 (Novocastra) demonstrated intranuclear labeling confined to spindle cells, like nodular stage of KS.
Nested polymerase chain reaction, assayed in duplicate on paraffin-embedded samples, showed the presence of HHV-8 DNA in
all the specimens.
Our report could be of interest because only few previous reports
are present in the literature and because in case of PG-like lesions
with sindle-cell areas showing only scanty positivity for CD31/
CD34/FVIII/SMA, HHV-8 detection should be always performed, independently from the location. This is particularly true
for lesions on the hand, that, being an unusual location of true KS,
Sezione di Anatomia Patologica, Dipartimento di Area Critica MedicoChirurgica; **Dipartimento di Fisiopatologia Clinica; ***Sezione di Dermatologia Clinica, Preventiva e Oncologica, Dipartimento di Area Critica
Medico-Chirurgica, Università di Firenze, Firenze, Italia
O. Schillaci, B. Belmonte, C. Guarnotta, V.Franco, D. Cabibi
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comunicazioni orali
increases the diagnostic challenge. We think that PG-like KS is
an underdiagnosed entity, mainly when localized on uncommon
sites and a follow-up could be advisable, as a true KS.
References
1
Fukunaga M. Kaposi’s sarcoma-like pyogenic granuloma. Histopathology 2000;37:192-3.
2
Ryan P, Aarons S, Murray D, et al. Human herpesvirus 8 (HHV-8) detected in two patients with Kaposi’s sarcoma-like pyogenicgranuloma.
J Clin Pathol 2002;55:619-22.
3
Urquhart JL, Uzieblo A, Kohler S. Detection of HHV-8 in pyogenic
granuloma-like kaposi sarcoma. Am J Dermatopathol 2006; 28:31721.
Patologia Infettiva
encephalitis and aracnoiditis have been detected as well and several cryptococcal yeasts have been found within hepatic, renal e
myocardial vessels. The patient’s blood has been tested for HIV
antibodies and found to be reactive.
Thus, a diagnosis of cryptococcal setticemia in HIV+ patient was
posed. The diagnostic aid given in the present case by the quite
asymptomatic, rapid fatal course of a disseminated cryptococcal
infection needs to be underlined.
References
1
Satish S, Rajesh R, Shashikala S, et al. Cryptococcal sepsis in small
vessel vasculitis. Indian J Nephrol 2010;20:159-61.
2
Taniguchi T, Ogawa Y, Kasai D, et al. Three cases of fungemia in
HIV-infected patients diagnosed through the use of mycobacterial
blood culture bottles. Intern Med 2010;49:2179-83.
A case of systemic cryptococcosis
Sudden death for a disseminated cryptococcal
infection: a case report
G. Abbona*, D. Bellis*, L. Viberti*.
L. Sollima , G. Calvisi , I. Cicchinelli , G. Crisman , P. Leocata*
Cryptococcosis is one of the most common fungal infections; the
fungus is present in our surroundings, and is particularly common
in bird feces. It is generally accepted that cryptococcal infection
is acquired through the inhalation of dust particles. The lung
is the primary route of infection with subsequent spread of the
fungi to the other organs via the haematogenous route. Although
the yeast can invade apparently immunocompetent hosts, predisposed patients are more frequently infected. Cryptococcosis is an
important cause of morbidity and death in immunocompromised
patients. Currently, AIDS is the predisposing factor in approximately 90% of cryptococcal infections; in patients with lymphoma Cryptococcus is the fourth most common death-causing
agent. Nonetheless reports of cryptococcosis are rare in patients
with hematologic malignancies due to diagnostic difficulties and
low frequency of the infection.
We report a case of disseminated cryptococcosis diagnosed at
autopsy.
The patient, a 61-year-old man with refractory anemia with excess blasts, was admitted to our hospital with dispnea and only
a small amount of sputum production; it was initially thought to
have disease progression with blastic transformation and pulmonary thromboembolism. Chest X-ray revealed pulmonary interstitial non specific infiltrates, most evident in right lower lobe.
On sputum cytology the presence of sporadic fungal spores in
alveolar macrophages was observed and a diagnosis of suspected
fungal infection was performed. The patient died of respiratory
failure five days later regardless of therapies.
Autopsy revealed diffuse consolidation and marked congestion
of the lungs; the spleen weighted 1070 g and showed diffuse
areas of necrosis on cut surface. Histologically, the lungs showed
diffuse areas of necrosis lacking granulomatous reactions; sheets
of fungal organisms were present within necrotic areas and the
alveolar spaces. The yeast cells appeared pale-blue, round to oval
in H-E stained sections and were best demonstrated with silver
stain and PAS. Similar lesions were found in the spleen, lymph
nodes, bone marrow, liver and kidneys.
Autopsy studies of cryptococcosis are very rare. In the autopsy
study of 13 cases reported by Benesova, only two cases were
diagnosed ante mortem. This may be taken as a proof that the
clinical diagnosis of cryptococcosis is difficult. The false-negative
diagnoses of cryptococcosis may be explained by its low frequency
as well as by limited knowledge and non specific symptomatology
of this infection. Disseminated cryptococcosis is invariably fatal if
untreated and the prognosis remains poor even when treated.
Antigen detection of capsular antigens in body fluids by latex
agglutination is the first and most sensitive diagnostic method
in patients suspected of having cryptococcal infection. Cytology
is a rapid test for pulmonary cryptococcosis, though small fungi
*
**
*
*
Anatomia Patologica, Dipartimento di Scienze della Salute, Università
degli Studi dell’Aquila, L’Aquila, Italia; ** Unità Operativa di Anatomia
Patologica, Ospedale Civile “San Salvatore”, L’Aquila, Italia
*
Cryptococcus neoformans and C. gattii are basidiomycetous fungal pathogens that represent the most common cause of meningoencephalitis in immunocompromised hosts, which results fatal
without treatment. The incidence of cryptococcal meningitis has
recently increased. According to the literature, some cases of
cryptococcal meningitis in immunocompetent patients have been
reported, due to corticosteroid therapy, sarcoidosis, organ transplant and chemotherapy related immunosuppression, even though
in some cases any cause could be identified.
We report on a case of a 29-year-old male presented to the
neurology department of “San Salvatore” Hospital of L’Aquila
(L’Aquila, Italy) complaining monolateral strength’s deficit. He
referred a previous recovery in another hospital two weeks before due to a low-grade fever, intermittent, lasting 20 days. The
patient referred any history of head trauma, seizures, earache, nor
any weight loss, chronic cough history of tuberculosis or other
malignancy.
On examination, neck rigidity and Kernig’s sign were negative.
Examination of other systems was unremarkable. Laboratory investigations resulted normal as well as serum electrolytes, renal
function tests, and liver function tests. Immunoglobulin levels
(IgG, IgA, IgM), complement levels, CD3 and CD4 cell counts
were found to be within normal limits.
A MRI investigation highlighted several multifocal pointed lesions of the semioval centers and a diagnosis of a chronic cerebral
vasculopathy was posed.
Few hours later, the patient’s health’s conditions worsened for
an acute and severe dyspnea. High D-dimer levels have been
detected and a computerized tomography (CT) scan revealed a
pulmonary embolism of the right pulmonary artery and several
emphysematous bullae.
Subsequently, the patient developed cough, tachycardia, hypertension and ingravescent dyspnea; there was no evidence of
meningitis. The patient expired on the 36th hours from the recovery and a postmortem examination was performed in the aim to
determinate the cause of the sudden death.
Two interesting findings were found at the gross investigation:
irregular cysts, lined by brownish walls, of the upper pulmonary
lobes bilaterally and pulmonary edema, and a severe meningeal
congestion with cerebral edema.
Interestingly, histological examination of all organs’ tissues
revealed a disseminated cryptococcal infection, involving lungs
and ilar lymph nodes bilaterally. Multiple foci of cryptococcal
S.C. Anatomia Patologica - Ospedale Martini-Valdese ASL TO1 Torino
*
144
may be overlooked during routine microscopic screening unless
a large number of organisms are present or there is a clinical suspicion of infection. Staining of specimen with PAS or Silver stain
may increase its sensitivity. Definitive diagnosis requires specific
histopathological examination and positive culture detection. Combined therapy consisting of amphotericin B with flucytosine is recommended as first line therapy for disseminated cryptococcosis.
References
1
Benesova P, Buchta V, Cerman J, et al. Cryptooccosis: A review of
13 autopsy cases from a 64-year period in a large hospital. APMIS
2007;115:177-83.
2
Pagano L, Fianchi L, Caramatti C, et al. Cryptococcosis in patients
with hematologic malignancies. A report from GIMEMA infection
program. Haematologica 2004;89:852-6.
Epidemiology and neuropathology of human
prion diseases in Piemonte and Valle d’Aosta
districts from 2002 to 2010
S. Taraglio*, D. Imperiale**, C. Buffa**, R. Testi***, G. Natale****
*
SC Anatomia Patologica, Ospedale Maria Vittoria di Torino ** SC Neurologia, ***. SC Medicina Legale, ****. SC Lab. Analisi, Ospedale Maria
Vittoria di Torino. Centro Interdipartimentale DOMP (Diagnosi Osservazione Malattie da prioni) ASLTO** Torino
Aims. 1) To analyse the epidemiology of human prion diseases
in Piemonte and Valle d’Aosta from 2002 to 2010. 2) To classify
the prevalent cases according to the clinico-pathological criteria
of Parchi and Gambetti.
Methods. Clinical, laboratory and autopsy findings of patients
with suspect prion disease in 2002-2010 years were retrospectively evaluated.
Results. A diagnosis of definite/probable of prion disease was
made in 92 cases. Pathological confirmation was available in 76
of 92 cases (82.6%). The annual mortality rate (CI 95%) for all
prion diseases per million of inhabitants was 2.13 (0.79-3.45) in
2002, 0.83 (0.01-1.65) in 2003, 1.67 (0.50-2.84) in 2004 and 2.88
(1.35-4.41) in 2005, 2.08 (0.77-3.39) in 2006, 1,85 (1.35-4.41)
in 2007, 2,70 (1.21-4.19) in 2008, 3.27 (1.65-4.90) in 2009, 1.66
(0.49-2.83) in 2010. Sporadic Creutzfeldt-Jakob disease (CJD)
was diagnosed in 71 patients while sporadic fatal insomnia in 2
ones. No cases of iatrogenic or variant CJD was reported.
Discussion. Since 2002, all autopsies and laboratory investigations in human prion diseases in Piemonte and Valle d’Aosta
have been unified in an unique reference center. The observed
mortality rates in 2002-2010 paralleled the rates observed in the
whole Italy.
Conclusions. The unification of autopsies and specific laboratory investigations in one reference center may be considered an
efficient model to improve case ascertainment and active surveillance on human prion diseases.
Patologia tiroidea
Braf mutation and RASSF1a expression in thyroid
carcinoma: a genetic and epigenetic study
G. Pannone*, A. Santoro*, R. Franco***, G. Botti***, S. Cagiano*,
G. De Rosa**, P. Bufo*
Department of Surgical Sciences, Section of Anatomic Pathology and Cytopathology, University of Foggia, Foggia, Italy; **Dipartimento di Scienze
Biomorfologiche e Funzionali, Sezione di Anatomia Patologica, Università
degli Studi di Napoli ‘Federico II’, Napoli, Italy; ***Istituto Nazionale per lo
studio e la cura dei tumori. Fondazione ‘G Pascale’, Napoli, Italy
*
Introduction. The role of aberrant tumor suppressor gene in the
biology of thyroid cancer has not been well documented. Aim
of this work is to to provide a detailed comparison of clinical-
pathologic features between thyroid tumors according to their
BRAF and RASSF1A status.
Materials and methods. We analyzed RASSF1A methylation by
MSP and BRAF mutation by MASA in neoplastic thyroid tissues
and in respective controls of non-neoplastic thyroidal parenchyma. Immunohistochemical evaluation of RASSF1A expression
was also performed by standard LSAB-HRP technique.
Results. An overall higher degree of RASSF1A overexpression
than normal peritumoral thyroid parenchyma (p<0.05) has been
found in all neoplasias. Moreover, higher levels of RASSF1A
expression were observed in more differentiated cancers, also associated to an inflammatory background (p<0.05).
Amplifiable DNA was obtained only in 9 cases of PTC and
in 1 control. The V600E mutation was found in 66% tumors.
BRAF was mutated in 4 of 6 (66%) classical PTC, and in 2 of
6 (33%) follicular variant PTC. According to TNM stage, we
found V600E mutation more frequently (66%) in early cancer.
The MSP analysis has shown that the epigenetic methylation for
RASSF1A have not a carcinogetic role in our study cases.
Discussion.We showed that there was generally good agreement
between absence of RASSF1A methylation status and RASSF1A
protein over-expression. Finally, by supporting the importance
of the BRAF pathway alterations in papillary thyroid carcinoma
pathogenesis, our study confirms that BRAF mutation do not
coexist with RASSF1A epigenetic alterations in any of the papillary cancers.
Class III β-Tubulin and cell-cell adhesion protein
expression in papillary thyroid carcinoma: a
preliminary report
C. Colato*, A. Parisi*, P. Brazzarola**, M. Ferdeghini*, M.
Chilosi*
*Department of Pathology and Diagnostic, University of Verona, Verona,
Italy; **Department of Surgery, University of Verona, Verona, Italy.
Background. Tubulin, the major component of microtubules,
is a multi-functional protein involved in many essential cellular roles, including cell movement, intracellular transport
and mitosis.
Class III β-tubulin (TUBB3) is expressed in neural tissue and
in neuroendocrine cells and also in several human malignancies, including ovary, breast, prostate, and non-small-cell lung
carcinomas. Over-expression of TUBB3 in these tumours is
associated with an unfavourable outcome and resistance to
taxane-based therapies. In thyroid tissue, TUBB3 immunostaining remains relatively uncharacterised.
Claudins (CLDNs), a family of tight junction proteins, play a
role in adhesion, cell proliferation, and tumorigenesis. Recently,
CLDN1 was found to be up-regulated in papillary thyroid carcinoma (PTC), both at the gene and protein level. CLDN7 is also
expressed in the thyroid, both during embryonic development
and in the adult, and its expression is modulated in thyroid
cancer.
E-cadherin (E-CD) is a homophilic cell surface adhesion protein
that plays a critical role in the establishment of cell polarity and
maintenance of the epithelial phenotype. In PTC, reduced E-CD
expression has been associated with a poorer outcome.
Aim. To test TUBB3 protein staining in various thyroid neoplasms and in the normal thyroid tissue in an attempt to clarify
the role of TUBB3 in thyroid gland, comparing its expression
pattern with that of molecules with cell-cell adhesive role.
Methods. The study included 40 papillary thyroid carcinomas
(PTC), 7 follicular adenomas and 5 nodular hyperplasias. Immunohistochemical analysis was performed using a panel of
monoclonal (TUBB3; CLDN7; E-CD) and polyclonal (CLDN1)
antibodies.
Results. In the normal thyroid, TUBB3 immunoreactivity was
detected both in the nerve fibres and C-cells, but not in the
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follicular epithelium. Moreover, no positivity was observed in
nodular goiters and follicular adenomas. In the PTC samples,
the reactivity was heterogeneous and demonstrated strong cytoplasmic staining in widely infiltrating PTC associated with
fibrous stroma, particularly at the invasive front of the tumour,
or in moderately differentiated PTC with loss of cellular polarity/cohesiveness. In these areas the CLDN1 and 7 expression
were decreased or less intense in comparison with the center of
the neoplasia. Decreased E-CD staining was also observed at
the invasive front of the tumour.
In contrast, the encapsulated variant PTC or PTC with well developed papilla or follicles were constantly negative.
Conclusions. We report for the first time TUBB3 expression in
thyroid tissue.
In analogy with various carcinomas of other sites, TUBB3
expression appears to be increased in PTC with “aggressive”
histological features, thus suggesting a possible role of this
cytoskeleton protein in the invasive activity and metastatic
potential of cancer cells. At the molecular level, the epithelialto-mesenchymal transition involves the loss of the expression
of structural adhesion proteins and the gain of mesenchymal
markers.
The decreased CLDN1 and 7 and E-CD expression in combination with the TUBB3 positivity, at the invasive front of
the tumour, could be one of the morphological indicators of
epithelial-to-mesenchymal-transition in PTC.
Further investigations are needed to determine whether our findings may have clinical implications.
FNA cytology of multicentric papillary oncocytic
neoplasm (EPON) of thyroid with histological,
immunohistochemical and molecular correlations
C. Bellevicine, V. Varone, U. Malapelle, G. Pettinato, G. Troncone
Dipartimento di Scienze Biomorfologiche e Funzionali, Università Federico II, Naples, Italy
Background. Encapsulated Papillary Oncocytic Neoplasm of the
Thyroid (EPONT), is a benign papillary growth of oncocytic cells
lacking papillary thyroid cancer (PTC) nuclear features 1.
Case. A 41-year old woman presented with a long standing
multinodular goiter. Ultrasound (US) highlighted three nodules
with two nodules clinically evident; one was in in the right lobe
and the other was paraisthmic. Both nodules were aspirated. In
both samples, the smears showed high cellularity; this was almost completely composed by oncocytic (Hurthle) cells. These
were mostly arranged in papillary groups with a clear vascular
core with peripheral cellular palisading. At higher magnification,
the cells showed abundant granular eosinophilic cytoplasm and
appeared to be regularly spaced without crowding and overlapping. The nuclei showed little variations in size and shape, with
a small nucleoli. No PTC nuclear features were observed. A final
diagnosis of Hurthle cell neoplasm with papillary features was
rendered for both nodules and surgical excision was recomended.
Surgical specimen showed on the three nodules EPONT. Immunohistochemistry (CD56+ CK19-) was consistent with this
diagnosis. DNA was extracted from the three nodules tissue and
mutational analysis of BRAF and NRAS oncogenes was carried
out by Sanger Sequencing. Both gene were found wild-type, confirming the diagnosis of benign. Conclusion. To our knowledge this is the first description of a
multicentric EPONT diagnosed by FNA. Care should be taken to
not overdiagnosis FNA from EPONT.
Reference
1
Woodford RL, Nikiforov YE, Hunt JL, et al. Encapsulated papillary oncocytic neoplasms of the thyroid: morphologic, immunohistochemical, and molecular analysis of 18 cases. Am J Surg Pathol
2010;34:1582-90.
Patologia mammaria
CD1a expression in primary breast cancer and
lymph nodes: correlation with clinicopathological
parameters
F. Rappa*, F. Cappello*, G. La Rocca*, R. Anzalone*, S. David*,
S. Corrao*, E. Unti, G. Zummo*, N. Scibetta**
*Dipartimento di Biomedicina Sperimentale e Neuroscienze Cliniche, Sezione Anatomia Umana; **Servizio di Anatomia ed Istologia Patologica,
A.R.N.A.S. Civico, Palermo
CD1a is a molecule belonging to the highly conserved group of
CD1 proteins. Its expression on dendritic cells (DCs) was supposed to be functionally important for presentation of tumourderived glycolipid antigens to T cells, and therefore the development of a successful antitumoral response. We have recently
shown that CD1a may be expressed on the cells of Barrett’s
metaplasia (BM) and its expression may predict its evolution
toward oesophageal adenocarcinoma 1. Interestingly, we showed
that CD1a may be expressed not only by DCs but also by transformed epithelial cells in BM 2.
We now focused our attention on the presence of CD1a positive
cells in both primary tumours and LN metastases of a series of invasive ductal carcinomas. In N0 lesions CD1a is highly expressed
intratumorally, and this result reaches statistical significance if
compared to the low expression of the molecule in N1 primary
lesions (p<0.0001). Moreover, we show for the first time in breast
cancer the presence of epithelial elements expressing CD1a, as
observed in Barrett’s metaplasia. Parallel correlation analyses
allowed us to significantly correlate CD1a expression in primary
tumours to positivity to estrogen (p=0.0025) and progesterone
(p=0.0226) receptors, which are markers of well-differenced
cancers, related to a favourable prognosis. CD1a may exert an
antitumoral role stimulating an immunitary antitumoral response.
The contemporary positivity to CD1a and ER and PR at the
primary tumour level, strengthens the idea of a positive role of
CD1a expression in breast cancer. Moreover, the lack of a high
number of CD1a+ cells in lymph nodes with micrometastases (vs
N0 ones) suggests that the evaluation of this antigen could help
in the detection of micrometastases at nodal level, showing that
the correct immune response is compromised.
146
Invasive breast cancer: a significant correlation
between histological parameters and molecular
types
Identification of new molecolar pathways
associated with “triple negative phenotype”
of breast cancer
A. Caldarella1, S. Bianchi2, E. Crocetti1, V. Vezzosi2, T. Intrieri1,
P. Apicella3, M. Biancalani4, A. Giannini5, C. Urso6, F. Zolfanelli7, E. Paci1
M. Di Bonito, M. Cantile, F. Collina, G. Scognamiglio, M. Cerrone, G. Liguori, G. Botti
Clinical and Descriptive Epidemiology Unit, Institute for Study and Cancer Prevention (ISPO), Florence, Italy; 2Section of Pathological Anatomy,
Department of Medical and Surgical Critical Care, University of Florence,
Florence, Italy; 3Anatomic Pathology, Pistoia Hospital, Italy; 4Pathologic
Anatomy Unit, Empoli Hospital, Italy; 5Anatomic Pathology, Misericordia
e Dolce Hospital, Prato, Italy; 6Anatomic Pathology, S. M. Annunziata
Hospital, Azienda Sanitaria di Firenze, Italy; 7Anatomic Pathology, S.
Giovanni di Dio Hospital, Azienda Sanitaria di Firenze, Italy
1
Background. Breast cancer can be categorized in several ways,
according to histological type or according molecular type based
on expression of tumor markers. The aim of this study was to
evaluate the correlation between hystological type and molecular
subgroups in a population based series of female breast cancer
patients.
Materials and methods. Through the Tuscan Cancer Registry
all histological reports of invasive breast cancer cases diagnosed
during the period 2004-2005 in the provinces of Florence and
Prato, central Italy, were retrieved and information on age at diagnosis, tumor size, lymph node status, histological type, grade of
differentiation, pathological stage, immunohistochemical expression of hormonal receptors and HER2 were collected. Molecular
subtypes were defined by immunohistochemical expression
of hormonal receptors and HER2 as: luminal A subtype (ER/
PR+HER2-), luminal B (ER/PR+HER2+), triple negative (ER/
PR-HER2-), HER2+ (ER/PR-HER2+). Histological type was categorized as ductal, lobular, mixed ductal + lobular, tubular, mucinous, cribriform, papillary, medullary, other types (carcinoma
nos and rare types). According to histological grade, carcinomas
were classified as well, moderately and poorly differentiated
carcinoma. The association of each variable with molecular subgroups was assessed by a chi square test.
Results. Out of 1487 patients with available immunohistochemical results 69.1% were luminal A subtype (ER/PR+HER2-),
16.8% luminal B (ER/PR+HER2+), 7.9% triple negative (ER/
PR-HER2-) and 6.2% HER2+ (ER/PR-HER2+). Invasive ductal
carcinoma NOS was the most frequent histotype (58.5% of total),
followed by lobular (13.9%), mixed (ductal and lobular or invasive ductal carcinoma NOS + other histotype) (12.4%), mucinous
(2.9%), tubular (2.9%), cribriform (2.6%), papillary (1.4%), NOS
+ comedocarcinoma (0.6%) and medullary (0.3%). A statistically significant association of these molecular subgroups with
histological type was found (p<0.000). All tubular, over 70% of
lobular, 60% of ductal and 50% of NOS + comedocarcinomas
were in luminal A group, while 9% of ductal, and 30% of NOS +
comedocarcinomas were in HER2+ group. Almost all medullary
carcinomas were in triple negative subgroups and no tubular and
cribriform histological types were found in HER2+ and triple
negative subgroups. A significant correlation between molecular
type and histological grade was also found (p<0.000): compared
to luminal A cases, women with HER2+ and triple negative cancers tended to have lower grade tumors.
Conclusions. Our data from a population based cancer registry
revealed a significant correlation between histological parameters
(histological type and histological grade) and molecular types in
invasive breast cancers.
SC Anatomia Patologica e Citopatologia, INT Fondazione G. Pascale,
Napoli
Triple-negative (ER-negative, PR negative, HER2/neu not overexpressed) breast cancer has distinct clinical and pathologic
features, and mostly comprise the basal-like molecular subtype
of breast cancer. Since triple-negative breast cancer is resistant
to current HER2-targeted therapies such as trastuzumab, and
hormonal therapies such as tamoxifen and aromatase inhibitors,
chemotherapy is the mainstay of treatment. This lack of targeted
therapies has intensified the interest in this group of patients
and in the research of new molecular signatures tailored to this
specific subtype.
One such pathway is centered on cancer stem cell surface marker
CD133/Prominin 1 linked to more aggressive cellular behaviour,
including resistance to chemotherapy and radiotherapy.
Moreover, previous reports have demonstrated that Geminin, a
nuclear protein that functions by inhibiting DNA replication, and
SPARC/Osteonectin, a matricellular glycoprotein, are frequently
alterated, in vivo, in a variety of human tumors (Kidney, colon,
breast, lung cancer and lymphoma) and their expression rises
with increasing tumor grade, leading to a poor prognosis.
In this study we have built a Tissue MicroArray containing
180 “Triple Negative” breast cancer samples and investigate
the expression of stem cell surface marker CD133/Prominin 1
to identify CSCs, geminin and osteonectin proteins. Gene expression of all three markers was further investigated at mRNA
level for selected tumor types through real-time quantification on fresh-frozen biological samples from our Institutional
BioBank.
Preliminary results show the strong association between all examinated markers and clinicopathological parameters related to
“Triple Negative” breast cancer phenotype.
Breast lumps and fine-needle aspiration cytology:
a 5-year retrospective study on 307 cases
G. Crisman*, F. Marra*, F. Brunelli**, S. Discepoli**, P. Leocata*
Anatomia Patologica, Dipartimento di Scienze della Salute /Università
Patologica/ Ospedale “SS Filippo e Nicola”, Avezzano (AQ), Italia.
*
Fine needle aspiration cytology (FNAC) represents an important
tool in preoperative assessment of breast lumps. High sensitivity,
specificity and accuracy as well as its less invasive, fast and easy
approach with little complications leads this tecnique to achieve a
pivotal role in the initial pathological investigative methods in the
aim to differentiate benign from malignant lesions.
The purpose of this study is to evaluate our experience with
Fine Needle Aspiration Cytology (FNAC) and to correlate histopathologic and cytological FNAC diagnoses of palpable and
non-palpable breast lesions.
We retrospectively analyzed 1063 cytological smears obtained
from breast FNAC over a period of five years (2005–2009),
performed at the Department of Pathology of Avezzano Hospital
(Avezzano, L’Aquila, Italy). The patients ranged in age from 13
to 89 years (mean, 53 years). The aspirations were performed
by cytopathologists, using 22-gauge needles, and subsequently
alcohol-fixed and air-dried smears were prepared. The former
were stained with Hematoxylin and Eosin. The diagnosis from
the cytologic evaluation ranged from inadequate (C1) to cancer
(C5) and a histopathological evaluation was available for 307
cases (28,88%).
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The results of 307 FNACs were reported as benign in 180 cases
(59%), as malignant in 127 (41%).
The histolopathological diagnosis of each nodule confirmed the
diagnosis of benignity in 176 cases and confirmed the diagnosis
of malignancy in 126 cases; thus, only four false negative diagnoses and one false positive case have been detected.
According to the literature, the FNAC sensitivity, specificity
and accuracy in our study are close to 100% being, respectively,
96,92%, 99.44%, and 98,37%. The positive predictive value is
99,26%, the negative predictive value is 97,77%, false-negative
rate is 0.22%, and false-positive rate is 0.78%.
Our results underline once again the reliable value of this tecnique as an accurate preoperative diagnostic procedure for the
evaluation of breast lumps.
FNAC is considered to be a safe method for screening purposes.
Although moderately less sensitive than core needle biopsy,
FNAC is most accurate when experienced cytopathologists are
available to assess the adequacy of the aspirated material and
advise on additional aspirations for ancillary tests when needed.
References
1
Mendoza P, Lacambra M, Tan PH, et al. Fine needle aspiration cytology of the breast: the non malignant categories. Patholog Res Int
2011;2011:547580.
2
Yan L, Qiang S, Wei-Xuan Z. Role of the fine needle aspiration cytology in surgical treatment of breast cancer. Zhongguo Yi Xue Ke Xue
Yuan Xue Bao 2011;33:80-2.
Resection margins in breast-conserving surgery
E. Orvieto, L. Alessandrini, M. Lo Mele, G. Marchelle, E. Privitera, V. Belardinelli, M. Rugge
UOC Anatomia Patologica & Clinica Chirurgica II-Breast Unit Azienda
Ospedaliera - di Padova, Università degli Studi di Padova
Objectives. Margin status is a major issue in conserving surgery
for breast cancer. Cancer-positive margins as assessed in primary
excision specimens frequently do not match with residual cancer
found on re-excision. The pathology of primary cancers was
assessed in a consecutive series of breast cancers with positive
surgical margins to identify parameters capable of predicting
residual tumor in re-excisions.
Methods. Seventy-five consecutive invasive breast cancers treated with the same conserving surgery approach (years 2001-2009)
were considered. In all cases, there was tumor on at least one
resection margin (on the cut edge), so re-excision was performed.
The following morphological features of the primary tumor were
considered: size, multifocal disease (grossly and/or histologically assessed), growth pattern (infiltrative, diffuse and pushing),
linear extension of cancer on resection margins (> 5 versus <5
mm), cancer histotype and peritumoral vascular invasion (present
versus absent). The prevalence of an intraepithelial component
(DCIS) in the primary cancers was also considered and scored
as: 1 (DCIS: <5%); 2 (DCIS:6%- 25%); 3 (DCIS:26%- 75%);4
(DCIS >76%).
Results. Primary tumors were a median 1.8 cm in size (range=
0.2-10). Invasive ductal carcinomas prevailed (74%) over the
other histotypes, though the prevalence of the lobular histotype
(26%) was higher than generally reported in breast cancer populations (10-15% of cases). Residual cancer was found in 62.5%
of re-excision specimens, and invasive cancer significantly prevailed over the intraepithelial histotypes (63.8% versus 36.2%).
Residual tumor was associated more frequently with lobular than
with ductal carcinoma (75% versus 60%; p= NS). The cancer’s
linear extension along the resection margins (cut-off = 5 mm)
did not predict residual tumor (66% of cases >5 mm versus 58%
<5mm). Eleven (85%) of 18 cases of multifocal cancer featured
residual tumor in the re-excision specimen (p=0.067). Pushing,
infiltrative, or diffuse growth patterns were seen in 34/75 (45%),
33/75 (44%), and 8/75 (11%) cases, respectively. When infiltra-
tive and diffuse cases were grouped together, residual tumor
re-excision specimen was documented in 30/41 cases (71%;
p=0.047). The score for DCIS coexisting with an invasive component correlated significantly with residual tumor (p=0.014).
At multivariate analysis, an infiltrative/diffuse growth pattern
(OR 4.48; 95%CI= 1.10-18.18; p=0.036) and an extensive in situ
component (score 3-4) (OR 2.00; 95%CI= 1.17-3.41; p=0,011)
both correlated significantly with the presence of residual tumor
in the re-excision specimen.
Conclusions. In conserving surgery for breast cancer, positive
margins warrant surgical re-excision. Infiltrative/diffuse growth
pattern, multifocal cancer, lobular histotype and an extensive
intraepithelial component correlate significantly with residual
tumor.
Interregional fish her2 quality control on breast
cancer: three years experience
L. Verdun di Cantogno1, P. Gugliotta1 2, C. Botta1 2, S. Vigna1 2, A.
Andreozzi3, L. Baron4, L. Casorzo5, G. De Maglio6, M. Flora7, A.
Gianatti8, E. Leonardi9, C. Lagrasta10, C. Lo Cunsolo11, M. Paglierani12, L. Pecciarini13, S. Salvi14, A. Santinelli15, G. Tallini16, A.
Zangrandi17, L. Zanatta18, A. Sapino1
Anatomia Patologica III, Az. Osp. Univ. San Giovanni Battista di Torino,
Italia; 2 Az. Osp. Univ. San Giovanni Battista di Torino, Dip. di Scienze
Biomediche ed Oncologia Umana, Università di Torino, Italia; 3 Anatomia
Patologica, Osp.S. Luigi Gonzaga, Torino, Italia; 4 Anatomia Patologica,
Osp.S. Leonardo ASL-NA3sud, Castellammare di Stabia, Napoli, Italia; 5
Laboratorio Citogenetica-Servizio Anatomia Patologica IRCC Candiolo,
Torino, Italia; 6 Anatomia Patologica, Az. Osp. Univ. S M della Misericordia, Udine, Italia; 7 Anatomia Patologica, Dip Oncologico Osp. S. Maria
Nuova, Reggio Emilia, Italia; 8 Anatomia Patologica, Az. Osp. Ospedali
Riuniti, Bergamo, Italia; 9 S.S. Patologia Molecolare, Anatomia Patologica/Osp.S. Chiara, Trento, Italia; 10 Dipartimento di Patologia e Medicina
di Laboratorio, Sez. di Anatomia ed Istologia Patologica, Parma, Italia;
11
Lab.Citogenetica-Serv. Anatomia Patologica, Osp.S. Martino, Belluno,
Italia; 12 Dipartimento di Patologia Umana e Oncologia, Laboratorio di
Immunoistochimica, Az. Osp. Univ. Careggi, Firenze, Italia;13 Anatomia
Patologica-DIBIT2, Osp. S. Raffaele di Milano, Italia; 14 IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italia; 15 Anatomia Patologica,
Az. Osp. Univ. Ospedali Riuniti, Ancona, Italia; 16 Università di Bologna
Anatomia Patologica Ospedale Bellaria, Bologna, Italia; 17 Anatomia
Patologica, Osp. Guglielmo da Saliceto di Piacenza, Italia; 18 Anatomia
Patologica, Osp. Generale di Treviso, Italia
1
Background. Quality Controls (QC) of HER2 FISH testing
(FISH) on breast carcinoma are more complicated to be performed than QC for immunocytochemical procedures due to the
fading of the immunofluorescent test.
Aim. Desire of comparing and evaluating the accuracy of the preanalytical (fixation, embedding and sectioning) and of the analytical
(performance and interpretation) phases of the FISH test for HER2
in breast cancer in different Pathology laboratories in Italy.
Material and methods. The FISH QC originates in March 2008
from the Quality Control of the Regione Piemonte for the Prognostic and Predictive Factors of breast cancer. The centers initially participating were 9 and increased to 18 during 14 months and
now participants are divided in two groups A and B of 9 centers.
The project is designed as a “Ring Quality Control study” so that
once a month, the assigned center (on a rotating base) sends to all
the other participating centers two slides of formalin fixed paraffin embedded sections of breast cancer. With this approach, every
center contributes equally without overloading of a single center
for preparing the slides. Each center performs the FISH testing
following its own method and reagents. The results obtained and
their interpretation are inserted in the website created appositely.
Results. Up to now 35 cases have been analyzed. The results obtained have been evaluated in terms of assessment of CEP17 and
HER2 gene number, final interpretation of the HER2 gene status
and evaluation of the quality of the section (fixation, cutting). The
148
diagnosis given by the majority of the centers was considered as the
gold standard. In particular, the diagnosis (amplified, not amplified,
polysomy) reflected the evolution of the interpretation of HER2
FISH testing on breast carcinoma. The first 18 cases performed
during 2008/2009 showed 79% of concordance. The second group
of cases (2009/2010) reflected the difficulties of this period of transition on the interpretation of polysomy; the agreement decreased
to 38%. In the third group of cases (2010/2011), were 2 subgroups
of centers were created and after the adoption of the guidelines
originated from the Consensus Conference AIOM/SIAPEC Catania 2010, the percentage of concordance increased again 77% for
subgroup A and 67 % for subgroup B.
In conclusion, this experience reflects the interest of the participants for an inter-laboratory sharing of experience on FISH. The
work load for the participating centers is manageable and the
objectives have been reached. The need of QC FISH is demonstrated by the good, but not perfect concordance of the results,
that may depend more from the biology of tumor that from the
technical performance of the centers. We hope therefore that this
experience will be extended to set up interregional QC FISH
networks for other solid tumors.
External quality control assessment (EQC) study
for the immunohistochemical determination of
HER2 in breast cancer: an experience on regional
scale
I. Terrenato1, S. Pizzamiglio4, L. Perracchio2, L. Costarelli5, E.
Bonanno6, V. Arena7, S. Buglioni2, D. Baldini8, S. Candia9, A.
Crescenzi10, A. Dal Mas11, C. Di Cristofano12, V. Gomes13, L.R.
Grillo14, P. Pasquini15, M.N. Pericoli16, M.T. Ramieri17, L. Ruco18,
S. Scarpino18, D. Vitolo19, G. D’Amati19, P. Muti3, A. Paradiso20,
P. Verderio4, M. Mottolese2
S.C. Epidemiologia, 2Anatomia e Istologia Patologica e Citodiagnostica,
Direzione Scientifica Istituto Nazionale Tumori Regina Elena, Roma, Italia; 4S.C. Statistica medica, biometria e bioinformatica, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italia; 5Anatomia e Istologia
Patologica, Ospedale San Giovanni Addolorata, Roma, Italia; 6Anatomia
Patologica, Università Tor Vergata, Roma, Italia; 7Anatomia Patologica
Macroscopica, Policlinico Universitario Gemelli, Roma, Italia; 8Anatomia Patologica, ACO San Filippo Neri, Roma, Italia; 9 Anatomia Patologica, Ospedale Sandro Pertini, Roma, Italia; 10Anatomia Patologica,
Ospedale Regina Apostolorum, Albano Laziale, Roma, Italia; 11Anatomia
Patologica, Ospedale San Salvatore, L’Aquila, Italia; 12Anatomia Patologica, Università La Sapienza Polo Pontino ICOT, Latina, Italia; 13Anatomia Patologica, ASL Viterbo, Italia; 14Anatomia Patologica, Ospedale
San Camillo Forlanini, Roma, Italia; 15Anatomia Patologica, Ospedale
Militare Celio, Roma, Italia; 16Anatomia Patologica, Ospedale Santa Maria Goretti, Latina, Italia; 17Anatomia Patologica, ASL Frosinone, Italia;
18
Anatomia Patologica, Azienda Ospedaliera Sant’Andrea, Roma, Italia;
19
Anatomia Patologica, Policlinico Umberto I, Roma, Italia; 20Direzione
Scientifica, Istituto Nazionale Tumori Giovanni Paolo II, Bari, Italia.
1
3
An accurate assessment of HER2 status in Breast Cancer (BC)
is of paramount importance to establish patient eligibility for
HER2-tailored therapies. Nevertheless, up to now, about 20% of
current HER2 testing may be inaccurate. Within the framework of
the Italian network for Quality Assessment of Tumor biomarkers
(INQAT), an External Quality Assessment (EQA) program was
developed to investigate the state of the art of immunohistochemical
(IHC) HER2 determination within 16 Pathology Departments, defined as Participating Center (PC), in the Lazio region. A two-phase
study was conducted, after defining a strict protocol, aimed to evaluate both the staining and interpretative reproducibility of IHC HER2
determination. The Regina Elena Cancer Institute (Rome) was the
Coordinating Center (CC) and one of the four Reviser Centers (RC)
that selected the cases and defined the reference distributions of
HER2 IHC score for both phases (reference scores).
In the first phase, (EQA HER2 immunostaining), four BC paraffin embedded slides were distributed to each PC who had to stain
each specimen using their own procedures, previously reported
in a dedicated questionnaire. In the second phase (EQA HER2 interpretation) thirty slides, chosen by RC and stained by CC, were
codified and rotated, in set of ten, among the 16 PC which had
been randomly divided into three groups. Each PC had to report,
for each of the ten received slide, the qualitative evaluation of
membrane staining using the ASCO-CAP score (0, 1+, 2+, 3+).
In the EQA HER2 immunostaining phase, the agreement was
evaluated within each scoring category by comparing all the
scores obtained on the slides stained by each PC with the
reference scores. To this end the unweigheted Kappa statistic (K),together with its Jackknife 95% Confidence Interval
(95%CI), was calculated.
In the EQA HER2 interpretation phase, for each PC in addition to
the agreement within each scoring category versus the reference
values, it was also evaluated the overall agreement versus the
reference values by means of the weighted Kappa statistic (Kw)
and its Jackknife 95%CI.
According to the Landies and Koch classification criteria (Landis
R, Koch G. Biometrics 1977; 33: 117-27), in the EQA HER2
immunostaining phase it was observed a substantial agreement
for score 0 (K = 0.80; 95%CI[0.64-0.97 ]), and an almost perfect
agreement for score 3+ (K = 0.84; 95%CI[0.70-0.99 ]). In contrast, moderate and fair agreement ware observed for score 1+ (K
= 0.54; 95%CI[0.31-0.78 ]) and 2+ (K = 0.37; 95%CI[0.06-0.70
]), respectively.
In the EQA HER2 interpretation phase the agreement was considered satisfactory if the lower limit of the 95% CI of the Kw was
higher than the usual threshold value of 0.80. According to this
criteria, a satisfactory agreement was observed for 6 out of 16 PC
(37.5%) and a quite satisfactory for the remaining 10 PC (62.5%).
In addition by considering the K values within each scoring category
calculated for each CP, we confirmed a lower agreement for score 1+
and 2+ (median K value of 0.67 and 0.52, respectively).
Finally, by jointly considering the results of the two phases, 9
PC reached a satisfactory level in at least one phase of the whole
process.
Our findings confirmed that the two intermediate scoring categories (1+ and 2+) are less reproducible, both in the staining and in
the interpretation phase, than the other two classes (0 and 3+).
These findings are relevant in clinical practice where the treatment choice is based on categories defined by this assay, suggesting the need of adopting sharing procedures within laboratories,
educational programs and/or new reference materials to improve
the assay performance.
Supported by Roche.
Prognostic-predictive factors in breast cancer:
comparision of mRNA level (TargetPrint®) and
immunohistochemistry expression
L. Zandonà1, A. De Pellegrin1, E. Ober1, A. Zacchi1, F. Martellani1, A. Romano1, E. Leonardo1, T. Al Omoush1, F. Giudici1, M.
Bortul2, G. Pellis3, A. Dell’Antonio4, G. Mustacchi5, L. Torelli6,
C. Convertino7, L. Di Bonito1, F. Zanconati1
U.C.O. Anatomia e Istologia Patologica, Dipartimento Clinico di Scienze
Mediche, Chirurgiche e della Salute, Università di Trieste, Italia; 2 U.C.O.
Clinica Chirurgica, Dipartimento Clinico di Scienze Mediche, Chirurgiche e della Salute, Università di Trieste, Italia; 3 U.O. Chirurgia Generale,
Casa di Cura “Sanatorio Triestino”, Trieste, Italia; 4 U.C.O. Chirurgia
Generale, Dipartimento Clinico di Scienze Mediche, Chirurgiche e della
Salute, Università di Trieste, Italia; 5 Centro Sociale Oncologico, Dipartimento Clinico di Scienze Mediche, Chirurgiche e della Salute, Università
di Trieste, Italia; 6 Dipartimento di Matematica e Informatica, Università
degli Studi di Trieste, Italia; 7 S.C. 1° Chirurgica, AOU “Ospedali Riuniti” Trieste, Italia.
1
Introduction. TargetPrint® (TP®) is a microarray-based gene expression test which offers a quantitative assessment of the patient’s
comunicazioni orali
level of estrogen receptor (ER), progesterone receptor (PR) and
HER2/neu overexpression within her breast cancer. As compared
to Immunohistochemistry (IHC), TP® delivers an added benefit to
the diagnostic process. IHC provides a semi-quantitative positive
or negative result, whereas the gene expression result provided by
TP® allows to integrate the absolute level of ER, PR and HER2
gene expression into treatment planning. It is known that differences in tissue fixation, choice of antibody, and interpretation can
severely affect IHC accuracy and reproducibility.
Aim. In this study we have assessed the association between
mRNA expression measured by TP® and protein expression
evaluated by IHC for ER, PR, HER2 and the IHC diagnostic reliability of our laboratory.
Methods. Microarray expression data for ESR1 (ER), PGR (PR),
and ERBB2 (HER2) were obtained by TP® from 143 not selected
invasive breast carcinomas observed from November 2008 to
November 2009, out of 311 women with breast carcinoma diagnosed in the Pathology Institute of Trieste. The analysis is carried
out with fresh tissue, placed immediately after biopsy or surgery
in a solution that preserves the mRNA at room temperature
(RNARetaind®). ER, PR and HER2 read-outs are determined by
measuring the level of gene expression for the three biomarkers
by Agendia BV. For IHC assessment of ER and PR, a tumor was
deemed positive when more than 10% of the tumor cells showed
positive staining. Tumor HER2 IHC status was scored as 0, 1+,
2+, or 3+: a tumor was considered negative for HER2 for score 0
and 1, and HER2 positive for score 3. For 2+ samples, FISH test
was performed to assess final HER2 gene amplification status.
Results. We have compared the results of 143 cases obtained from
the assessment of ER, PR and HER2 by immunohistochemistry
and TP®: 142 of 143 (99.3%) matched for ER (117 ER positive
and 25 ER negative), 123 of 143 matched for PR (86%) (83 PR
positive and 40 PR negative) and 140 of 143 (97.9%) matched for
HER2 (15 HER2 positive and 125 HER2 negative). These results
indicate an excellent agreement between microarray readout and
IHC for ER and HER2, and a good agreement for PR. In addition
to this assessment, we also compared the numerical values (expression levels) of ER and PR, subjected to percentage normalization,
obtained with IHC and TP®, exclusively on the share of positive
cases. Wilcoxon-test demonstrates that the two methods are different: IHC shows greater intensity than TP® in the evaluation of
positivity (IHC-ER, median=0.85 and TP®-ER=0.65, p<0.001;
IHC-PR, median=0.70 and TP®-PR=0.40, p<0.001).
Discrepant cases. Analyzing the discrepant cases, we note that the
largest share affects the evaluation of PR. It must be stressed that
the PR are present with a more inhomogeneous distribution then
ER; so the microarray sample could not be representative of the
entire lesion. Another important consideration is that the presence
of an active gene is not necessarily associated with the expression
of a particular protein (epigenetic phenomena). This may explain the
positive cases at TP® and negative at IHC. Finally the neoadjuvant
therapy could influence the results; in fact two of the three discrepant
cases for HER2 have received pre-operatory treatment.
Conclusions. Our study has allowed to verify the reliability of
immunohistochemistry diagnostic performed in our laboratory
and it suggests the possible role of gene signature as an external quality control. The study also emphasizes the limits of the
microarray technique to obtain material representative of the
entire lesion, especially for the evaluation of PR.
Intratumoral heterogeneity of HER-2 in breast
cancer
V. Arena, I. Pennacchia, F.M. Vecchio, A. Carbone
Institute of Pathology, Catholic University of Sacred Heart, Roma
Pathologist s involved in HER-2 characterization in breast
cancer could face with cases of HER-2 heterogeneity (H).
149
The recently proposed guidelines prompt all officer works to
contribute in realization of the best way in reporting results of
HER-2 characterization in breast cancer. A reasonable way to
document HER-2 H could be to attach to the report the FISH
cells count results and to close the report with a final interpretation of results obtained at one or more tumor sites. According to
Albarracin et al. we think that an analytical report completed by
a critical valuation of results about HER-2 H should be worldwide promoted. In adherence to guidelines, FISH report should
describe: a) the number of cells analyzed; b) the copy number
of HER-2 gene per nucleus; c) the copy number of CEP17 per
nucleus; d) the ratio HER-2/CEP17 for each nucleus; e) the
overall average ratio and standard deviation; f) the number
(and the percentage) of cells, if any, with ratio >2.2, and g) the
average ratio in this group. But what could be the simplest and
most practical way to report such analytical results? For this
purpose the use of a spreadsheet form, e.g. an Excel form can
be adopted. Experimental data can be introduced and results are
automatically calculated. FISH, as first test or as assessment
of HER-2 in case of borderline IHC result (2+), is a molecular
exam, and lab investigators do rely in molecular tests, as natural. Absence of clear-cut results, e.g. a ratio close to cut-off, can
be confusing. Furthermore, the finding of not aligned results
from different tumor sites in the same case, or the finding of
an important genetic H in the same slide can become cause of
irresolution in writing the final report. Regarding HER-2 H,
moreover, we can not underestimate the fact that in situ hybridization is a method prone, in some extent, to register heterogeneous events, since it works on thin tissue sections and tissue
sections are, de facto, much thinner than tumour nuclei are.
CEP17 can be entirely or poorly represented in nuclear sections
and proportions of HER-2 and CEP17 may vary from nucleus to
nucleus and, consequently, HER-2/CEP17 ratios may vary, even
significantly. So, HER-2 assessment can become a tough task,
especially in those cases with average number of HER-2 spots
around cut off value over CEP17 dots. Cases whose results are
at or near the cut off point and, therefore, “should be interpreted
with caution”, can become a real interpretative nightmare. PCR
could represent an alternative molecular approach to asses
HER-2 status and in cases of H, its use (especially on laser
capture microdissected samples) should be encouraged. Finally,
we believe that also HER-2 phenotipic H (PH) should be taken
in the due consideration in reporting a HER-2 characterization
test. For example, we think that the PH as that observed in Fig.
1, with about 10% of cells with 3+ score and the rest of cells
with incomplete, faint membrane decoration, might rise the
same questions as a case of HER-2 genetic H. We suggest that,
as proposed for the genetic H, the IHC report should contain the
score observed and percentages of cells presenting each score.
Following the Dako score, percentages of cells with 0, 1+, 2+
and 3+ scores might be reported. The biological relevance of
HER-2 PH will be established comparing response to therapy
of patient with different HER-2 PH types. It would be very
interesting, in fact, to evaluate how patients with focal 3+ cells
may benefit from trastuzumab therapy, since recently has been
shown that patients with HER-2 GH can take advantages from
trastuzumab adjuvant therapy.
Reference
Albarracin C, Edgerton ME, Gilcrease MZ, et al. Is it too soon to start
reporting HER-2 genetic heterogeneity? Arch Pathol Lab Med.
2010;134:162-3; author reply 163.
150
Ginecopatologia
Role of p16 INK4a staining as an adjunct specificity
and diagnostic accuracy enhancer in HPV+ women
within a program of organized cervical cancer
screening
D. Gustinucci*, B. Passamonti*, E. Cesarini*, M. Staiano**, D.
Butera**, A. Gioioso**, F. Fulciniti**
Azienda Sanitaria Regionale dell’Umbria USL n.2 – U.O.C. Diagnostica di Laboratorio-U.O. Citologia- Head Basilio Passamonti; **S.S.D. di
Citopatologia, U.O.C di Anatomia Patologica e Citopatologia, Istituto
Nazionale Tumori “Fondazione G. Pascale”, Napoli
*
Background. It was our intention in this paper: 1) to evaluate
the usage of the p16INK4a immunostaining within an organized cervical screening program in some cytologic diagnostic
categories, as ASCUS and LSIL after triage with HR-HPV test
and in the ASC-H and HSIL categories, also independently from
HR-HPV triage; 2) to verify whether the routinary introduction of
p16INK4a staining might be useful in enhancing specificity and
positive predictive value vs. CIN 2+ lesions of the cytological
screening test to guarantee an always more tailored follow-up,
aimed at the most possible precise identification of these preneoplastic changes.
Methods. performances of the p16INK4a test were compared to
the cytological diagnoses in 578 cytological samples obtained
from a screening population between 25 and 64 years of age
(medium age 38) followed by the Operative Unit of Screening
Cytology based in Perugia. 213 patients were HR-HPV+ ASCUS,
186 HR-HPV+ LSIL, 74 ASC-H and 105 HSIL. The 105 HSIL
were sub-classified into HSIL-favor CIN2 (HSIL-CIN2) (n=56)
and into HSIL favor severe dysplasia/CIN3/carcinoma in situ
(HSIL-CIN3) (n. 49). For the categories ASCUS and LSIL, pap
smears were obtained as a parallel test from liquid based cell samples (Thin Prep, TM) vials sent to our Laboratory for HR-HPV
test (Digene High-Risk HPV Hybrid Capture 2 (HC2 DNA Test,
Hologic TM), the remaining Thin Prep samples were obtained
during colposcopy from women sent to second level diagnostic
examination. In all cases taken by the liquid based technology,
when atypical cells consistent with the original cytological diagnosis made on traditional smear were lacking, p16INK4a immunocytochemistry was performed on the original (traditional)
smears. All samples in this study had histological follow-up.
Results. In the ASCUS category, p16INK4a sensitivity was 91%
for CIN2+ and 100% for CIN3, specificity was, respectively,
64% and 58%, the NPV was, respectively 96% and 100% and the
ratio of p16INK4a positivity was 47%; in the LSIL category the
sensitivity was 77% and 75% respectively, with a specificity of
64% and 57%, a NPV of 93% and of 98% and a ratio p16INK4a+
samples of 43%. By using p16INK4a staining there was an
increase of the PPV vs CIN2+ of 19% in the ASCUS category
and of 13% in the LSIL category. p16INK4a positivity ratios for
ASC-H, HSIL-CIN2 and HSIL-CIN3 were, respectively, 90%,
87% and 90%; sensitivity both for ASC-H and HSIL-CIN3 was
100%, either vs CIN2+ or CIN3, for HSIL-CIN2 it was 91% and
95% respectively. The NPV was 100% either vs. ASC-H and
CIN2+ or vs. CIN3, of 43% and 86% respectively, for HSILCIN2; specificity was, respectively, 26% and 17% for ASC-H
and of 23% and 17% for HSIL-CIN2. No cases diagnoses as
HSIL-CIN3 resulted p16INK4a negative. The increase of PPV vs
CIN2 was of 6% for the ASC-H category, of 3% for HSIL-CIN2
and 0% per HSIL-CIN3. Subsequent follow-up examinations of
8 cases originally diagnosed as ASC-H and of HSIL-CIN3 that
were p16INK4a positive, but histologically negative or CIN1 on
the first biopsy, showed 4 CIN 2 and 4 CIN3 lesions.
Conclusion. the obtained values of sensitivity, specificity, TPV
and NPV confirm the importance of the utilization of p16INK4a
in the categories ASCUS and LSIL after triage with HR-HPV test
as an indicator of CIN 2+ lesions, permitting to design tailored
models of colposcopic follow-up which, keeping into account
also the expression of p16 protein, could permit to reduce the
psycological pressure of repeated testing. As far as ASC-H and
HSIL-CIN 3 lesions are concerned, p16INK4a showed to be an
exceptionally good marker in picking-up CIN 2+ lesions, especially in controversial cases with cyto-histological discordance.
The obtained results in the category of HSIL-CIN 2 lesions, notwithstanding good sensitivity data, do not seem to encourage to
usage of p16INK4a to enhance the diagnostic specificity vs. CIN
2+ lesions in this category.
p16/ki-67 expression in precancerous and
cancerous cervical lesions
M.A. Caponio, S. Petroni, T. Addati, M. Centrone, O. Popescu,
G. Giannone, V. Rubini, G. Simone
Anatomic Pathology Unit, National Cancer Institute “Giovanni Paolo II”,
Bari, Italy
Background. Cervical cancer is the most common HPV-associated cancer. Molecular detection of HPV DNA as a diagnostic test
to cervical carcinogenesis gave a low positive predictive value as
compared to the use of biomarkers, therefore use of modulators
involved in the cell cycle as markers of HPV infection may be an
important tool in patients who could develop cervical carcinoma.
p16INK4a is a cyclin-dependent kinase-4 inhibitor which plays an
important role in the mechanism of cell cycle regulation. This
protein provides an anti-proliferative effect when expressed during regular cellular progression. Diagnostic application of p16
has been investigated in cervical pathology being expressed in
HPV-associated lesions: in low–grade cervical intraepithelial
neoplasia (CIN) and in high-grade CIN.
On the contrary, Ki-67 is a proliferation-associated protein which
can be detected in the nucleus exclusively of proliferating cells.
Thus, concomitant expression of p16INK4a and ki67 in atypical
cervical sample may be used as a marker of deregulation of the
cell cycle 1.
In this study we analyzed the diagnostic utility of p16/ki67 dual
stain with the aim to evaluate: 1) clinical utility of this dual test
for the identification of precancerous cervical lesions and 2) the
agreement between cyto-histological p16/ki67 expression and
HPV DNA test, using Hybrid Capture technology 2.
Methods. In our prospective study, 32 female patients were enrolled. P16/ki-67 were assessed on 29 out of 32 evaluable LBCs
and on 32 out of 32 histological samples. Cytological were diagnosed as follow: 5 out of 32 were negative for cervical lesions, 4
ASCUS, 1 AGUS, 12 L-SIL and 7 H-SIL. Moreover, 13 out of
32 histological specimens were classified as negative for cervical
lesions, 12 as CIN1, 5 as CIN2-3 and 2 as CaIS.
Immunohistochemistry was performed, using CINtec® PLUS
Kit (mtm, Laboratories), an immunohistochemical assay for the
simultaneous qualitative detection of the p16 INK4a and Ki-67 proteins in cervical preparations.
Moreover, HPV DNA test was performed on all 32 sample, using
Hybrid Capture II (HCII.) which uses a molecular technology to
detect the DNA of 13 high-risk types of HPV.
Results. Our analysis showed that simultaneous p16 and ki-67
immunoreactivity was found in 0/7 H-SIL, 3/12 L-SIL (25%),
0/1 AGUS, 1/4 ASCUS (25%), whereas it was absent in all 5
negative cytological samples. On the contrary, on histological
samples p16/ki-67 immunostain was detected in 2/2 (100%) CIS,
4/5 (67%) CIN2-3, 3/12 (25%) CIN1 and in 1/13 (8.33%) negative histological samples.
In relation to HCII detection, 4 out of 32 cervical specimens resulted negative to the HPV infection, whereas in 28 LBC, HPV
infection was evidenced, with a high prevalence of HR-HPV
subtypes. Moreover, our analysis showed that p16/ki-67 immu-
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noreactivity was absent in 25/28 (89%) HPV (+) cytological samples, whereas it was present in 3/28 (11%) cases. It is important
to note that all the 3 p16/ki-67 (+) and HPV (+) specimens were
histologically classified as high grade lesions.
Conclusions. Immunohistochemistry for p16/ki-67 detection
could be considered an useful assay to evaluate high grade lesions. P16/ki-67 reaction improves cervical lesion analysis becauseit evidence viral genome integration in proliferative cells. Our
preliminary data show that the specificity and sensibility rates
of this biomarker result better in histological than in cytological
samples and HCII test was less specific than p16/ki67 in atypical
cervical samples.
References
1
Schmidt D, Bergeron C, Denton KJ, et al. p16/ki-67 dual-stain cytology in the triage of ASCUS and LSIL papanicolaou cytology: results
from the European equivocal or mildly abnormal Papanicolaou cytology study. Cancer Cytopathol 2011;119:158-66.
2
Petry KU Schmidt D, Scherbring S, et al. Triaging Pap cytology negative, HPV positive cervical cancer screening results with p16/Ki-67
Dual-stained cytology. Gynecol Oncol 2011;121:505-9.
Typing of immunophenotypic P16INK4α Cyclin E,
Cyclin A and KI67 in the differential diagnosis
of endometrial adenocarcinomas
E. Tallarico, A. Nocita, S. Mazza, N. Papaleo, V. Rossano, F.
Tallarigo
U.O.C. Anatomia Patologica e Citodiagnostica, Ospedale San Giovanni
di Dio, Crotone
Introduction. The endometrial cancer is one of the most common malignant neoplasm affecting women in industrialized countries. Known risk factors for this disease include: obesity, hypertension, diabetes mellitus, late menopause and exogenous estrogen use.
In 1983, Bokhman proposed a dualistic model of endometrial tumorigenesis based on the clinicopathologic correlations. The majority
endometrial cancers (80%) designated as type 1 carcinomas, while
another 20% of endometrial cancers, designated as type II carcinomas. Histologically, most of the type I tumors have endometrial differentiation and generally have a good prognosis with immunophenotypic expression of estrogen-progesterone receptors. In contrast
type II tumors do not express estrogen-progesterone receptors, and
have the clinical point of view a more aggressive disease course.
Aim. The objective of this study is to make a typing immunofenotpica with new antibodies, a large number of endometrial
carcinomas with different degrees of differentiation so that they
can add to the panel of antibodies already used, so that they can
provide useful information in setting diagnostic chemotherapy
protocols. The biomarkers that we have taken into account in
this study are proteins that regulate and control “checkpoints” of
the cell cycle and therefore have a key role in the mechanisms
of tumor progression and tumoregenesis and are: P16INK4α, Cyclin E, Cyclin A. Also in the study were compared the expression of these three antibodies with that of Ki67, which is an
expression of ‘index of cell proliferation.
Material and methods. Immunohistochemical staining for
cyclin E, cyclin A and Ki67 was perfomed and detected by the
destran method (Dako Envision+Dual Link System) while for
P16INK4α was used the CINtecHistology Kit.In this study, as tissue samples, we were analyzed endometrial biopsies from 63
patients, 43 with adenocarcinomas of endometrioid type1, 12
with well-differentiated grade(G1), grade 21 with moderately differentiated (G2), 10 poorly differentiated (G3)and 20 diagnosed
with papillary Serous type 2. The histological material was fixed
in 10% formalin and embedded in paraffin and then were sectioned at 4μm thick and deparaffinized through the Pit-link.
Results. Profile immunophenotypic of the markers P16INK4α, cyclin
E, cyclin A and Ki67 was considered differently in the two types of
endometrial cancer, as are the different molecular events responsi-
ble for the process of carcinogenesis. The monoclonal antibodies
cyclin E, cyclin A and Ki67 showed a nuclear expression pattern
with percentages increase significantly and in proportion to the
degree of histological differentiation for isotype endometrioid and
the serous papillary. With regard to the percentage of cyclin E expression was as follows: 35.41% in cases of G1 (8 of 12), 37.17%
in G2 cases (15 of 21), 48.43% and 51, 84% respectively in both
cases G3 (8su 10) and in cases Serous papillary (16 of 20). For
the cyclin A the expression was 30.22%, respectively, in welldifferentiated (G1), moderately differentiated in the 33.66% (G2),
and finally 45.89% in the poorly differentiated (G3) and in Serous
Papillary is 55.80%. Most interesting results were obtained by
analyzing the immunohistochemical scores of the marker P16INK4α,
who had a low expression for the endometrioid carcinomas, in fact,
are the immunohistochemical scores 14.41% (4 of 12) of 17.54%
(6 of 12) and 30.67% (5 / 10) respectively in grades G1 and G2
and G3 while in Serous papillary the immunoreaction was 92.92%
(20/20). Also interesting is the distribution that has the p16INK4α
in neoplastic endometrial tissue, in fact Serous papillary pattern of
expression has a carpet with an intense and diffuse staining both
nuclear and cytoplasmic compared to FIGO grade 3 endometrioid
carcinoma which is distributed in patches, while in FIGO grades
1 and 2 endometrioid carcinomas the distribution is sporadic with
weak and focal expression pattern in the nuclei of the cancer cells.
Finally we went to see if there is a statistical correlation between
markers p16INK4α, cyclin E, cyclin A with the monoclonal antibody Ki67.The expression of Ki67 in the cases investigated were
respectively: 39.74% G1,44.36% G2, 66.85% G3 and 67.15% in
Serous papillary. Statistical analysis showed that the index of each
marker expression correlates with Ki67, and are also statistically
significant (P <0.05).
Conclusions. The results allow to underline that the endometrial adenocarcinoma type endometrioid and Serous papillary have a
different immunophenotypic profile that is correlated with the
histopathological features Also testing the different monoclonal antibodies P16INK4α, cyclin E, cyclin A and placing them in
comparison with Ki67 was noted that these markers could be
used not only in the differential diagnosis between the two different isotypes of cancer but may also form the basis for new protocols chemotherapy. As Cyclin E provides useful information on
the progression of the disease, and thus be considered a prognostic indicator of endometrioid tumors, whereas cyclin A emphasizes the progression of neoplastic transformation of the endometrium and thus the proliferative activity of cancer cells, and therefore
it is considered an important prognostic parameter for classifying
a subtype of patients who must follow a targeted therapy in correlation with tumor aggressiveness. P16INK4α unlike cyclin E and
A, can be added to the panel of antibodies used in the differential
diagnosis of adenocarcinoma endometrial. In fact, the strong and
intense expression in Serous papillary, emphasizes both its inactive state that the loss of expression from a biological point of
view function as a promoter of cell cycle control.
Future prospects. Sequencing the gene located on chromosome 9p21 CDK2NA encoding P16INK4α in tumors where there was
a high expression, associated with a high proliferation index (Ki67),
and therefore delineate a new guidelines for chemotherapy.
Detection of P16(INK4A) in serous ovarian
neoplasms
C. Manini*, E. Bar**, S. Mazzola***, P.L.Montironi**
S.C. Anatomia Patologica, Osp. S. Giovanni Bosco, ASLTO**, Torino; ** S.C.
Ostetricia e ginecologia, Osp. Santa Croce, ASLTO5, Moncalieri (TO);***
S.C. Ostetricia e ginecologia, Osp. Maggiore, ASLTO5, Chieri (TO)
*
Background. defects of the “Rb/cyclinD1/p16 pathway” have
been shown to play a critical role in the development of human
malignancies. The aim of the study was to investigate p16(ink4a)
expression in serous ovarian neoplasms.
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Methods. immunoreactivity of p16(ink4a) was investigated using
paraffin sections from 21 serous high-grade ovarian carcinoma, 7
low-grade ovarian carcinoma and 5 borderline serous tumour of
the ovary. A composite staining score (∑% positive cells x intensity) was calculated for each case.
Results. diffuse p16-staining was a common finding in all
neoplasms. A strong expression was found in 11 (52%) cases of
high-grade carcinoma; weak expression was found in 9 (42%)
cases of high-grade carcinoma and in all low-grade and borderline neoplasms.
Conclusions. p16(ink4a) immunostaining is widespread involving most tumour cells in serous ovarian neoplasms, but the intensity of the staining seems to be directly related to histological
grade. A weak p16(ink4a) expression is a common feature in
low-grade ovarian carcinoma and borderline tumour.
Morphologic differential diagnostic criteria
between two synchronous primary ovarian and
endometrial cancer and endometrial cancers
metastatic to ovaries
V.G. Vellone*, G. Chiarello*, E.D. Rossi*, G. Fadda*, S. Moncelsi*, G. Scambia**, G.F. Zannoni*
*
Division of Surgical Pathology, Policlinico A.Gemelli, Università Cattolica del S. Cuore, Roma; **Division of Gynaecologic Oncology, Policlinico
A.Gemelli, Università Cattolica del S. Cuore, Roma
For prognostic and therapeutic purposes, it is important to distinguish those cases where there is a metastasis to ovary from a
primary endometrial cancer (metastatic cancers) from those cases
where two primary cancers are present (synchronous cancers).
The computer database of the histopathology service of the A.
Gemelli Hospital of Rome was reviewed retrospectively in the
period 2000-2008 in search of endometrial carcinomas with
metastases to the ovaries and concurrent endometrial and ovary
cancers. a total of 52 cases were selected and compared: 13 have
been considered as synchronous; 39 as metastatic.
There was no significant difference in age between the groups.
Metastatic cancers, considering the endometrial component, showed a significant (p<0,05) larger size, percentage of
myometrial infiltration, and detection of neoplastic emboli.
Considering the ovarian component, there were no significant
differences in size between the two groups although metastatic cancers were more heterogeneus in size, ranging from
large masses (up to 30 cm) to subcentimetric nodules Ovarian
metastases showed significantly (p<0,05) more frequent histologic and grade concordance, multinodular appearance, dirty
necrosis, bilaterality, and incidence of metastases in other sites.
A histological discrepancy between endometrial and ovarian
component resulted as characteristic of synchronous tumors.
Complete congruence is more frequent in metastatic tumors,
however, the correlation may be biased in particular if the primary
endometrial cancer show mixed features. In these cases, ovarian
metastasis is generally made up of only one cell subpopulation of
primary endometrial cancer. When present, metastases in other
sites in patients with synchronous cancer resulted consistent with
an ovarian origin. The synchronous cancers patients showed a
better survival trend if compared to metastatic cancer patients.
In conclusion, the distinction between the two populations requires a careful examination of both endometrial and ovarian
components: congruence, at least partial, of the two components
for histologic type and grade, a large and deeply infiltrating
carcinoma with numerous neoplastic emboli in endometrial component; multinodularity bilaterality, dirty necrosis in the ovarian
component, are diagnostic for an endometrial cancer metastatic to
the ovaries resulting in worse prognosis.
Tyrosine kinase receptors expression and
mutations as predictive markers for target
therapies in endometrial stromal sarcoma
P. Cossu-Rocca*, M. Contini*, M.G. Uras*, A. Mura*, M.R. Muroni*, F. Pili*, C. Carru***, E. Maricosu*, R. Olianas*, A. Zinellu***,
F.F. Nogales**, G. Massarelli*, M.R. De Miglio*
*
Department of Clinical, Experimental and Oncologic Medicine, University of Sassari, Italy; ** Department of Pathology, University of Granada, Spain; *** Department of Biomedical Sciences, University of Sassari,
Italy
Purpose. Endometrial stromal sarcomas (ESS) are rare malignant mesenchymal neoplasms, which are currently treated by
surgery, whereas effective adjuvant therapies have not yet been
established. Tyrosine kinase inhibitors have been rarely applied
in ESS therapy, with few case reports describing Imatinib responsivity. Aim of our study was to analyze different tyrosine kinase
receptors status in a ESS series, to evaluate their potential role as
molecular targets.
Experimental Design. A series of 28 cases of ESS, including 23
LG-ESS and 5 UES, was selected from the archives of the Departments of Histopathology of the Universities of Sassari (Italy)
and Granada (Spain). From formalin-fixed, paraffin-embedded
(FFPE) specimens, 3 micron sections were obtained for haematoxylin and eosin stains and immunohistochemical analyses.
Consecutive sections were also obtained for genetic analyses. We
performed immunohistochemistry for EGFR, c-KIT, PDGFRalpha, PDGFR-beta, and ABL. On the same series, we screened
for ‘hot-spots’ mutations on EGFR, c-KIT, PDGFR-alpha, and
PDGFR-beta genes, by sequencing. We also investigated for
EGFR, PDGFR-alpha, and PDGFR-beta gene expression by
qRT-PCR on 14 selected cases.
Results. EGFR expression was appreciable in 14 out of 28 cases,
(50%), with staining intensity ranging from 1+ to 3+, and percentages of positive cells ranging from 20 to 80%. No immunoreactivity was recognizable in normal, peritumoral tissues. PDGFRalpha expression was detected in 19 out of 28 cases (68%), with
staining intensity ranging from 1+ to 3+, and percentages of
positive cells ranging from 30 to 70%. PDGFR-beta expression
was detected in 10 out of 28 cases (25%), with staining intensity
ranging from 1+ to 2+, and percentages of positive cells ranging
from 10 to 70%. Nuclear-cytoplasmic immunoreactivity for both
PDGFR-alpha and PDGFR-beta was also observed in normal
vessels endothelial cells. ABL expression was recognizable in 7
out of 28 cases (25%), with staining intensity ranging from 1+
to 3+, and percentages of positive cells ranging from 10 to 70%.
ABL expression was also recognizable in glandular structures of
normal endometrium. CD117 (c-KIT) expression was detected
only in 1 UES out of 28 cases with 1+ intensity in 10% of neoplastic cells.
Expression of 2 or more tyrosine kinases receptors was observed
in 18 out of 28 cases (64 %), with at least 2 receptors expressed
simultaneously in 10 cases, 3 receptors in 7 cases, and 4 receptors in a single UES case. Only 5 LG-ESS out of 28 cases were
consistently negative for all the antibodies.
RT-PCR analysis did not show any statistical significance between tumor and normal tissues expression levels, with P-values
of 0.95, 0.85, and 0.89 for EGFR, PDGFR-alpha, and PDGFRbeta, respectively. Gene expression profiles did not show significant correlations between gene and protein expression levels,
apart from a single case displaying high mRNA levels and protein
overxepression for PDGFR-alpha and PDGFR-beta. No activating mutations were found on the cases included in the study.
Conclusions. Our study demonstrated that tyrosine kinase receptors are often expressed in ESS, and, in a majority of cases (64%),
the simultaneous expression of 2 or more receptors was appreciable, even in the absence of activating mutations or gene overexpression, suggesting that TKRs anomalous activation should
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be related to post-translational abnormalities. Nevertheless, TKIs
should not be ruled out for ESS patients with TKRs overexpression, even in the absence of genetic abnormalities, and appropriate clinical trials with specific or multi-targeted TKIs should be
proposed for patients with advanced disease.
Further investigations are needed to identify specific post-translational abnormalities, potentially useful to further select patients
who might benefit from current targeted therapies.
Undifferentiated endometrial carcinoma; report
of three cases with therapeutic implications
N.S. Losito*, S. Greggi**, C. Scaffa**, C. Pisano***, S. Pignata***,
G. Scognamiglio*, G. Botti*
*
S.C. Anatomia Patologica, INT “G.Pascale” di Napoli, Italia; **S.C.
Oncologia Chirurgica Ginecologica, INT “G.Pascale” di Napoli, Italia;
***
S.C. Oncologia Medica Uro-Ginecologica, INT “G.Pascale” di Napoli,
Italia.
Endometrial carcinoma is the most common malignant tumor of
the female genital tract. It is generally considered a low grade
malignancy because the vast majority (80%) of newly diagnosed
endometrial carcinomas in western countries fits in type I category, exhibiting endometrioid histology. A minority of cases
(20%) is represented by high grade endometrial carcinomas, a
heterogeneous group of clinically aggressive tumors, including
FIGO 3 endometrioid carcinoma, serous and clear cell carcinoma,
undifferentiated carcinoma, special histotypes that require different therapeutic regimens. We describe three cases of undifferenziated endometrial carcinomas. Case 1 occurred in a middle-aged
woman was diagnosed as dedifferentiated endometrial carcinoma
because of the combination of an undifferentiated component
(45%) with FIGO 2 endometrioid areas (30%) and FIGO 3 spindle endometrioid areas (25%). All neuroendocrine markers were
negative and CK was only focally but strongly expressed. The
two other cases arose in old, postmenopausal women and were
associated with variable expression of neuroendocrine markers.
Case 2 was represented by a small cell neuroendocrine carcinoma
mixed with FIGO 2 endometrioid carcinoma, arising in endometrial polyp; CK20 dot-like, sinaptophysin and CD56 stainings
were diffusely positive.
Case 3 was an advanced-stage tumor, involving uterine sierosa
and both ovaries. This tumor was diagnosed as large cell neuroendocrine carcinoma; CK7 and CD56 were diffusely and strongly
expressed while sinaptophysin dot-like positivity was focal and
faint.
Differential dagnosis was not easy; FIGO 3 endometrioid carcinoma was excluded because every case of ours lacked trabecular
or focally glandular areas, while a diffuse, non-cohesive growth
pattern was present. The epithelial immunophenotype was confirmed in every case and the absence of muscle and lymphoid
markers was sufficient to exclude undifferentiated sarcoma
and lymphoid diseases. Positivity of neuroendocrine markers
confirmed morphologically based diagnosis in case 2 and 3,
nothwithstanding the fact that many endometrial carcinomas may
show indeed this positivity, but only faintly and focally.
Pure neuroendocrine carcinomas of the endometrium are extraordinary rare and there are occasional reports of combined neuroendocrine carcinomas and adenocarcinomas. Their prognosis is
very poor and, therefore, they need more aggressive therapies and
close follow-up. Case 2 and case 3 showed an optimal response to
chemiotherapy with cislatin and etoposide and have no relapse at
the moment. Extensive surgical sampling is mandatory to avoid
to miss undifferentiated components very often underdiagnosed.
Human papillomavirus genotype attribution in
adenocarcinoma of the uterine cervix
F. Rivasi*, S. Venturoli**, S. Silvano Costa***, D. Barbieri**, E.
Mataca*
Department of Pathologic Anatomy and Forensic Medicine, Section of
Pathological Anatomy, University of Modena and Reggio Emilia, Modena; **Department of Haematology, Oncology and Laboratory Medicine,
Section of Microbiology, S.Orsola-Malpighi Hospital, University of Bologna, ***Department of Obstetrics and Gynecology, S. Orsola-Malpighi
Hospital, University of Bologna
*
Background. Over the past 50 years, the relative proportion,
compared with squamous cell carcinoma (SCC), as well as absolute incidence of invasive and preinvasive glandular lesions
of the uterine cervix have been changing in Western Countries.
Reports from the 1950’s and 1960’s indicated that adenocarcinomas (AC) accounted for only 5% of cervical cancer cases, while
in the 1990’s AC represented 20-25% of all cervical carcinomas.
Moreover registries from 1975 to 2000, showed that contrary to
SCC where the raising carcinoma in situ (CIS) rates corresponded
to a decrease of invasive SCC rates, raising adenocarcinoma in
situ (AIS) rates paralleled an increase in invasive adenocarcinoma
rates, mainly among young women. This change in the ratio of
AC relative to all cervical cancers may be due to several reasons.
The introduction of the PAP test as a screening tool allowed
early detection of cervical squamous cell lesions, but despite
well organized screening programmes, many studies have shown
that AIS and AC are frequently missed by conventional cytology. Reasons for missing AC and its precursors might be a) their
location and topography in the endocervical canal with less accessibility by the spatula and brush, or b) failure of cytologists to
recognize these lesions. Difficulty in diagnosing glandular lesions
even in biopsy is further increased by the fact that this pathologic
entity does not always show any evident abnormalities in colposcopy, and minimally invasive biopsy procedure or scraping of
endocervical lesions may be inadequate, leading to false negative
diagnoses. As pointed out by several authors, all this contributes
to the fact that only 40% to 60% of AIS cases are correctly diagnosed before surgery (conisation or hysterectomy). Furthermore,
AC and its precursor lesions have been recently included in the
growing list of Human Papillomavirus (HPV) associated genital
tumors. Indeed, HPV DNA has been detected in 95% of invasive
AC lesions using polymerase chain reaction (PCR) based assays
and serologic tests, suggesting that the increase of this disease
may be a consequence of the increasing incidence of HPV infections. Nevertheless while AC and SCC share many similar risk
factors, it appears that they act differently in epidemiology, prognostic factors, and patterns of failure after similar treatments.
Aim. The purpose of this study was to estimate the distribution
of human papillomavirus (HPV) genotypes in AC lesions derived
from the cervical cancer screening in the city of Modena and surrounding neighbourhoods in the period 1991–2010.
Methods. Paraffin-embedded specimens from 58 patients (aged
24-87 years; median 41; mean 45.2) were obtained from pathology archives. All the samples were reviewed for a pathological
confirmation of a primary cervical cancer of epithelial glandular
origin and analysed for HPV DNA presence with INNOLiPA
Extra Genotyping Assay.
Results. The overall positivity for high-risk (HR) HPV DNA
was 96.6%, while only 15.5% were mixed infection. HPV types
16, 18, and 45 were the three most common types identified with
a prevalence of 71.8%, 23.1% and 10.3% respectively. Furthermore these were the only three genotypes detected as single infections, as the others HR-HPV genotype were always associated
with HPV 16 and 18. Moreover patients with lesions related to
HPV18 and HPV 45 were younger (median 39.0 yrs., mean 40.6
yrs.) than those with HPV16 (median 43.0 yrs., mean 45.9 yrs).
Conclusions. These data confirm the predominant role of HPV
154
16, 18 and 45 in cervical AC, showing restricted genotype
contribution in the pathogenesis of adenocarcinoma. The early
presentation of lesions related to HPV18 and HPV 45 might
be indicative of a short time of progression from preinvasive to
invasive cancer.
Does Clear cell carcinoma really belongs to Type I
ovarian carcinoma?
V.G. Vellone, F. Morassi, E.D. Rossi, G. Chiarello, G. Fadda, D.
Gallo, G.F. Zannoni
Division of Surgical Pathology, Policlinico A.Gemelli, Università Cattolica del S. Cuore, Roma; **Division of Gynaecologic Oncology, Policlinico
A.Gemelli, Università Cattolica del S. Cuore, Roma
*
Ovarian cancer is one of the most lethal cancers in women and
is a major public health problem. Epithelial ovarian tumors are
divided into different subtypes, the research group of RJ Kurman,
Johns Hopkins University, Baltimore, in 2004 proposed a new
dual-type model for tumorigenesis of ovarian cancer. The clearcell carcinoma represents 4 to 12% of cases in Western countries,
is usually included in type I cancers, but has unique characteristics and in most of the cases, a worse prognosis.
The aim of the study is to test in the new two-tier model of ovarian cancer, the possible location of clear-cell carcinoma considering a series of clinicopathological and immunohistochemical
parameters.
The electronic archive of the Pathology Service of the Gemelli
Hospital - Catholic University of the Sacred Heart was examined
retrospectively in the period January 2000 to December 2009.
272 cases of ovarian cancer were considered suitable for the study
and divided in three groups: 71 Type I (Endometrioid Low Grade:
24, Serous Low Grade: 46 Squamous: 1); 157 Type II (Serous
High Grade: 128; Endometrioid High Grade: 14; Undifferentiated / Poorly Differentiated: 7; MMMT: 5; Transitional: 3) and
21 Clear Cell.
The clear cell carcinomas showed significant differences (p
<0.05) with carcinomas of type II with more frequent expression of C-ErbB2 and less frequent bilaterality, infiltration of the
capsule, lymph node metastases, peritoneal metastases, stage,
expression of ER, PR, Ki67, p53. Also significant differences (p
<0.05) with type I cancers were observed with increased expression of C-ErbB2 and less frequent bilaterality, infiltration of the
capsule, peritoneal metastases, stage, expression of ER and PR.
In conclusion, clear cell carcinomas showed only partially overlapping features of type I carcinomas with an even milder clinical presentation but with a lower expression of ER and PR and
increased expression of C-erbB2 and the latter aspect may have
important implications for future treatments.
Glandular lesions of the cervix uteri: can they be
diagnosed cytologically?
E. Macciocu*, E. Menia*, E. Giarnieri**, M.R. Giovagnoli**, G.
Negri*
*
Department of Pathology, Central Hospital Bolzano; **U.O.D. Citopatologia (E.C., A.P.), IIa Facolta` Medicina e Chirurgia La Sapienza, Rome
Background. Cytological diagnosis of neoplastic lesions of the
glandular epithelia of the cervix uteri is often difficult. Inflammatory or hyperplastic changes in the cervical epithelia may
mimic neoplastic lesions. Moreover, most cytologists only rarely
see true neoplastic lesions of glandular epithelia and may feel
their experience inadequate. The Bethesda System (TBS) 2001
distinguishes between adenocarcinoma in situ (AIS) and atypical
glandular cells (AGC), which are categories that should have significantly different predictive values for endocervical neoplasia.
In Italy, however, most cytologists believe that the cytological
finding of glandular atypia, including AIS, is not sufficiently
reproducible, and include these categories in a generic AGC. In
this study we evaluated the feasibility of the cytological diagnosis
of glandular lesions using the TBS 2001.
Materials and methods. The study included 29 AIS or AIS with
early stromal infiltration (AIS+) which had been histologically
diagnosed on surgical specimens and had a previous pap-test
as well as a biopsy or scraping. The cytological, bioptical and
definitive histological diagnosis of these cases was compared.
Moreover, the positive predictive value (PPV) of the cytological diagnosis of glandular lesions was calculated evaluating the
follow-up of 36 pap-test with the cytological diagnosis of AIS
(15) or AGC (21).
Results. In 12 out of the 29 surgical specimens (41.4%) a squamous lesion (CIN) was associated. The overall original pap-test
diagnosis was AIS in 12 (41.4%), AIS with associated SIL in 1
(3.4%), AGC in 3 (10.4%), AGC with SIL in 3 (10.4%), SIL in 8
(27.6%), carcinoma in 1 (3.4%) and ASC-US in 1 (3.4%) of the
cases. Taking only the 17 AIS or AIS+ without associated CIN
into consideration, the original cytologic diagnosis was AIS in
11 (64.7%), AGC in 4 (23.5%), SIL 1 (5.9%) and ASC-US in 1
(5.9%) of the cases. Twenty-one (72.4%) of the biopsies showed
a CIN or AIS, 5 (17.3%) were negative and 3 (10.3%) were
doubtful or not diagnostic. The PPV for AIS and AGC in the 36
pap-test was 0.87 and 0.47, respectively.
Conclusions. Our study shows that the cytological differentiation between AIS and AGC is feasible and may have a clinically
relevant impact on the management of women with glandular
lesions of the cervix.
This study was carried out as part of the Tesi di Master di I livello
in Citologia Diagnostica e Screening di Popolazione, Anno Accademico 2009-2010, Università degli Studi Sapienza, Rome,
Italy.
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Sabato, 29 ottobre 2011
Patologia polmonare
Braf mutations in lung adenocarcinomas:
correlations with clinicopathological parameters
and prognosis
L. Felicioni1, M.G. Sciarrotta2, S. Malatesta2, P. Viola3, A. Chella4, L. Guetti5, F. Mucilli5, A. Marchetti2, F. Buttitta1
Medicina Molecolare Oncologica e Cardiovascolare/ FondazioneUniversità “G.d’Annunzio”Università di Chieti-Pescara, Chieti, Italia; 2 Centro di Medicina Molecolare Predittiva /Fondazione-Università
“G.d’Annunzio”Università di Chieti-Pescara, Chieti, Italia; 3 Unità Operativa di Anatomia Patologica/ Ospedale Clinicizzato di Chieti, Chiet,
Italia; 4 Dipartimento di Chirurgia, Università di Pisa, Pisa, Italia; 5 Dipartimento di Chirurgia/ Università “G.d’Annunzio”Università di ChietiPescara, Chieti, Italia
1
Purpose. In recent years, the pharmacological treatment of nonsmall cell lung cancer (NSCLC) has undergone a major contribution by the introduction of new molecular targeted drugs whose
effectiveness is closely dependent on the presence of specific
genetic mutations in the tumor context. Somatic mutations of
BRAF gene could represent important targets for newly developed biologic treatments. The vast majority of the BRAF mutations correspond to the hotspot transversion mutation T1799A, at
exon 15, that causes the amino acidic substitution V600E. A wide
range of other missense mutations (non-V600E) have been detected in the glycines of the G-loop in exon 11 or in the activation
segment in exon 15 near the codon V600. The actual prevalence,
distribution and prognostic role of BRAF mutations in NSCLC
patients is still unclear.
The present study was devised to investigate the prevalence,
distribution and prognostic role of BRAF mutations in a large
cohort of Caucasian Non-Small Cell potential clinicopathological parameters that could help in the selection of patients to be
subjected to mutational screening.
Patients and methods. A retrospective series of 1046 NSCLCs,
comprising 739 adenocarcinomas (ADC) and 307 squamous cell
carcinomas (SCC) was investigated for BRAF mutations. High
resolution melting analysis followed by sequencing and a strip
hybridization assay were used to screen the samples. All cases
were also analyzed for KRAS and EGFR mutations.
Results. BRAF mutations were present in 36 (4.9%) ADC and
1 (0.3%) SCC. None of the matching normal samples showed
evidence of mutation, indicating the somatic nature of all mutational events. Twenty-one (56.7%) of the mutations were V600E,
and 16 (43.3%) were non-V600E. The two main types of BRAF
mutations, V600E and non-V600E affected different patients and
were associated with different pathological features of lung ADCs. V600E mutations were more prevalent in females (16 of 187
cases, 8.6%) than in males (5 of 552 cases, 0.9%) as documented
by univariable (P<0.0001) and multivariable analysis (HR= 0.09,
P<0.0001). Tumors affected by BRAF mutations were revised
histologically according to the new International Multidisciplinary Classification of Lung ADC (IMCLA). Based on the
IMCLA classification, the tumors with V600E mutation showed
micropapillary features, in 80% of cases. BRAF mutated tumors
were significantly associated with a shorter overall survival at
both univariable (P<0.0001) and multivariable Cox regression
analysis (HR: 0.46; P=0.013). All non-V600E mutations were
found in smokers (P=0.015) and were associated with neither
clinicopathological parameters nor prognosis. This series of lung
ADC was also investigated for EGFR and K-ras mutations. K-ras
mutations were observed in 203 (27%) cases and EGFR mutations in 86 (12%) cases. All of the tumors with BRAF mutations
were found to be negative for K-ras mutations, whereas 2 tumors
with V600E BRAF mutations showed concomitant EGFR mutations (in both cases a deletion in exon 19).
Conclusion. We report for the first time that the V600E BRAF
mutation represent a negative prognostic factor in NSCLC patients. Moreover, we identified a number of clinicopathological
parameters potentially useful for the selection of patients carrying
BRAF mutations. Patients with these highly aggressive tumors
could benefit of new therapeutic strategies based on inhibition of
BRAF signaling.
Diagnosis of synchronous primary lung
adenocarcinomas based on EGFR and KRAS gene
status: a case report
F. Castiglione, G.L. Taddei, L. Messerini, D. Rossi Degl’Innocenti,
M. Pepi, A.M. Buccoliero, M. Rotellini, L. Novelli, C.E. Comin
Dipartimento di Area Critica Medico Chirurgica, Sezione di Anatomia
patologica, Università degli Studi di Firenze, Firenze, Italia
Introduction. The incidence of multiple primary lung cancers has
been reported to be increasing; this is most likely due to the availability of high-resolution thoracic imaging techniques and a rising
incidence of adenocarcinoma histology among nonsmall cell lung
carcinomas (NSCLCs). The distinction between multiple primary
lung cancers and intra-pulmonary metastasis may be challenging, especially when tumours with the same histologic type are
identified. In such situation, molecular analysis may contribute to
obtaining an accurate dignosis. This issue is clinically important
for staging, management plan, and prognosis. We describe a case
of synchronous double primary lung adenocarcinomas diagnosed
by mutational analysis of the EGFR and KRAS genes.
Case report. A 66-year-old man presented with two separate
tumours in two different lobes of the left lung, one in the upper
lobe (UL) and one in the lower lobe (LL). Video-assisted thoracic
surgery was performed and both nodules and mediastinal lymph
nodes were resected. The UL and LL tumours measured 1.5 cm
and 2.5 cm in greatest dimension, respectively. Microscopic examination revealed similar morphologic features in both tumours:
the UL tumour was classified as adenocarcinoma, acinar type,
whereas the LL lesion was classified as adenocarcinoma, mixed
subtype (with acinar and papillary components). Metastasis in the
hilar lymph node was observed. Immunohistochemical studies revealed positive staining for thyroid transcriptor factor 1 (TTF-1)
and napsin A in the UL tumour, whereas, the LL adenocarcinoma
and the metastatic lymph node were TTF- 1-negative and napsin
A-positive. Both tumours and the positive hilar lymph node were
analyzed for EGFR and KRAS mutation status by direct sequencing. The UL tumour was found to harbour a point mutation within
exon 21 (L858R) of the EGFR gene, whereas the LL tumour
harbored a point mutation in codon 12 (G12C). The lymph node
was found to be wild-type EGFR gene and wild-type KRAS gene.
Based on the differing mutation status, the tumours were characterized as synchronous primary adenocarcinomas.
Discussion. Recent evidences have confirmed that gene mutations of EGFR and KRAS and downstream molecules in the
signaling pathways define different subsets of NSCLCs. Thus, it
is reasonable to consider the two lung adenocarcinomas oncodevelopmentally indipendent, based on different mutational status.
Moreover, despite similar morphology, the immunophenotype
156
of the two tumours was different. In fact, the EGFR-mutated
adenocarcinoma was found to be TTF-1-positive, whereas the
KRAS-mutated adenocarcinoma was TTF-1-negative. This result
supports the novel concept of “the terminal respiratory unit”
(TRU) that recognizes distinct subsets of TTF-1-positive adenocarcinomas in which particolar molecular pathways (such as
EGFR, BRAF, and ERBB2 mutations) are involved. Moreover,
this case confirms the evidence that a considerable proportion
of NSCLCs show discrepancy in EGFR and KRAS mutational
status between primary tumours and corresponding metastasis. In
conclusion, mutational analysis seems to be very usefull not only
in the diagnosis of synchronous primary lung cancers but also in
providing informations on prognosis and management decisions.
ALK rearrangements in non small cell lung
carcinoma
P. Dell’Orto, V. Stufano, S. Pessina, H. Fara Tanjona, C. Fumagalli, M. Manzotti, P. Possanzini, M. Barberis.
Division of Pathology, Unit of Histopathology and Molecular Diagnostics,
European Institute of Oncology, Milano.
Introduction. In 2007 Soda et al reported the EML4-ALK fusion
gene in non small cell lung cancer (NSCLC). EML4-ALK fusions
result in protein oligomerisation and constitutive activation of the
kinase. Other genes such as KIF5B and TFG were rarely found as
fusion partners for ALK.
Even if ALK rearranged cases represent less than 7% of the
NSCLC, they may define a molecular subgroup of tumors that
is susceptible to targeted kinase inhibition. The therapeutic efficacy of inhibiting ALK in such tumors with the available smallmolecule crizotinib (PF-02341066) has been recently described
in different studies. The clinical and pathological characteristics
of these tumors are not completely known therefore in this study
we report our experience correlating the morphological data with
the molecular pattern of the tumors.
Patients and methods. The study concerns 125 patients with advanced NSCLCs potentially candidates to tyrosine kinase inhibitors. There were 70 males and 54 females. The age ranged from
33 to 83 years with a mean of 63.
There were 119 cases of adenocarcinoma, 3 large cell carcinomas, 2 adenosquamous carcinomas and one squamous cell
carcinoma. Routine histological slides from surgically resected
tumor specimens (15 wedge resections, 28 lobectomies and 7
pneumonectomies) and tumor biopsies (49 cervical or mediastinal lymph nodes, 25 pleural biopsies and one bronchial biopsy)
were reviewed.
Activating mutations in exon 18,19,20 and 21 of the EGFR gene
and mutations in codon 12,13 and 61 of K-ras gene were screened
by Sanger bidirectional sequencing. Unstained slides from
formalin-fixed, paraffin embedded (FFPE) tumor samples were
analyzed by means of FISH with the use of an ALK break-apart
(or split-signal) probe (Abbott Molecular, Les Plaines, IL, USA).
Samples were deemed to be FISH-positive if more than 15% of
scored tumor cells had split ALK 5′ and 3′ probe signals or had
isolated 3′ signals (11). All the ALK rearranged cases were confirmed to be FISH-positive by a central laboratory under standards and conditions certified according to the Clinical Laboratory
Improvement Amendments. This confirmation was mandatory to
obtain crizotinib by Pfizer Inc.
Results. Alk rearrangement was detected by FISH in 18 cases
(14.4%; 11 females and 7 males), 11 patients were never or
former light smokers (< 8 pack/year); 7 were smokers (more than
35 pack/year). The age ranged from 33 to 77 years with a mean
of 53.
EGFR and k-ras mutations were not found.
There were 17 adenocarcinomas and 1 squamous cell carcinoma.
Adenocarcinoma were prevalent solid pattern adenocarcinomas
with signet ring cell component observed in 10/18 cases (55.5%).
This component ranged from 10% to 90% of the tumor cells, but
in 6 cases it was prevalent (more than 50% of the tumor cells).
Conclusions. The incidence of alk rearrangements in our series
(14.4%) doesn’t reflect the real incidence in unselected patients
that remains rare in our country, probably less than 4%.
The patients were not smokers or light smokers with female
prevalence and with a relatively low mean age.
Our data reinforce the view that alk-rearranged tumors are not
restricted to adenocarcinomas. The presence of squamous cell
carcinomas has been rarely but consistently reported. Anedoctically cases of adenosquamous carcinomas have bee reported
too. The absence of EGFR and k-ras mutations confirmed prior
observations that alk alterations are mutually exclusive with these
events. The frequent presence of cohesive clusters of signet ring
cells is a morphological characteristics of these tumors that in
association with clinical data could indicate in which cases the
FISH test could be performed.
In conclusion alk-rearranged tumors seem to represent a specific
subgroup of NSCLC with specific characteristics.
Epidermal growth factor receptor gene analysis
from cytological samples of lung adenocarcinoma
by means of highly sensitive molecular assay
S. Allegrini*, J. Antona*, R. Mezzapelle*, U. Miglio*, A. Paganotti**, C. Veggiani**, O. Alabiso***, M. Frattini****, G. Monga * **,
R. Boldorini * **
* Dipartimento di Scienze Mediche /Università del Piemonte Orientale,
Novara,Italia; ** SCDU di anatomia Patologica/Ospedale Maggiore della Carità, Novara, Italia; *** SCDU di Oncologia/Ospedale Maggiore
della Carità, Novara, Italia; **** Istituto Cantonale di Patologia/ Ospedale Cantonale di Locarno, Switzerland
Background. Epidermal growth factor receptor (EGFR) gene
mutations are known to predict the response to EGFR tyrosine
kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancers
(NSCLC). Somatic mutations in EGFR gene have been found
in ~10% of NSCLC, with higher frequency in adenocarcinoma
(AC). Over 80% of mutations are small in-frame deletions on
exon 19 or L858R mutation in exon 21; other clinically significant mutations are G719X of exon 18, S768I of exon 20 and
L861Q of exon 21. The diagnosis of AC is usually achieved by
means of cytologic procedure which most often is able to supply
only scarce material. This occurrence could actually limit the use
of molecular diagnostic proceeding. The aim of this study is to
evaluate the feasibility of EGFR analysis on cytologic samples,
in particular in relation with the fixative used and the number of
cancer cells available.
Methods. Ninety-four cytological samples from AC (identified
by morphology and positive immunostaing by antibodies antiTTF1 and cytokeratin 7) were prospectively analyzed for EGFR
mutations by TheraScreen® EGFR29 Mutation Kit (QIAGEN,
Manchester, UK). This kit enables the detection of the following
mutations against a background of wild-type genomic DNA by
Real-Time PCR assay based on Scorpions® technology: in-frame
deletion on exon 19, insertions on exon 20 and G719X, S768I,
L858R and L861Q mutations. A suitable cancer area for genetic
analysis was selected in all the samples by an expert pathologist
and manually dissected. DNA was extracted by MagneSil® Genomic Fixed Tissue System (Promega, Madison, USA). EGFR
results were reported as positive (presence of EGFR mutation),
negative (wild-type EGFR) or not amplified (no DNA amplification). Both in positive and not amplified samples the percentage
of cancer cells and the mean cancer cells were retrospectively
evaluated to estimate the “adequate” cellularity for EGFR analyses in cytological samples.
Results. The cytological samples included: fine needle aspirations (n=74), pleural fluids(n=9), bronchoalveolar lavage (n=6),
bronchial brushing (n=2), bronchial aspirate (n=2) and ascitic
comunicazioni orali
fluid (n=1). As some cytologic samples came from different
hospitals, several type of fixative were used: ETOH95% (n=
45), ThinPrep® (n=22), Duboscq-Brazil (n=13),CytofixTM (n=5);
in one case, the sample was unfixed. In 8 samples the fixative
used was unknown. EGFR mutations were found in 17 samples
(18,09%; deletions exon 19, G719X, L858R, L861Q), whereas
63 (67,02%) were negative and 14 (14,89%) were not amplified.
Among amplified samples 37 cases were fixed by ETOH95%,
22 by ThinPrep®, 8 by Duboscq-Brazil, 4 by CytofixTM and one
was unfixed. The percentage of cancer cells in the area selected
for analysis in mutated samples ranged from 15% to 80% (mean
62,5%); in these cases a mean of 210 cancer cells was analyzable.
Notably, mutations were detectable also in one sample with less
than 15% of cancer cells and in another one with only 16 cancer
cells. The percentage of cancer cells in not amplified samples
ranged from 10% to 80% (mean 46%); among them 8 cases were
fixed by ETOH95%, 5 by Duboscq-Brazil and one by CytofixTM.
Notably, in all samples fixed with ThinPrep®, the EGFR status
was detectable.
Conclusions. This study provided evidence that cytologic material is most often suitable for detecting EGFR status, even in
samples with few neoplastic cells, if a sensitive and specific assay is used. The type of fixative does not preclude the success of
analysis, even if a better chance is given by ThinPrep®. The lack
of DNA amplification could depend more on the time of fixation
or on other unpredictable factors than the different methodologies
and preparation used.
Rapid on-site cytologic evaluation of imprint
from transbronchial needle aspiration (TBNA) lung
biopsies
C. Scacchi, L. Chiapparini, E. Bresaola, M. Lusiardi, C. Di
Tonno, C. Casadio
Unit of Diagnostic Cytology, European Institute of Oncology, Milan,
Italy
Background. TBNA biopsies taken during bronchoscopy under
fluoroscopy guidance is a minimally invasive nonsurgical procedure to reach the diagnosis of primary or metastatic lung cancer
and to inform the sistemic treatment. Ensuring adequacy of tumor
specimens is particularly important for the management of those
patients with advanced disease at the time of first presentation
and, therefore, not eligible for surgery.
Since March 2011, in our Institute, TBNA biopsies are performed
by thoracic surgeons supported by a cytopathologist for on-site
cytologic evaluation of adequacy of the specimens.
Materials and methods. Since March 21st 2011 to June 20th
2011, 56 patients were submitted for TBNA lung biopsies in our
Institute.
The specimens were first used for delicate imprints, then formalin
fixed. The first two to four smears were quickly ethanol fixed
and stained with a rapid H&E staining for immediate evaluation
of adequacy. The procedure is continued until adequate sampling
is confirmed, pending patient tolerance. Adequacy was defined
when lung parenchimal cells and/or cancer cells were present,
while bronchial cells alone were considered as inadequate.
Results. In 28 cases (50%) the samples were judged as adequate
at the immediate evaluation which was confirmed by subsequent examination of all the smears: 27 were positive for tumor
cells, 1 was negative. The corresponding lung biopsies confirmed
the diagnosis in 24 cases, while in 4 of them the tissue was
crushed and an histological diagnosis was not achievable.
15 out of the 18 inadequate cases (32,2%) were confirmed both
on definite cytology and corresponding biopsies; in 2 of the
remaining cases, the additional smears and the related biopsies
were adequate for diagnosis (1 positive case and 1 negative). In
the last of inadequate cytologic case, both on rapid stained and on
remaining smears, the biopsy was diagnostic for lung cancer.
157
In 10 cases (17,8%) the cytologic diagnosis on rapid stained
smears was inconclusive, but 4 of them were judged atypical on
definite smears: 2 had positive biopsies, 1 had an inflammatory
biopsy and 1 had an inconclusive biopsy.
In 17 cases (9 adequate, 5 not adequate, 3 inconclusive) the patients underwent surgery with the following diagnoses: 12 non
small cell lung cancer (NSCLC), 1 metastatic adenocarcinoma, 4
non neoplastic lesions.
In 7 cases the biopsies were submitted also for molecular analysis
and in 1 case a K-ras gene mutation was found.
Conclusions. Rapid on-site cytology is invaluable in the assessment
of specimens during bronchoscopy for TBNA. Ensuring adequacy
of the specimens is particularly important for those patients with
advanced disease at the time of diagnosis and, therefore, not eligible
for surgery. Adequate samples obtained through TBNA for pathologic diagnosis as well as molecular analysis will be of immediate
importance for personalized management of lung cancer patients.
In our short experience, on-site cytologic diagnosis during TBNA
was confirmed by definitive cytology and histology in 39 cases
(24 adequate and 15 not adequate). Two more cases were inadequate on rapid stained smears, but adequate on the remaining
smears and on histology.
Some technical errors can be responsible for inadequacy both
in cytology and in histology: a too delicate imprint of the lung
biopsy can release only superficial bronchial cells on the smears,
while a heavy imprint can damage the tissue with crushing artifacts.
More practice, a continuous monitoring of data and a good clinical selection of patients will allow us to improve our results.
Heterogeneity of large cell carcinoma of the lung:
an immunophenotypic and mirna based analysis
M. Barbareschi MD 1,2,3, C. Cantaloni PhD 1,3, V. Del Vescovo
PhD 4, A. Cavazza MD 5, R. Carella MD 7, G. Rossi MD 8, G.
Pelosi MD9, P. Graziano MD 10, M. Papotti MD 6, A.M. Denti
PhD 4
1
Unit of Surgical Pathology, 2 Laboratory of Molecular Pathology, 3 Trentino Biobank, Unit of Surgical Pathology, S.Chiara Hospital, Trento, Italy;
4
Centre for Integrative Biology, University of Trento, Trento, Italy; 5 Unit
of Pathologic Anatomy, Arcispedale S. Maria Nuova, Reggio Emilia, Italy;
6
Unit of Pathologic Anatomy, San Luigi Hospital and University of Turin,
Orbassano, Italy; 7 Unit of Surgical Pathology, S.Maurizio Hospital, Bolzano, Italy; 8 Section of Pathologic Anatomy, Azienda Ospedaliera-Universitaria Policlinico, Modena, Italy; 9Unit of Pathology and Laboratory
Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;
10
Unit of Pathologic Anatomy, Forlanini Hospital, Rome, Italy
Introduction. Large cell carcinoma (LCC) of the lung are heterogeneous and may be of different cell lineage.
Material and methods. We analyzed 56 surgically resected
lung tumors, classified as LCC on basis of pure morphological
grounds, using a panel of immunophenotypic markers (TTF-1,
citokeratin 7, napsin A; SQCC, p63, cytokeratin 5, desmocollin
3 and Δnp63) and the quantitative analysis of miR-205 (mi-R
sample score method, mRSS).
Results. Based on immunoprofiles 19 (34%) cases were reclassified as adenocarcinomas (ADC), 14 (25%) as squamous
cell carcinomas (SQCC), and 23 (41%) cases were unclassifiable. According to the mRSS 36 cases were classified as ADC
and 20 as SQCC. Of the 23 cases unclassifiable on the basis of
the immunoprofiles, 18 were classified as ADC and 5 as SQCC
according to mRSS.
Discussion. Our data show that an extended panel of immunohistochemical markers, can re-classified around 60% of LCC as
ADC or SQCC. However a relevant percentage of LCC may escape convincing immunohistochemical classification, and mRSS
could be used for further typing, but its clinical relevance needs
further confirmation.
158
This study has been supported by grants of the Provincia Autonoma di Trento and of the Fondazione Cassa di Risparmio di
Trento e Rovereto.
EGFR mutations and squamous cell carcinoma
of the lung
P. Viola*, M.G. Sciarrotta**, S. Malatesta**, L. Felicioni***, L.
Guetti****, F. Mucilli****, T. D’Antuono*, F. Buttitta***, A. Marchetti**
*
Unità Operativa di Anatomia Patologica, Ospedale Clinicizzato di Chieti, Chieti, Italia **Centro di Medicina Molecolare Predittiva, Fondazione
- Università “G. d’Annunzio” Università di Chieti - Pescara, Chieti, Italia; *** Medicina Molecolare Oncologica e Cardiovascolare, Fondazione Università “G. d’Annunzio” Università di Chieti - Pescara, Chieti, Italia;
****
Dipartimento di Chirurgia, Università “G. d’Annunzio” Università di
Chieti - Pescara, Chieti, Italia
Background. EGFR mutations have been reported in NSCLC
patients with major responsiveness to specific EGFR tyrosine
kinase inhibitors (TKIs), such as Gefitinib and Erlotinib. Several
studies have been conducted in order to correlate EGFR mutations with clinicopathological data. Result from these studies indicate that EGFR mutations are more frequent in asiatic patients,
patients affected by lung adenocarcinoma (ADC), never smokers,
and females.
In recent studies, conducted mainly on East-Asian patients and
biopsy material, EGFR mutations have been reported in a minority (2-3%) of SCC, suggesting that mutational analysis should be
performed not only in adenocarcinoma, but also in SCC to allow
accurate diagnosis and treatment.
Since data about the relationship between EGFR mutations and
SCC in white patients are poor, we decide to address this point
by a multiple approach on large series of resected tumors from
Caucasian patients affected by NSCLC.
Matherial and methods. Two hundred and eight resected
SCC from Caucasian patients were investigated for EGFR
mutations with two different high sensitive mutation detection
techniques:Single-Strand Conformation Polymorphism (SSCP)
and High Resolution Melting (HRM) analysis. At the same time,
we decided to accurately evaluate the presence of solid areas in
a series of 54 resected lung ADC known to harbour EGFR mutations and performed immunohistochemical staining with thyroid
transcription factor 1 (TTF1) and p63 for a more precise characterization of these areas. In addition, we decided to investigate
a series of 10 resected lung tumours with histological diagnosis
of adenosquamous carcinomas by immunohistochemistry and
EGFR mutational analysis.
Results. No EGFR mutations were found in the series of 208
SCC. Morphological examination of 54 adenocarcinomas carrying EGFR mutations displayed the presence of solid areas in 10
(19%) cases and in 4 (7%) of them, focal squamous-like patterns
with intensive p63 staining were seen. The 10 adenosquamous
tumors were all confirmed immunohistochemically by TTF-1 and
p63 and one of them carried an EGFR mutation (del E746_T751
Ins A).
Conclusions. Based on the literature, current guidelines suggest
EGFR mutational analysis in lung cancer patients with adenocarcinoma, large cell carcinoma, adenosquamous carcinoma and
NSCLC not otherwise specified. SCCs were not included among
the tumors to be tested because of the low frequency of EGFR
mutations in this histotype. Our data confirm, in a large series
of Caucasian patients, the extremely low prevalence of EGFR
mutations in resected SCC. The adenosquamous carcinomas in
our series showed EGFR mutations in 10% of cases, in agreement
with previously reported data. In addition, we have observed that
lung ADC harbouring EGFR mutations can rarely show focal
squamous-like patterns within solid areas. On the basis of our
results, we speculate that when specimens are obtained from
surgical resections, the histologic diagnosis is accurate because
of the abundant material available for the morphological and
immunohistochemical analysis, allowing to differentiate ADC
from SCC in the vast majority of cases. In this type of samples,
EGFR mutations are extremely rare or not present at all. On the
other hand, when specimens for mutational analysis are obtained
from small biopsies, the accuracy of histopathological diagnosis
is necessarily lower due to the paucity of the samples and tumor
heterogeneity. Therefore, EGFR mutations in biopsy samples
with diagnosis of SCC could be present because the biopsy has
been performed in areas with squamous patterns within an ADC
or adenosquamous carcinoma. We think that in future guidelines
these considerations should be taken into account and the possibility of testing EGFR mutations in patients with diagnosis of
SCC should be considered, if not always, at least in particular
subsets of patients such as non-smokers, and females.
EGFR testing in NSCLC: the first year of experience
G. De Maglio1, G. Fasola2, A. Sibau2, A. Iop3, C. Rizzi4, A. Del
Conte5, S. Sulfaro6, G. Adami7, E. Vigevani8, V. de Pangher
Manzini9, A. Brollo10, A. Colonna11, S. Cernic1, E. Masiero1, J.
Menis2, S. Pizzolitto1
SOC Anatomia Patologica, Azienda Ospedaliero-Universitaria Santa Maria della Misericordia, Udine, Italy; 2Dipartimento di Oncologia,
Azienda Ospedaliero-Universitaria Santa Maria della Misericordia, Udine, Italy; 3SOC Oncologia, A.S.S. n.5 «Bassa Friulana», Italy; 4Servizio
di Anatomia ed Istologia Patologica, A.S.S. n.5 «Bassa Friulana», Italy;
5
SOC Oncologia Medica, Azienda Ospedaliera Santa Maria degli Angeli,
Pordenone, Italy; 6SOC Anatomia Patologica, Azienda Ospedaliera Santa
Maria degli Angeli, Pordenone, Italy; 7Oncologia, Ospedale di San Daniele, Italy; 8SOC Oncologia, ASS N°3 Alto Friuli, Italy; 9SOC di Oncologia,
ASS2 «Isontina», Italy; 10Anatomia Patologica, Ospedale San Polo, Monfalcone, Italy; 11Anatomia Patologica, Ospedale San Giovanni, Gorizia,
Italy.
1
Background. EGFR gene mutations have a key predictive role
to guide molecular targeted-therapy with anti-EGFR treatment in
non small cell lung cancer (NSCLC). Since July 2010 EGFR mutation status assessment has become mandatory in the selections
of NSCLC patients suitable for gefitinib treatment according to
national and international guidelines.
Methods. In the period July 2010-June 2011, 244 NSCLC
patients from several Medical Oncology Departments of Friuli
Venezia Giulia were screened for EGFR mutations in exons 1819-20-21. Samples were obtained from surgery (90; 37%), bronchoscopy (117; 48%), CT-guided FNAB (29; 12%) or effusion
aspiration (8; 3%). They resulted in small endoscopic biopsies
(117; 48%), surgical specimens (94; 39%) and cytological specimens (33; 13%), either paraffin-embedded pleural fluids (15; 6%)
or smeared washing/brushing (18; 7%). Among all samples, 177
(73%) derived from primary tumors, 37 (15%) from metastatic
sites and 30 (12%) from loco-regional and non regional lymphnodes. Cyto-histologic diagnosis were distributed as follows: 131
(54%) adenocarcinoma, 23 (9%) squamous carcinomas, 8 (3%)
mixed adeno-squamous carcinomas and 82 (34%) NSCLC NOS
(Not Otherwise Specified).
DNA extraction was performed using QIAamp DNA Mini kit
(QIAGEN, Germany). Gene status was assessed by pyrosequencing technology, accordingly to manufacturer’s instructions with
EGFR TKI response® (sensitivity) (Diatech Pharmacogenetics,
Italy) for the assessment of activating EGFR mutations in exons
18, 19 and 21 and EGFR TKI response® (resistance) (Diatech
Pharmacogenetics, Italy) for resistance related mutations in exon
19 and 20. Pyrosequencing analysis was performed on PyroMark
Q96 ID instrument (QIAGEN, Germany).
Results. Among the 244 tested patients, 42 (17%) harbored
EGFR mutations and 39 (93%) of them were activating. There
were distributed as follows: 5% in exon 18 (G719S/D: 1 case and
G719C: 1 case), 45% in exon 19 (E746-A750del: 12 cases; L747-
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S752del: 1 case; L747-A750>P: 1 case; complex mutation CDS
2234-2258: 1 case; suspect of mutation/indeterminate result: 4
cases) and 43% in exon 21 (L858R: 14 cases; L861Q: 3 cases and
suspect of mutation/indeterminate result; 1 case) and 7% in exon
20 (T790M: 1 case; D770>GY: 1 case and indeterminate level of
D761Y mutation: 1 case).
In 39 (16%) cases -31 (13%) small histologic biopsies and 8 (3%)
cytological specimens- sampling was not optimal because of too
low cancer cells enrichment/not enough cellularity for efficient
DNA genomic extraction.
Results were accessible to the oncologist in a medium time of 6,5
consecutive days from the availability of the sample.
Conclusions. Our data confirmed both frequency and distribution
of mutations reported in literature.
Various types of samples have been tested and all of them, even
cytologic specimens and very small biopsies, revealed diagnostic
results.
To reduce the inadequate/indeterminate rate of results, better
compliance should be reached with endoscopists in order to have
more representative samples to analyse.
A multidisciplinary discussion between pathologists, molecular
biologists, medical oncologists, radiotherapists, surgeons, pneumologists and radiologists is warranted.
Pyrosequencing techniques demonstrated a good performance in
term of sensitivity and allowed an efficient turn-around-time for
clinical purposes.
Ultrastructural microanalysis for metal
contaminants assessment in pleuro-pulmonary
cancer
M. Scimeca, E. Bonanno, A. Volpe, A. Colantoni, L.G. Spagnoli
Dipartimento di Biopatologia, Università di Roma Tor Vergata, Roma,
Italia
Background. In the large urban centers of industrialized countries a strong correlation between mortality caused by cardiorespiratory diseases and atmospheric concentrations of particulate
(from particles below 10 um, called PM 10, to the smaller ones
found till now, the so called PM 2.5) has been demostrate.
Although threshold values of pollution are well established in
environment, there are few data on the particles accumulation
and thresholds within the tissues particularly with reference to the
pathogenesis of pulmonary and cardiovascular disease.
Among the elements which take part of PM 10 and PM 2.5 classification, heavy metals are considered very interesting particles
due to their possible toxic effects induced by bio-accumulation.
Toxicity of these elements is well supported by in vitro experimental data and it could come from metal poisoning that is accompanied by a set of recognizable acute manifestations.
Anyway, the long term effects induced by bioaccumulation of
these elements and their involvement in chronic diseases are still
controversial and subject of many recent studies.
In this work lung and pleural tissue samples have been studied
through ultrastructural (electron microscopy) and atomic-molecular investigations (microanalysis) in order to identify tissue
storage of elements by bioaccumulation.
Due to the potential pathological impact of many air particles on
the human health, this method of investigation could be enlightening about the unknown role of the pollution long term effects.
Methods. In this study were included 16 cases of mesothelioma,
6 cases of pleural fibrosis, 5 lung cancers, 8 control tissue from
both lungs and pleuras of subjects without pleuropulmonary
diseases. For the ultrastructural and microanalysis study the tissues were embedded in hepoxidic resin, slices of 1250 nm and
70-100 nm respectively for light microscopy visualization (after
toluidine blue staining) and electron microscopy visualization
(after uranium and lead heavy salts staining).
All samples were displayed on TEM Hitachi H-7100 paying
attention to acquire digital images at different degrees of magnification.
Elementar analysis have been carried out doing 15 microanalitical
acquisitions for each section at 12000x of magnification keeping
the light spot to the maximum allowed diameter.
Results and conclusions. The tissue contaminants that we observed were: chrome, manganese, alluminum, palladium and
cobalt in mesothelioma; vanadium, cadmium and zinc in lung
cancer; manganese, alluminum, silver, cadmium in pleural fibrosis. Tin, in low concentrations, was detected in all pathological
tissues.
Mesothelioma, pleural fibrosis and lung cancer samples explored
by ultrastructural microanalysis showed a so surprising variability of elemental composition that let us to suppose that these
tissues could be have an anatomo-functional predisposition for
the elements bioaccumulation.
Unexpectedly, the multidisciplinary study of mesothelioma
through conventional histology, electron microscopy and microanalysis did not detect the presence of classic asbestos fibers in our
pathological tissues.
We can conclude that ultrastructural microanalysis is a suitable
technology for the study of the tissue sites where elements bioaccumulation takes place.
The presence of metallic elements in the examinated tissues rises
two fundamental questions:
Is bioaccumulation of pollutants to be considered as a primary
insult for the tumorigenesis or rather is it to be related to intrinsic
characteristics of tumor tissue that, for unknown reasons, tend to
behave as a storage tissue of the elements?
Could bioaccumulation of these elements represent the ‘”missing
link” for the understanding of the relationship inflammation /
tumor, or vice versa?
The data presented in this work, if they were supported by other
experimental evidence, could shed new light on the relationship
between environment, genetics and human health.
The pathologist’s role in the asbestos
investigation in Italy
D. Bellis * **, D. Antonini *, D. Belluso
Rinaudo ****, M. Musa ****, A. Croce ****
***
, S. Capella
, C.
***
*
ASLT01, Ospedale Martini, Servizio di Anatomia Patologica, Torino; **
Centro Interdipartimentale “G. Scansetti” per lo studio degli Amianti e
di Altri Particolati Nocivi dell’Università di Torino; ***Dipartimento di
Scienze Mineralogiche e Petrologiche dell’Università di Torino; ****Dipartimento di Scienze dell’Ambiente e della Vita – Università del Piemonte
Orientale, Alessandria
The last few years have witnessed an increase in the interest in the
selection of appropriate criteria able to recognise asbestos related
diseases (pleural plaques, pulmonary fibrosis, mesothelioma and
lung cancer), in particolar in the medico-legal elements involved.
As these criteria are of a clinical instrumental and morphological
nature, the pathologist role has become more crucial in the cytohistological phase. The request to determine wheter there is a link
between the disease observed (on biopsy and/or surgical specimens
and/or autopsy) and the work activity where occupational exposure
to asbestos has been confirmed, is constantly increasing on the part
of magistrates, I.N.A.I.L. and other such entities.There are several
phases involved: a) establishing an accurate work anamnesis along
with an environmental and paraoccupational one to define the
original source of the asbestos exposure, b) to make the correct diagnosis of the disease reported, c) to identify morphological markers of professional asbestos erxposure (pleural plaque, fibres and
particles in biological material) and c) assess the causal link. All of
which requires having the relevant clinical information and medical history (including set questionnaires e.g. those used in Italian
National Mesothelioma Register), be in possession of radiological
160
evidence and examine biological material for the presence of any
asbestos fibres, so a to evaluate any causal factors. The cytohistological analysis does not only involve an accurate diagnosis of
the neoplasia (mesothelioma, lung cancer vs secondary metastatic
lesions) but also the exclusion or confirmation of asbestos-induced
pulmonary fibrosis in the lung not involved in the neoplasia. In
case of a non-neoplastic disease, a differential diagnosis is to be
made between the different forms of pulmonary fibrosis to identify
any asbestos-induced fibrosis. The main point is that of examining the biological material for the presence or absence of mineral
fibres. Light microscopy does not suffice to this aim and is to be
supplemented with more complex techniques such as Transmission Electron Microscopy (transmission and scanning fitted with
microanalysis and analysis of the crystallinity of the mineral material). This technique requires the removal of the organic component
from the biological material to obtain a concentration of the mineral component and more dust. However, there is a lack of national
guidelines that standardize this type of sampling and mineralogical
analysis. To this aim, a technical workgroup, “Analisi biologiche
dell’amianto” which involves “equipment and analytical methods
for the mineralogical analysis of asbestos fibres and asbestos bodies in tissues and biological fluids” was set up, in 2010 within
the “Dipartimento della Prevenzione e Comunicazione Direzione
Generale della Prevenzione Sanitaria del Ministero della salute”.
However, this type of analysis requires digestion of the organic
material to obtain a higher concentration of organic material and,
therefore, the loss of relationship with the underlying pathological
lesion. This prompted interest in an alternative technique to be used
directly on tissue without the destruction of the organic material.
This technique is Raman spectroscopy, where histological section
by paraffin embedded specimens can be used to obtain a qualitative
analysis of the mineral present in the biological material e.g. lung
and neoplastic tissue. The pathologist has also come up against new
challenges related mainly to environmental exposure (i.e. tunnel
construction at sites with green serpentine, or sites adjacent to the
cement-asbestos industry and/or asbestos mines). These activities
have forged an alliance between the pathologist and other professionals, like mineralogists, geologists and veterinaries and it was
only thanks to these multidisciplinary teams that innovative data
for the evaluation of the causal link often required by judicial bodies came to light.
Pleural fluid mesothelin for the differential
diagnosis of malignant pleural mesothelioma
effusions and its contribution to cytology
examination
S. Roncella*,**, P.A. Canessa*, S. Colli*, B. Bacigalupo*, P. Ferro*
E. Battolla*, C. Manta*, M.C. Franceschini*, M. Sivori *, A.
Vigani*, V. Fontana***, MP. Pistillo***, F. Fedeli*
**,
*
ASL5 “Spezzino”, La Spezia; ** AIL F. Lanzone, La Spezia; *** National
Institute for Cancer Research (IST), Genova
Background. Malignant pleural mesothelioma (MPM) is a
highly aggressive tumor with a poor survival rate. Tumor-related
biomarkers, such as soluble mesothelin-related peptide (SMRP),
can be useful aids in the diagnosis of MPM.
It has been reported that the serum levels of SMRP are higher
in patients with MPM than in other patients. Therefore, serum
SMRP was proposed as a marker for mesothelioma diagnosis
1
. Few studies have shown the potential value of SMRP for the
differential diagnosis of pleural effusions (PE) 2-5. In the present
study we assessed the PE levels of SMRP from a large panel of
patients and investigated the value of SMRP as an adjunctive test
to cytology in the diagnostic evaluation of MPM PE.
Methods. We evaluated SMRP in a total of 211 PE (38 from
MPM, 71 from non-MPM pleural metastasis (MM) and 102 from
benign PE by means of MesoMark enzyme-linked immunosorbent assay kit 6 (Fujirebio Diagnostic, Malvern, PA). Mesothelin
dignostic performance parameters were estimated through the receiver operating characteristic (ROC) analysis. In particular, the
area under the ROC curves (AUC) was used as an index of pure
accuracy, namely the overall proportion of correctly classified patients. Youden’s index was applied to obtain the biomarker’s cut
off level of maximum discrimination between patient groups. For
each cut off, empirical accuracy (Ac), positive (PPV) and negative (NPV) predictive values, along with sensitivity (Se) and specificity (Sp), were calculated. Finally, the degree of correlation
between SMRP levels and patients’ disease stutus was estimated
using the Diagnostic Odds Ratio (DOR). For each index 95%
confidence limits (95% CLs) were also computed and, wherever
appropriate, chi-square test was performed to assess the statistical
significance (P) of the study comparisons.
Results. The mean PE SMRP level was higher in MPM
(41.7±49.4 nM) than in patients with MM (8.8±14.1 nM) or
benign PE (4.8±8.4 nM). We found a statistically significant difference between SMRP levels in MPM vs benign PE (DOR=30.8,
P<0.001), vs MM (DOR=11.8, P<0.001) and vs all other PE
(DOR=15.7, P< 0.001).
The AUC for SMRP-differentiating MPM PE and benign PE
was 79.8 (cut off=10.8 nM, Se=65.8%, SP=94.1%, Ac=86.4%,
PPV=80.6% and NPV=88.1%), the AUC for SMRP-differentiating MPM PE and MM PE was 75.6 (cut off=11.8 nM, Se=63.2%,
SP=87.3%, Ac=78.9%, PPV=72.7%, NPV=81.6%). Finally, the
AUC for SMRP-differentiating MPM PE and all other disease PE
was 78.1 (cut off=10.9 nM, Se=63.2%, SP=90.2%, Ac=85.3%,
PPV=58.5%, NPV=91.8%).
At the cut off of 10.9 we found higher SMRP values in 25/38
(66%) MPM PE, 13/71 (18%) of MM PE and 8/102 (8%) of benign diseases PE. Sixteen out of 38 MPM (42%) (12 epithelial, 1
sarcomatoid, 3 others) were cytology-positive with 11 specimens
(68%) showing high levels of SMRP (8 epithelial, 3 others). In
contrast, 22 MPM, (9 epithelial, 8 sarcomatoid, 5 others) (58%)
were cytology-negative with 11 samples (50%) showing high
levels of SMRP.
Conclusion. Our findings show that SMRP level in PE, as reported in serum, is a promising diagnostic marker to distinguish
MPM PE from benign and MM PE. SMRP test may be useful in
adjunct to cytology for the routine screening of MPM PE.
Aknowledgement
Supported by grants from Ricerca Sanitaria Regione Liguria
2009.
References
1
Robinson BW, Creaney J, Lake R, et al. Mesothelin-family proteins
and diagnosis of mesothelioma. Lancet 2003;362:1612-6.
2
Scherpereel A, Grigoriu B, Conti M, et al. Soluble mesothelin-related
peptides in the diagnosis of malignant pleural mesothelioma. Am J
Respir Crit Care Med 2006;173:1155-60.
3
Creaney J, Yeoman D, Naumoff LK, et al. Soluble mesothelin in effusions: a useful tool for the diagnosis of malignant mesothelioma.
Thorax 2007;62:569-76.
4
Pass HI, Wali A, Tang N, et al. Soluble mesothelin-related peptide
level elevation in mesothelioma serum and pleural effusions. Ann
Thorac Surg 2008;85:265-72.
5
Davies HE, Sadler RS, Bielsa S, et al. Clinical impact and reliability of
pleural fluid mesothelin in undiagnosed pleural effusions. Am J Respir
Crit Care Med 2009;180:437-44.
6
Beyer HL, Geschwindt RD, Glover CL, et al. MESOMARK: a potential test for malignant pleural mesothelioma. Clin Chem 2007;53:66672.
comunicazioni orali
Patologia gastrointestinale
Gastric ulceration after selective internal radiation
therapy: a quite new entity in Italy, needing for
fast recognition
F.M. Bosisio**, M.E. Dinelli***, G. Cattoretti**, G. Bovo*,
Department of Pathology and Azienda Ospedaliera San Gerardo, Monza,
Italy; **Department of Pathology, Department of Surgical Science, Università degli Studi di Milano-Bicocca, Milano, Italy and Azienda Ospedaliera San Gerardo, Monza, Italy; ***Department of Digestive Endoscopy,
Azienda Ospedaliera San Gerardo, Monza, Italy
*
Introduction. Selective internal radiation therapy (SIRT) is a
modality of internal beam radiotherapy. Its use has shown a great
efficacy in treating both primitive and secondary unresectable
hepatic malignancies, obtaining a good response according to
the RECIST criteria 1 2. At the same time, its complications are
becoming more evident as this technique is utilized by a growing
number of centres.
Case report. A 71-year-old man, underwent selective radioembolization with Yttrium-90 microsphere for an unresectable
hepatocellular carcinoma. Gastroscopy showed an ulceration of
the gastric mucosa located in the angulus. Endoscopic samples
were taken from the whole gastric antrum and from the angulus.
Histological examination showed in three samples alterations
compatible with peri-ulcerative specimens, characterized by a
subacute flogistic infiltrate and gastric glands with regenerative
changes with some reactive atypia. In some areas, a few number
of neutrophils could be seen into the surface epithelium. Helicobacter pylori infection was not present.
The fourth biopsy was the one more referable to an ulcerative
area. Only in this specimen, a single violet sphere was recognisable in the lamina propria, circumscribed by some giant cells
with a foreign-body pattern of inflammation. Subsequent seriate
sections were cut, that showed more violet rounded bodies in the
lamina propria, with a total number of three spheres histologically
documented.
Due to this findings, the diagnosis of ulceration of the gastric
mucosa associated with Yttrium-90 microspheres was made.
Conclusion. As soon as new therapeutics techniques develope
and are put into quotidian practice, pathologists can meet new
histological entity due to the effects of such therapies. While
some of this effects are already well-known, rarer ones can lead
to misdiagnosis. One of the complication of the selective internal
radiation therapy (SIRT) with Yttrium-90 microspheres, although
not very frequent, is gastro-duodenal ulceration. Its histopathologic features are for the most aspecific, excepted the presence of
the microsphere in the tissue, that must be searched.
HER2 status is consistent in primary and
metastatic esophagogastric junction
adenocarcinomas
M. Fassan* **, K. Ludwig*, M. Pizzi*, V. Guzzardo*, M. Balistreri*, A. Ruol***, G. Zaninotto***, L. Giacomelli*, E. Ancona***
****
, M. Rugge* ****
*
Department of Medical Diagnostic Sciences & Special Therapies, University of Padova, Italy; **Department of Oncology & Surgical Sciences,
University of Padova, Italy; ***Department of Gastroenterological & Surgical Sciences, University of Padova, Italy; ****Istituto Oncologico Veneto
- IOV-IRCCS, Padova, Italy
HER-2-targeted therapy has recently been shown to be beneficial
for patients with advanced gastro-esophageal adenocarcinomas.
Differences in HER-2 dysregulation in primary tumors and metastases may help to explain therapeutical inconsistencies. The
aim of this study was to examine HER-2 status in primary and
paired nodal metastatic adenocarcinomas of the esophagogastric
161
junction (EGA). A series of 47 radically-treated EGA (38 male,
9 female; mean age 67.9, range 49-87; 7 Barrett’s adenocarcinomas) was considered. None of the patients had received radio- or
chemotherapy before surgery. For each case, 4 tissue samples
were obtained from each surgical specimen: 2 from the primary
adenocarcinoma and 2 from their paired metastatic lymph nodes.
HER-2 status was assessed by both immunohistochemistry (IHC;
PATHWAY® HER-2/neu [4B5]; Ventana Medical Systems) and
dual chromogenic in situ hybridization (CISH; duoCISHTM;
DAKO). No immunohistochemical staining was detected in 22
tumors (46.8%), 1+ score in 10 (21.3%), 2+ score in 6 (12.8%;
3 amplified at CISH), whereas 9 cases (19.1%) had a score 3+.
HER-2 amplification (25.5%) was significantly associated with
advanced tumor grades (Fisher’s; p=0.030) and stages (Fisher’s;
p=0.015). IHC scoring significantly correlated with CISH status
(agreement 93.6%; k=0.84; p<0.0001). In terms of intra-tumor
variability, the different tumor samples showed an excellent
concordance for both IHC (agreement 89.9%; k=0.75; p<0.0001)
and CISH (agreement 95.6%; k=0.88; p<0.0001) evaluation. In
44 cases, there was an excellent agreement between the primary
and metastatic cancers, while for three tumors with a score of 3+,
one of the corresponding lymph node metastasis featured no IHC
stain. Our results confirm, albeit in only a small series of cases,
that HER-2 is consistently expressed in primary EGA and their
nodal metastases. CISH evaluation is an adequate standard for
assessment of HER-2 amplification in adenocarcinomas of the
esophagogastric junction.
HER2 status in gastric cancer: concordance
between primary and distant metastatic lesions
G. Perrone*, M. Amato*, M. Callea*, D. Righi*, P. Crucitti**, R.
Coppola**, A. Onetti Muda*
*
Anatomia Patologica, ** Chiurgia generale/Università Campus Bio-Medico di Roma, Roma, Italia
Trastuzumab in combination with chemotherapy is being considered as a new standard therapeutic option for patients with
HER2-positive metastatic gastric or gastroesophageal junction
cancer (Bang et al, Lancet 2010). Therefore, an accurate assessment of HER2 status is essential to determine which patients
might benefit from trastuzumab. Although trastuzumab-based
therapy is used to treat metastatic disease, HER2 status is usually evaluated in primary lesions since metastatic sites are rarely
removed or biopsied before treatment. However, it is still unknown whether HER2 status differs in metastases compared with
primary tumours.
Aim of our work was to evaluate the concordance rate between
primary gastric cancer and distant metastastic lesions in terms
of HER2 status. Twenty-four gastric cancer patients with distant
metastatic disease were retrospectively selected on the basis of
availability of tissue from primary and metastatic lesions obtained
at surgery. Biopsy specimens from primary tumour were also
included in 12/27 cases. HER2 immunostaining was performed
using the Bond Oracle HER2 IHC System (Leica). The HER2
immunoreactivity was evaluated by an experienced pathologist
according to the scoring system of Hofmann et al (Histopathology 2008). Equivocal cases (IHC 2+ score) were subsequently
analyzed by the FISH method to detect HER2 amplification, using the SPEC HER2/CEN 17 Dual Color Probe Kit (Zytovision).
FISH images were processed with an Olympus MX60 fluorescence microscope (Olympus, Hamburg, Germany) equipped with
a 100-W mercury lamp.
HER2 amplification was observed in 6/24 (25%) specimens from
metastatic lesions, while it was present in 4/24 (17%) primary
tumours (surgical and/or biopsy specimens).
Concordance between primary and metastatic HER2 status was
91% (k-value 0,82). In particular, two patients with metastatic
HER2 positive lesions were found HER2 negative in the respec-
162
tive primary cancers. Interestingly, when analyzing HER2 status
on biopsy and surgical specimens from the same patient, concordance was 83% (k-value 0,66): two HER2 positive lesions on
biopsy resulted negative on surgical specimens. In addition, these
two patients resulted HER2 positive on their metastatic disease.
Our results show a good concordance rate in terms of HER2 status between primary and metastatic lesions, as well as between
biopsy and surgical specimens. However, an higher rate of HER2
positivity was found in metastatic lesions (25%) rather than in
the related primary cancer (17%). These results are in line with
data recently reported on breast cancer (Fabi A, et al. Clin Cancer
Res. 2011) and suggest that HER2 positive distant metastasis may
arise from a small, undetectable HER2 positive subclone within
primary disease. In conclusion, our data underline the importance
of testing, whenever possible, HER2 status of metastatic sites in
patients who are candidates for trastuzumab-based therapy.
Sessile serrated adenoma of the colon and
rectum: reassessment of 6 year cases of serrated
lesions according to 2010 who classification
(preliminary results)
F. Bono* **, G. Bovo* **, M.E. Dinelli***, G. Cattoretti* **
*
U.O. Anatomia Patologia, HSGerardo Monza, Italia; ** Dipartimento di
Scienze Chirurgiche, Università Milano-Bicocca, Monza, Italia; *** U.O.
Endoscopia Digestiva, HSGerardo, Monza, Italia
Introduction. In 2010 Snover et al edited the chapter about serrated lesions (SL) in WHO blue book. They categorize serrated
lesions in: hyperplastic polyp (HP), sessile serrated adenoma
without cytologic displasia (SSAC-), sessile serrated adenoma
with cytologic displasia (SSAC+), traditional serrated adenoma
(TSA), serrated poliposis (SP) and serrated carcinoma (SC). Morphologic characteristics of these lesions were argued for years
before this latest classification, some aspects remain uncleared, so
some differential diagnosis from other entities, like cloacogenic
polyp can be difficult.
Matherials and methods. Between 2005 and 2010 at our endoscopic unit were performed about 10,000 polipectomies afterwards sent to our pathologic department. 2 pathologists analized,
reviewed and reclassified 267 lesions (from 237 patients) located
in the colon and rectum removed between 2005 and 2010, searching in our electronic archive by their diagnosis with term as “sessile serrated adenoma”, “sessile serrated adenoma with low grade
dysplasia”, “serrate polyp”, “mixed serrated polyp”.
The same 2 pathologist reviewed further 1850 slides from 1061
patients who underwent to polipectomy with at least one diagnosis of HP, with the aim to find again other sessile serrated
adenoma underdiagnosed at first.
All the SL were reassessed according to 2010 WHO classification
proposed by Snover et al.
Results. According to literature, the prevalence of SL (excluding HP) in our case records is about 1,25% (0,86% SSA with
according by 2 pathologists, 0,08% TSA with according by 2
pathologists, 0,31% of not accorded SL, or SL needly of close
examination).
Mean size of these lesions was 0,86 cm, mostly located in right
colon (66%).
Reassessment according to 2010 WHO criteria of lesions at first
diagnosed as “serrated” (267 cases between 2005 and 2010) resulted as follows:
68 lesions were reclassified as SSAC- (25,5%), 10 SSAC+
(3,7%), 7 TSA (2,6%), 6 cloacogenic polyp (2,2%), 68 low grade
tubular adenoma (25,5%), 55 HP (20,6%), 1 high grade tubular
adenoma (0,4%), 1 DALM (0,4%), 23 cases had too much artefacts to be classified (8,6%) and on 28 cases there wasn’t agreement between the 2 pathologist and needed of more deepening
(10,5%).
Furthermore the review of the cases of polipectomy in which
there was at least one diagnosis of HP (about 1850 lesions) revealed the presence of 8 SSAC-, 1 TSA, 4 cloacogenic polyp, and
3 cases that need of more deepening; all these cases was at first
diagnosed as HP.
8 patients had multiple SL in the same endoscopic removing,
from these 1 patient had 3 SSA and another one had 4.
Discussion. The low prevalence of SL and their morphologic
characteristics makes particularly difficult to make the correct
diagnosis.
Moreover the detection of SL, with or without cytological dysplasia is crucial because the evolution of these toward (serrated)
carcinoma is faster than “classic” adenoma.
In our experience the most common pitfalls are: the wrong diagnosis as SSA of low grade tubular adenoma, expecially in right
colon (38 of 68 cases); the overdiagnosis of HP, mostly in the
left colon (35 of 55 cases) and the misdiagnosis with cloacogenic
polyp (6 cases) incorrectly defined as SSA, all located beyond
the sigma.
Similarly 9 cases out of 1850 (8 SSA and 1 TSA) were at first
erroneously classified as HP.
About cases in which there isn’t agreement, we explore the possibility to perform additional investigations, as immunoistochemical and molecular analysis to achieve a diagnosis more correct
as possible.
Analysis of the activating Kras mutations in
advanced colorectal tumors: a three-year
diagnostic experience
S. Mariani*, P. Francia di Celle**, C. Di Bello*, L. Bonello* **, D.
Toppino*, F. Tondat*, A. Barreca*, L. Molinaro*, P. Cassoni*, L.
Chiusa***, A. Sapino*, G. Inghirami * **
Dipartimento di Scienze Biomediche ed Oncologia Umana, sezione Anatomia Patologica, Università di Torino, Torino, Italia; ** S.C.D.U. Anatomia Patologica, CeRMS A.O. San Giovanni Battista di Torino, Torino,
Italia; *** S.C.D.U. Anatomia Patologica, A.O. San Giovanni Battista di
Torino, Torino, Italia
*
Introduction. The detection of activating KRAS mutations at codons 12 and 13 is mandatory to enrol advanced colorectal cancer
(aCRC) patients into selected anti-EGFR protocols.
Material and methods. A total of 839 formalin-fixed and
paraffin-embedded samples were studied at our institution during
the period October 2008-June 2011 for KRAS mutations. We
currently investigate the mutational status of the KRAS gene (at
codons 12 and 13) combining the results of two methods, previously validated in multiple inter-institutional programs: PCR
followed by direct sequencing (PCR-DS, sensitivity near to 20%)
and REMS-PCR, a technique for the selective enrichment of mutated sequences at codon 12 (sensitivity near to 0,1%). Recently,
two additional approaches were also investigated: PCR followed
by Pyrosequencing (PCR-Pyro, sensitivity near to 5%) and Fast
Cold-PCR followed by DS (FC PCR-DS, sensitivity near to 3%).
Pre-analytical, analytical steps and report formats are generated
in accordance to the SIAPEC-IAP recommendations.
Results. We have identified four categories: a) both REMS-PCR
and PCR-DS provided a concordant KRAS mutational status in
a total of 788 samples (94,6%), with 292 (37%) and 69 DNA
(8,8%) mutated at codon 12 and 13, respectively, and a total
of 427 (54,2%) wild-type (WT) samples. When the PCR-DS
was compared to the PCR-Pyro (22 samples), all data were
concordant; b) only 17 samples (2%) didn’t produce either PCR
or sequence products by REMS-PCR (n=10) or by PCR-DS
(n=7). This failure is principally due to technical limits of each
individual methods on DNA extracted from routine fixed tissues;
c) REMS-PCR detected mutations at codon 12 in otherwise WT
samples (24 DNA, 2,9%) by PCR-DS and based on the PCR band
intensities, on polyacrilammide gels, we estimated a mutation rate
under 1%. This result reflects the higher sensitivity of REMS-
163
comunicazioni orali
PCR approaches. Nevertheless, in accordance to the guidelines
of the European Society of Pathology, all 24 patients were defined as KRAS WT. Notably, ex-post analyses by FC PCR-DS
demonstrated the presence of mutated sequences in 4 of 15 DNA
previously defined as WT by PCR-DS; d) Finally, we identified
the presence of multiple mutated sequences in 7 DNA samples
(0,8%). These later findings were further confirmed by PCR-Pyro
and by FC PCR-DS in selected cases (n=3). Within these set, the
REMS-PCR could not identify the mutations, for the concomitant
and competitive presence of codon 13 mutated sequences.
Lastly, in 2010-2011, 20 of 480 samples did not meet the SIAPEC-IAP recommendations. Analysis of the adequate samples
demonstrated a rate of 55% WT (n=252) versus 45% mutated
(n=208) cases, similarly to un-adequate samples which were 60%
WT (n=12) versus 40% (n=8) mutated, respectively.
Discussion. Our results support the need of a multifaceted technical approach for KRAS mutational analysis. Nevertheless,
very different sensitivities among methods could produce non
concordant data, despite the physically enrichment of tumor areas
prior DNA extraction. Thus the selection criteria of tumor tissues
in relation of new and more sensitive techniques than PCR-DS
may be required. The clinical impact of anti-EGFR therapy in
patients with minor or multiple mutated tumor clones has to be
defined.
Conclusion. We believe that an integrated working group of
molecular biologists, pathologists and oncologists is necessary
to monitor the clinical follow-up in patients with minor mutated
clones and required to define new and more precise cut-off of
biological impact.
A multi-gene approach to better select colorectal
cancer patients for anti-egfr treatment
G. De Maglio*, G. Aprile**, G. Falconieri*, S. Lutrino**, E. Masiero*, M. Mazzer**, L. Foltran**, G. Fasola**, S. Pizzolitto*
SOC Anatomia Patologica, Azienda Ospedaliero Universitaria Santa
Maria della Misericordia, Udine, Italy; **Dipartimento di Oncologia,
Azienda Ospedaliero Universitaria Santa Maria della Misericordia, Udine, Italy
*
Background. Although KRAS status in codon 12 and 13 is the
only requested data for the selection of suitable candidates for
anti-EGFR therapy, it has emerged that other genes are involved
in the response to these agents. To verify if a multi-gene approach
may help in maximizing the benefit of anti-EGFR treatment, we
retrospectively investigated tumor gene status of KRAS, BRAF,
PIK3CA, and NRAS in 171 patients with colorectal cancer.
Methods. Formalin-fixed, paraffin-embedded samples of colorectal carcinomas, previously analysed for KRAS status were
tested for BRAF, PIK3CA and NRAS. Gene status was assessed
by pyrosequencing accordingly to manufacturer’s instructions
with Anti-EGFR MoAb response® (KRAS status), Anti-EGFR
MoAb response® (BRAF status), Anti-EGFR MoAb response®
(PIK3CA status), and Anti-EGFR MoAb response® (NRAS status) (Diatech, Italy) on PyroMarkTMQ96 ID instrument (Qiagen,
Germany).
Results. Among 171 tested patients, 99 (58%) harbored at least
one mutation, while 66 (39%) tumors had a KRAS mutations on
codon 12-13. We observed a mutation rate of 6%, 14% and 3%
for BRAF, PIK3CA and NRAS, respectively. In particular, mutations in KRAS were the following: G12V: 21; G13D: 15; G12D:
15; G12A: 5; G12S: 5; G12C: 3; G12F: 1; G13C: 1. On codon 61
and 146 of KRAS we found 2 cases with Q61H genotype, 1 case
Q61L, 1 case Q61R, 4 cases A146T and 1 case A146V. Among
KRAS wild-type cases, we reported 10 patients with tumors bearing mutated BRAF (exon 15, V600E). PIK3CA mutations were
distributed as follows: in exon 9 E545K: 13 patients; E542K: 4
patients; Q546P: 1 patient; and in exon 20 H1047R: 5 patients
and H1047L: 1 patient. NRAS mutations were mapped on codon
12 (G12D: 2 patients; G12A: 1 patient) and 61 (Q61L: 1 patient;
Q61R: 1 patient). NRAS mutations were always mutually exclusive with alteration in the other genes, while 15 carcinomas
carried PIK3CA mutations occurring with KRAS or BRAF mutations altogether. “All wild type” patients were 71 (42%).
Conclusions. In our series of consecutive colorectal cancer patients we found that the mutation rates of KRAS, BRAF, PIK3CA, NRAS were comparable to those reported in the literature.
Along to the paradigm of individualized treatment, identifying
“all wild type” cases may be useful to select patients with greater
chance to respond and those who may benefit from anti-EGFR
therapy. Further insights and prospectively validated investigations are however necessary before implementing a multi-gene
approach in clinical practice.
Characterization of MYC and MNT in colorectal
cancer patients
E. Curcio*, D. Romanelli*, E. Zanellato*, A. Bordoni**, L. Mazzuchelli*, M. Frattini*, V. Martin*, S. Crippa*
*
Institute of Pathology, Locarno, Switzerland;
Southern Switzerland, Bellinzona, Switzerland
**
Oncology Institute of
Background. Genetic deregulations involving the c-Myc oncogene have been observed in a lot of human tumors. Myc protein
plays a fundamental role in promoting cell proliferation and its
effect is finelly tuned by a series of repressors, of which the
most relevant is MNT. In colorectal cancer (CRC), Myc is altered by gene amplification in up to 25% cases and MNT loss of
expression has been associated with colorectal carcinogenesis.
As the precise interplay between Myc and MNT has not been
fully investigated in CRC specimens, the aim of this work is to
better characterize the roles played by Myc and MNT in CRC
pathogenesis.
Methods. We analyzed formalin-fixed paraffin-embedded tissues from 53 sporadic CRC patients. c-Myc gene status was
evaluated by FISH using c-Myc/CEP8 probes (Abbot Molecular). We considered c-Myc amplified tumors those cases showing a ratio between c-Myc gene and chromosome 8 centromere
> 2 in more than 10% of tumor cells. MNT gene status was
investigated by loss of heterozygosity (LOH) analysis by evaluating the status of six microsatellite loci spanning the chromosome region of 17p13.3 where MNT gene maps. We considered
MNT loss when we observed at least 30% signal reduction
intensity of one allele in the neoplastic tissue with respect to
the normal tissue in more than 30% of microsatellite loci tested.
Microsatellite instable or omozygous cases were considered non
informative.
Results. c-Myc gene status was not evaluable in 8 cases due
to poor hybridization signals. Out of the remaining cases, we
observed c-Myc amplification in 14/45 (31%) cases. MNT LOH
analysis was non informative in 6 cases. Out of the remaining 47
cases, MNT loss was found in 28/47 (58%) cases.
Thirty-nine cases were evaluable for both c-Myc and MNT. Out
of these, c-Myc amplification was found in 12/39 (31%) cases
and MNT loss in 25/39 (64%) cases. By comparing c-Myc and
MNT gene status, we observed that c-Myc amplification occured in 10/25 (40%) tumors with loss of MNT and only in 2/14
(14,28%) cases without MNT loss (p= 0,15).
Conclusions. In our cohort, c-Myc and MNT are deregulated in
a considerable number of CRC specimens confirming a key role
of these deregulations in the pathogenesis of CRC, although not
statistically significant (due to cohort size). Near the totality of
c-Myc amplified tumors showed MNT loss, possibly implying
that a synergistic effect of both these alterations is required in
CRC development. Further analysis are strongly recommended
to confirm these results on a larger series of cases.
164
Mast cells contribute to the engendering of a proinflammatory milieu towards mucosal damage in
coeliac disease
M. Cacciatore*, B. Frossi**, C. Guarnotta*, G. Gri**, A. Carroccio***, C. Pucillo**, M. Calvaruso*, A.M. Florena*, V. Franco*,
C. Tripodo*
Dipartimento di Scienze per la Promozione della Salute /Università di
Palermo, Palermo, Italia; ** Dipartimento di Scienze e Tecnologie Biomediche /Università di Udine, Udine, Italia; *** Unità Operativa di Medicina
Interna, Ospedali Riuniti Civili, Sciacca, Italia
*
Coeliac disease (CD), a common autoimmune disease of the
small bowel, is induced in genetically prone individuals by the
exposure to dietary gluten, and, particularly, to gliadin.
Although the pathogenesis of CD has been widely investigated,
the mechanisms underlying the engendering of a pro-inflammatory microenvironment towards tissue damage, and involving
a complex crosstalk between immune players of innate and
adaptive immunity, are mostly unknown. In this context, little is
known regarding the role of mast cells (MC).
More than mere effectors of allergic responses, MC have recently
come into focus as pleiotropic cells able to sense signals from the
surrounding microenvironment and to modulate their function
towards pro-inflammatory or regulatory outcomes 1. Such a multifaceted contribution of MC in inflammatory/immune responses
can be also envisaged for CD.
Aim of this study was to investigate the contribution of MC to
the pathological setting of CD. To this aim we tested the correlation between the presence and amount of infiltrating MC and the
degree of mucosal damage, as well as the correlation between
MC infiltration and that of other immune system components,
such as T- and B-cell subsets. Moreover, we investigated MC
response to antigenic challenging by gluten peptides in vitro.
We selected 63 consecutive cases of CD graduated according
to Marsh-Oberhuber sistem 2, and 5 normal control samples.
Immunohistochemistry was performed in order to highlight
the diverse cell populations: T lymphocytes (CD3; CD4; CD8;
FOXP3), B lymphocytes (CD20), plasmacells (CD138; IgA; IgG)
and MC (tryptase). Immunohistochemical analyses revealed that
the number of MCs in the lamina propria was significantly correlated with the severity of tissue damage, with MC increasing in
the late athrophic phase. We thus tested wether such correlation
between MC infiltration and damage was a specific feature of
CD. To this purpose, we extended our analyses in a group of 44
cases with variable degree of damage and comprising both CD
and non-CD samples according to serology. Notably, we detected
comparable pictures in both groups of cases, which suggested that
MC infiltration correlated with the degree of histological lesion
irrespectively of CD serology. Tissue damage proved also tightly
associated with T cell infiltration, regardless of their helper/
cytotoxic phenotype. Of note, Th17 proved mainly expressed
in the early phases of the inflammatory damage where they correlated with MC amount, in accordance with their alleged role in
the recruitment of immune effectors at sites of early infiltration.
This paralleled the dynamics of Th17 trafficking in other immune
diseases, such as experimental autoimmune encephalomyelitis.
No significant correlations were observed between B-cells and
damage or MC infitrates. Nevertheless, cases with higher MC
densities displayed a higher amount of IgA-expressing plasma
cells in the lamina propria. This was in line with our previous
observation of murine MC being able to affect B cell maturation,
towards plasma cell differentiation and IgA class switch 3.
MC are able to produce and release inflammatory mediators in
the mileu mainly through IgE-mediated activation. In CD setting,
MC activation might stem from stimuli potentially related to
gluten-derived antigens in an IgE-independent fashion. Following
ex vivo analyses highlighting the contribution of MC to damage, we assessed whether there was a direct interaction between
gluten-derived peptides and MC degranulation in vitro. We incubated human LAD2 MC with p31-43 peptides in vitro and found
a significant increase in IgE-indipendent MC activation and degranulation. Among factors released by MC was IL-6, whose role
in the engendering of the pro-inflammatory microenvironment of
CD deserves further investigation.
References
1
Frossi B, Gri G, Tripodo C, et al. Exploring a regulatory role for mast
cells: ‘MCregs’? Trends Immunol 2010,;31:97-102.
2
Oberhuber G, Granditsch G, Vogelsang H. The histopathology of coeliac disease: time for a standardized report scheme for pathologists.
Eur J Gastroenterol Hepatol 1999;11:1185-94.
3
Merluzzi S. Frossi B, Gri G, et al. Mast cells enhance proliferation
of B lymphocytes and drive their differentiation toward IgA-secreting
plasma cells. Blood 2010;115:2810-7.
Patologia dei tessuti molli
Overexpression of HOXC13 in the chromosomal
area 12q13-15 in human liposarcomas
F. Galletta*, M. Cantile*, F. Fazioli**, G. Liguori*, G. Aquino*, G.
Botti*, A. De Chiara*
Pathology Department, National Cancer Institute “Fondazione G. Pascale”, Naples, Italy: ** Orthopaedic and Sarcoma Department, National
Cancer Institute “Fondazione G. Pascale”, Naples, Italy
*
Introduction. Liposarcoma is the most common soft tissue mesenchymal neoplasm in adult and is characterized by a neoplastic
adipocytic proliferation.
Some subtypes of liposarcomas show aberrations involving the chromosome 12: the most frequent are the t(12;16) (q13;p11) in more
than 90% of myxoid liposarcoma and the 12q14-15 amplification in
the well-differentiated and dedifferentiated liposarcoma.
In this regions there are important oncogenes like CHOP
(DDIT3), GLI, MDM2, CDK4, SAS, HMGIC but also the HOXC
locus, involved in development and tumor progression. Preliminary data, obtained by MultiTumor Array (MTA) thecnology,
show that HOXC13 protein is absent in normal adipose tissue,
while is expressed in more than 25% of liposarcomas.
Aim. In this study we have built a Tissue Micro Array (TMA)
containing human adipose tumours, to analyze the chromosomal
area 12q13-15 amplification by FISH Thecnology, and correlate
this amplification with aberrant expression of HOXC13, included
in this chromosomal region.
Methods. We selected and inserted in a TMA 18 well-differentiated liposarcomas, 4 dedifferentiated, 11 myxoid, 6 pleomorphic
and 13 lipomas. We performed a FISH investigation with DDIT3
Dual Color Break Apart Rearrangement Probe to evaluate the
amplification of 12q13-15 region and its oncogenes. Then the
TMA was submitted to IHC analysis to evaluate the HOXC13
expression and these data were reevaluated with a Real-Time
PCR for gene expression.
Results and conclusion. FISH investigation showed the amplification of the 12q13-15 region where there are oncogenes like
MDM2, CHOP, CDK4 but also HOXC13 in almost all welldifferentiated and dedifferentiated liposarcomas. In the other
subtypes of liposarcomas and lipomas, no amplification of the
12q13-15 region was detected.
The IHC analysis showed the proteic expression of HOXC13 only
in well-differentiated and dedifferentiated liposarcomas. We performed also a Real-Time PCR to evaluated the gene expression
which confirmed the immunohistochemical results.
In conclusion, our data show that in well-differentiated and dedifferentiated liposarcomas the 12q13-15 amplification is associated
not only with the amplification of already well-known oncogenes,
but also with the HOXC13 gene overexpression.
165
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Cytoplasmic expression of WT1 in pediatric soft
tissue fibro-myofibroblastic tumor and tumor-like
lesions
G.M. Vecchio*, P. Greco*, F. Longo*, R. Alaggio**, P. Amico*, A.
Sabino*, F. Amore*, A. Bosco*, L. Salvatorelli*, G. Magro*
*
Dipartimento G.F. Ingrassia, Università di Catania, Catania, Italia;
Dipartimento di Patologia, Università di Padova, Padova, Italia
**
Wilms tumor transcription factor-1 (WT1) is encoded by Wilms
tumor suppressor gene located on chromosome 11p13. Nuclear
expression of WT1 is widely known in several tumors, including
nephroblastoma, ovarian and mesothelial neoplasms, Sertoli cell
tumor and desmoplastic small round cell tumor. Although WT1
cytoplasmic immunoreactivity was originally questioned, there is
increasing evidence that this staining truly reflects the presence of
the protein within the cytoplasm, suggesting its complex regulator activity in transcriptional/translational processes. Apart from
some tumors, such as vascular tumors and rhabdomyosarcomas,
WT1 cytoplasmic expression has also been documented in the
fibroblasts of some connective tissues and desmoplastic stroma
of some carcinomas. To the best of our knowledge, there are no
studies on WT1 expression in fibro-myofibroblastic lesions occurring in pediatric age.
Accordingly, the aim of the present study was to define whether
WT1 is expressed in pediatric soft tissue fibro-myofibroblastic
tumor and tumor-like lesions, to establish if it can be helpful in
the routine differential diagnosis. For this purpose, we investigated immunohistochemically the expression and distribution of
WT1 in nodular fasciitis, myofibroma/tosis, fibrous hamartoma
of infancy, lipofibromatosis, fibromatosis (desmoid tumor), and
congenital/infantile fibrosarcoma, using antibody clone 6F-H2
directed to the WT1 N-terminus of the protein.
Materials and methods. Tissues samples were collected from:
i) 5 cases of soft tissue nodular fasciitis (age: 8 to 17 years); ii) 6
cases of soft tissue myofibroma/tosis (age: 6 to 17 years); iii) 10
cases of soft tissue fibrous hamartoma of infancy (age: 2 months
to 2 years); iv) 2 cases of soft tissue lipofibromatoses (age: 5to
7 years); v) 4 cases of soft tissue fibromatosis (age: 10 to 18
years); vi) 4 cases of congenital/infantile fibrosarcoma (age: 2
to 4 months).
Results. Nodular fasciitis, fibrous hamartoma of infancy, and
fibromatosis (desmoid tumors) were negative to WT1, with the
exception of a focal cytoplasmic staining, ranging from 1 to 3%
of the cells, variably observed in the different lesions. In contrast,
all cases of myofibroma/tosis, lipofibromatosis and congenital/
infantile fibrosarcoma exhibited a strong and diffuse (more than
90% of cells) cytoplasmic staining for WT1. In all cases examined, WT1 was detected in the cytoplasm of endothelial cells of
intralesional blood vessels. No WT1 nuclear staining was seen in
any of the tissues examined.
Discussion. The present study first shows that WT1 is strongly
and diffusely expressed in the cytoplasm of some soft tissue
fibro-myofibroblastic lesions occurring in pediatric age. Interestingly, WT1 is expressed in myofibroma/tosis, lipofibromatosis
and congential/infantile fibrosarcoma, while it is absent or only
focally detectable in nodular fasciitis, fibrous hamartoma of infancy, and fibromatosis (desmoid tumor). These findings suggest
that WT1 cytoplasmic expression is not a reliable marker in
distinguishing benign from locally aggressive/low-grade fibromyofibroblastic lesions of soft tissue in pediatric patients. However, although we admit that histology remains pre-eminent in
the diagnosis of the above mentioned lesions, WT1 may assist in
the differential diagnosis between fibromatosis (desmoid tumor)
vs congential/infantile fibrosarcoma and fibrous hamartoma of
infancy vs lipofibromatosis.
Patologia pancreatica
The (changing) role of pathology in
defining resection margin status in radical
pancreaticoduodenectomy
G. Perrone*, D. Borzomati**, M. Callea*, M. Amato*, R. Coppola**, A. Onetti Muda*
Anatomia Patologica, **Chirurgia Generale, Università Campus BioMedico di Roma, Roma, Italia
*
Outcome of patients with pancreatic duct adenocarcinoma (PDAC)
is dismal, with an extremely poor 5-year survival rate below 5%.
As the response of this cancer to chemo- and radiotherapy is
limited, surgical resection currently represents the only chance
to improved survival. The rate of microscopic Residual Margin
(RM) reported in the literature varies markedly, ranging from
20% to 75%, thus severely hampering the prognostic significance
of the RM status. However, recent studies suggested that a careful and standardized examination of the surgical specimen may
significantly increase the rate of R1 resections. Consequently, R1
rates should be considered as a performance measure not only
for the surgeon, but also for the reporting pathologist. Aim of the
present study is to verify the efficacy and reliability of standardized axial slicing technique, compared with the “classical” (AFIP
2010) approach, in examining surgical pancreaticoduodenectomy
(PD) specimens.
We recently introduced at our Institution a standardized method
for the pathological examination of PD specimens. According
to this strategy, the entire surface of pancreatic head should be
examined as a surgical margin (circumferential margin). The
surface of the specimen was carefully inked using six different
colours in order to mark all margins: 1) anterior (green); 2) posterior (black); 3) SMV surface (blue); 4) SMA surface (red); 5)
neck (orange); 6) biliary (yellow).
The specimens were subsequently cut following the axial plane of
the pancreatico-duodenal block; a positive (R1) resection margin
was defined as the presence of tumour cells within 1 mm distance
by the inked margin. In order to evaluate the effective advantage
of the axial slicing approach, histological samples from 13 consecutive cases of PD resection operated at the beginning of 2010
and obtained with the “classical” slicing technique (Group 1)
were re-evaluated by a single pathologist and compared with 13
PD specimens processed according to the axial slicing technique
(Group 2). Histotypes, transection and circumferential resection
margins, total number of lymph nodes, number of metastatic
lymph nodes, T factor, N factor, differentiation grade, perineural
invasion, presence of non-invasive neoplastic lesions (IPMN and
PanIN) were recorded.
Group 1 specimens included 4 ampullary carcinomas, 4 pancreatic ductal adenocarcinomas, 2 endocrine carcinomas, 1
cholangiocarcinoma and 2 IPMN, while in Group 2 specimens
there were 11 pancreatic ductal adenocarcinomas and 2 cholangiocarcinomas. No differences were found between the two groups
in terms of resection margins status when considering a margin
as positive only when tumour cells were present at the surface,
i.e. clearance = 0 mm. Instead, using clearance <1 mm to define
the positivity of circumferential resection margins, axial slicing
technique showed a significantly higher number of positive cases
(p<0,05) than classical approach. Significant differences (Group
2 vs. Group 1) were also evident when considering total number
of lymph nodes (median 35 vs. 9), number of metastatic lymph
nodes (median 4 vs. 2), pN+ cases (11 vs. 5), extra-pancreatic
cancer growth (13 vs. 5) and perineural invasion cases (9 vs. 2).
Our results confirm that the axial slicing technique is a reliable and
effective approach in examining surgical specimens of PD and, more
generally, that the use of a meticulous histopathological approach,
as suggested in the recent literature, leads to an increased rate of R1
166
resections. It should be highly advisable that centres for pancreatic
cancer surgery adopt a common strategy about macro- and microscopic approach to PD specimens. A consensus meeting including
surgeons and pathologists should be soon realized to define common
guidelines with the goal to achieve comparable results.
5 years collection of pancreatic lesions eusfna
revision
V. Nirchio*, R. Lotta**, R. Gentile**, N. Muscatiello***, F. Tricarico****
SSD Cytopathology, departments of Pathology, Ospedali Riuniti, Foggia,
Italy; **U. O. Anatomic Pathology, ISMETT, Palermo, Italy; ***U.O.C. Gastroenterology Univ, Ospedali Riuniti, Foggia, Italy; ****U.O.C Emergency
Surgery, Ospedali Riuniti, Foggia, Italy
*
Background. Pancreatic fine needle aspiration citology is a rapid
safe accurate and cost-benificial modality of
investigation of pancreatic mass lesions. Cytodiagnosis rests as
much on morphological
examination and adequate spreading technique is important to
prepare cells for accurate microscopy
as well as is important the expertise of cytopathologist in pancreatic cytomorphology. In cases of
well-differentiated tumors the interpretation can be challenging.
Methods. The study is a retrospective audit of all pancreatic lesions FNA sampled from 2005 to 2010, at the
AOU «Ospedali Riuniti di Foggia». The series includes 80 patients, 34 women and 46 men.
All the smears were reviewed by one pathologist and classified as
in adequate, benign, borderline
and malignant. 15 cases between those diagnosed as malignant by
the internal cytopathologist were
reviewed by a second one from another institution for a double
check. The most representative
smears were demounted and stained again with immunoistochemical stain for KI67 and P16.
Results. The material was inadequate to provide the definitive
diagnosis in 10 cases (12%), cytological
examination provided a conclusive result in 70 cases. They were
classified as benign (27)
borderline (11) and malignant (32).
Regarding the double checked cases, the agreement between the
two pathologist was 80%. The
disagreement related to lesion classified as suspicious for malignancy by one of the pathologist and
as malignant by the other one.
In these cases ICC showed a low proliferation index and P16
negativity. Malignant lesions showed
high KI67 proliferation index and the positivity of P16 was unevenly expressed.
Conclusion. In the considered doubtful cases the double ICC for
KI67 and P16 was not so useful in
discriminating borderline from malignant lesions when diagnostic disagreement was there.
References
Bhatia P, Srinivasan R, Rajwanshi A, et al. 5-year review and reappraisal
of ultrasound-guided percutaneous transabdominal fine needle aspiration of pancreatic lesions. Acta Cytol 2008;52:523-9.
Different HER2 expression, gene amplification
and chromosome 17 copy number between
primary pancreatic adenocarcinoma, lymph node
metastasis and distant metastasis: implication for
HER2 status evaluation and therapy
S. Salvi*, S. Asioli**, S. Boccardo *, P. Ferro *** ****, N. Gorji***,
P. Dessanti***, M.C. Franceschini***, M. Truini*, S. Colli***, F.
Fedeli***, M.P. Pistillo*, S. Roncella***
*
National Institute for Cancer Research (IST) Genova; **Department of Biomedical Sciences and Human Oncology, University of Turin, Italy; ***ASL5
“Spezzino”, La Spezia; ****AIL Sezione “F. Lanzone”, La Spezia, Italy
Introduction. Pancreatic adenocarcinoma (PAC) remains an
incurable disease. The development of a new therapy with antip185HER2 monoclonal antibodies (Trastuzumab, Pertuzumab) has
been proposed although the status of HER2 gene in PAC and its
correlation with clinical history remain not completely defined.
We evaluated the expression of p185HER2, HER2 gene amplification and chromosome 17 centromere (CEP17) copy number in
PAC. In addition, we compared p185 HER2 expression and HER2
gene status of primary PAC, matched lymph node metastasis
(LMTS) and distant metastasis (DMTS).
Materials and methods. We analyzed 93 cases of PAC including
31 PAC at initial diagnosis, 22 LMTS and 40 DMTS. In particular, primary tumours were matched with hepatic DMTS in two
cases, with peritoneal and gastric DMTS in one case and with
LMTS in eleven cases.
We performed IHC, on paraffin-embedded tissues, using the
PATHWAY kit by Benchmark XT system (Ventana). To evaluate patterns of membranous immunoreactivity of p185HER2, a
score of 0 or 1+ was regarded as IHC negative and 2+ or 3+ as
IHC positive in 10% of the tissue section. FISH was performed
using the Pathvision® (Abbot) or ZytoLight kit (ZytoVision).
HER2 gene amplification was defined as a mean number > 6
signals per nuclei whereas CEP17 aneuploidy by a mean number
> 3 signals per nuclei.
Results. We found p185HER2 positive expression in 14/93 (15.0%)
(all with 2+ score), increased CEP17 with HER2 amplification
in 5/93 (5.4%), increased CEP17 without HER2 amplification
in 12/93 (12.9%) and disomic HER2 gene amplification in 2/93
(2.1%), of all PAC specimens analysed. In particular, p185HER2
expression was found in 1/31 (3.2%) of primary PAC, in 2/22
(9.1%) of LMTS and in 11/40 (27.5%) of DMTS. CEP17 aneuploidy was associated with HER2 gene amplification in 2/31
(6.4%) cases of primary tumours, in 1/22 (4.6%) cases of LMTS
and in 2/40 (5.0%) cases of DMTS. CEP17 aneuploidy status
without HER2 gene amplification was found in 1/31 (3.2%) of
the primary tumours, in 3/22 (13.6%) of the LMTS and in 8/40
(20.0%) of the DMTS. Finally, disomic HER2 gene amplifica-
Tab. I.
PAC specimens
Primary tumours (n=31)
Lymph node MTS (n=22)
P185
expression
score 2+
CEP17 aneuploidy
with HER2
amplification
CEP17 aneuploidy
without HER2
amplification
Disomic
HER2
amplification
Number (%)
Number (%)
Number (%)
Number (%)
1 (3.2)
2 (6.4)
1 (3.2)
0 (0.0)
2 (9.1)
1 (4.6)
3 (13.6)
0 (0.0)
Distant MTS (n=40)
11 (27.5)
2 (5.0)
8 (20.0)
2 (5.0)
Total (n=93)
14 (15.0)
5 (5.4)
12 (12.9)
2 (2.1)
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tion was restricted to 2/40 (5.0%) of DMTS (Table 1). Therefore,
we compared HER2 status between primary tumours, matched
LMTS and matched DMTS. Increased CEP17 was found in 3
cases of LMTS, but not in related primary PAC. One primary
PAC showed CEP17 aneuploidy, not found in matched hepatic
DMTS and, in one PAC, HER2 amplification detected in primary
tumour and in matched gastric DMTS was not found in matched
peritoneal DMTS.
Discussion. We found high heterogeneity of the HER2 status in
primary PAC and between primary PAC, LMTS and DMTS. Our
data suggest the need for the pathologist to evaluate more PAC
specimens to define the HER2 status finalized to carry out HER2targeted therapy.
Emolinfopatologia
PSGL-1 (CD162) as a potential target of
immunotherapy in anaplastic large t-cell
lymphoma
M. Calvaruso*, P. Macor**, P.P. Piccaluga***, N. Mezzaroba**, C.
Guarnotta*, M. Cacciatore*, A. Gulino*, G. Inghirami****, A.M.
Florena*, V. Franco*, S.A. Pileri***, C. Tripodo*
Dipartimento di Scienze per la Promozione della Salute /Università di
Palermo, Palermo,Italia; ** Dipartimento di Scienze della Vita/Università
di Trieste,Trieste, Italia; *** Dipartimento di Ematologia e Scienze Oncologiche/ Università di Bologna,Bologna, Italia; **** CERMS, Università di
Torino, Torino, Italia.
*
Anaplastic T cell lymphoma (ALCL) is an aggressive form of nonHodgkin’s lymphoma characterized by proliferation of large atypical lymphoid cells. ALCLs can be classified into two subsets based
on the presence or absence of the t(2,5) translocation generating the
oncogenic fusion tyrosine kinase NPM-anaplastic lymphoma kinase
(ALK). This different genetic hallmark is paralleled by a different
biological behaviour, since ALK+ patients have a more favourable
prognosis as compared with ALK- ones 1. The low incidence of
ALCL and the poor understanding of its pathogenesis contribute
to the lack of effective and standardized treatments. Monoclonal
antibodies (mAbs) has proved effective in the treatment of several
haematological neoplasms. Yet, their application to T-cell lymphomas has been so far limited by the lack of appropriate antigens for
targeting tumor cells and by the disappointing clinical responses 2.
We have recently reported the constitutive expression of P-selectin
glycoprotein-1 (PSGL-1/CD162) on normal and neoplastic plasma
cells. PSGL-1 proved to be a potential suitable target for mAb-mediated MM immunotherapy as demonstrated by in vitro cytotoxicity
of MM using a blocking mAb against PSGL-1 3.
In light of the known PSGL-1 expression on activated T-cells
and of its role in inducing activated T-cell apoptosis upon crosslinking, we aimed to test the suitability of PSGL-1 as a candidate
target of mAb immunotherapy in ALCL and other peripheral
T-cell lymphomas.
We first tested PSGL-1 expression by immunohistochemistry in
tissue microarrays obtained from 110 ALCL cases and 50 PTCLNOS and graded the staining intensity according to a four-grade
system (range 0 to 3). PSGL-1 was almost invariably expressed
by the neoplastic clone of ALCLs (104 cases, 94.5%) with a
median score of intensity of 3. Differently, 86% of PTCL-NOS
expressed PSGL-1 in the neoplastic clone with a median score of
2. These results were corroborated by GEP analysis on AITL (40
cases), ALCL (36 cases), ATLL (13 cases), and PTCL-NOS (67
cases) confirming the highest PSGL-1 expression in ALCL and
prompting us to further investigate the role of PSGL-1 in such
prototypical setting.
We therefore tested the expression of PSGL-1 in three human
ALCL cell lines (SU-DHL, TS and JB6) and in the PTCL cell line
(MAC1) using two different mAbs against PSGL-1: the blocking
KPL-1 and the TB5.
Following the evidence of elevated levels of PSGL-1 on the
surface of the three ALCL cell lines, we performed in vitro Complement Dependent Cytotoxicity (CDC), Antibody Dependent
Cell-mediated Cytoxicity (ADCC), and direct cytotoxicity assays. Complement activation following anti-PSGL1 mAbs binding resulted in the lysis of a fraction of ALCL cells ranging from
10% to 16% with the mAb KPL1 and from 4% to 15% with TB5.
Such limited ability of anti-PSGL-1 antibodies to kill ALCL cells
by complement fixation was likely due to the elevated expression
of the complement inhibitors CD46, CD55 and CD59 on target
cell surface, as determined by flow cytometry. Consistently,
neutralization of membrane-complement regulatory proteins significantly enhanced complement activity and ALCL cell killing.
ADCC induced tumor cell killing in a range between 7% and
17% with KPL-1 and between 15% and 47% with TB5, while
direct cytotoxicity ranged between 8% and 10% with KPL-1 and
37% and 92% with TB5. Altogether these data suggest a role for
PSGL-1 as a target of humoral immunotherapy in ALCL and support further in vivo studies assessing the effects of anti-PSGL1
blocking and agonist mAbs.
References
1
Rodig SJ, Abramson JS, Pinkus GS, et al. Heterogeneous CD52
Expression among Hematologic Neoplasms: Implications for
the Use of Alemtuzumab (CAMPATH-1H). Clin Cancer Res
2006;12:7174-9.
2
WHO Classification. Tumours of Haematopoietic and Lymphoid Tissues (IARC-October 2008).
3
Tripodo C, Florena AM, Macor P, et al. P-selectin glycoprotein
ligand-1 as a potential target for humoral immunotherapy of multiple
myeloma. Curr Cancer Drug Targets 2009;9:617-25.
Anaplastic large-cell lymphoma mimicking
a granulomatous lesion with necrosis
V. Tralongo, G. Becchina, C. Nagar, G. Ottoveggio, G. Scaglione, B. Giacalone, F. Genovese
Unità Operativa Complessa di Anatomia Patologica, Presidio Ospedaliero “G.F. Ingrassia”, Azienda Sanitaria Provinciale di Palermo, Palermo,
Italia
Introduction. When granulomas are present in association with
lymphomas, they are usually non necrotizing, sarcoid-like, although rarely granulomas with central necrosis occurs 1-3. Few
cases only are been reported in literature on the association of
granulomatous reaction with anaplastic large cell lymphoma
(ALCL); ALCL with histological features simulating a necrotizing granulomatous lesion, has not been reported previously in the
literature.
Case report. A 55-year-old man presented with a 3-month history of a painless, enlarged, lymph node localized on the left
inguinal region. Escissional biopsy was performed. The lymph
node was 2 cm in diameter and on cut surface it showed multiple,
round formations, yellow in colour.
At microscopic examination there was complete effacement of
nodal structure with multiple granulomas with central necrosis;
a diagnosis of granulomatous lymphadenitis with necrosis was
performed.
Two months later the patient returned because he had note the
appearance of a nodule in the right axillary region, which was
rapidly increasing in size. Escissional biopsy was performed.
At histological examination the normal lymph node architecture
was totally effaced by a diffused growth consisting of large,
highly pleomorphic lymphoid cells. Immunohistochemical stains
demonstrated that the neoplastic cells expressed CD45RB/LCA,
CD45RO/UCHL1, CD30 and focally CD-3. Approximately 80%
of the neoplastic cells reacted positively when stained with an
antibody to Ki-67. A diagnosis of ALCL ALK-1 negative was
168
performed. The first case was revaluated and immunohistochemical analysis demonstrated a positivity for CD45RO/UCHL1 and
CD-30 in the areas of necrosis and gene rearrangement analysis
showed a T monoclonal population.
Discussion. Preservation of antigenicity in some cases of lymph
node infarction associated with lymphoma has been reported in
literature 4-7.
Pallesen and Knudsen showed that immunoreactivity with a
wide number of leukocyte antigens is retained for up to 72 h post
mortem 4. Norton et al reported the role of immunohistochemistry
in the diagnosis of lymphoma in the presence of necrosis of the
entire lymph node 5. Vega et al reported a case of B lymphoma
with complete lymph node necrosis in which immunophenotypic
and immunogenotypic studies were performed using paraffin
embedded necrotic tissue 6.
In summary this report shows the importance of a careful assessment of any necrosis in lymph node and illustrated the potential
application and usefulness of immunohistochemical and gene
rearrangement analysis in the diagnosis of lymphoma in cases
with necrosis of neoplastic component.
References
1
Balamurugan S, Rajasekar R, Ramesh Rao R. Anaplastic large-cell
lymphoma with florid granulomatous reaction: a case report and
rewiev of literature. Indian J Pathol Microbiol 2009;52: 69-70.
2
Basu D, Bundele M. Angioimmunoblastic T-cell lymphoma obscured
by concomitant florid epithelioid cell granulomatous reaction: a case
report. Indian J Pathol Microbiol 2005;48:500-2.
3
Braylan RC, Long JC, Jaffe ES, et al. Malignant lymphoma obscured by concomitant extensive epithelioid granulomas. Cancer
1977;39:1146-55.
4
Pallesen G, Knudsen LM. Leukocyte antigens in post mortem tissues:
their preservation and loss as demonstrated by monoclonal antibody
immunohistological staining. Histopathology 1985;9:791-804.
5
Norton AJ, Ramsay AL, Isaacson PG. Antigen preservation in infarction lymphoid tissue. Am J Surg Pathol 1988;12:759-67.
6
Vega F, Lozano MD, Alcade J, et al. Utility of immunophenotypic
and immunogenotypic analisys in the study of necrotic lymph nodes.
Virchows Arch 1999;434: 245-8.
7
Strauchen JA, Miller LK. Lymph node infarction. An immunohistochemical study of 11 cases. Arch Pathol Lab Med 2003;127:60-3.
Primary cns lymphoma: prognostic role of STAT 6
R. Scamarcio, A. Cimmino, G. Ingravallo, G. Fiore, R. Ricco
Dipartimento di Anatomia Patologica,Università di Bari, Italia.
Primary central nervous system lymphoma (PCNSL) is a rare
non-Hodgkin’s lymphoma that has been increasing in past few
decades. Diffuse large B-cell lymhomas is the most common
type. Survival is reported very short. Treatment by means of
chemotherapy and/or radiation has improved survival but whole
brain radiation may be associated with neurotoxicity specially in
old patients; so methotrexate (MTX) alone is better in old patients
while in young patients may be recommended to use combined
treatment.
Recently, the activation of STAT 6, a transcriptional mediator
of IL-4 dependent gene expression was also recognize to be
expressed by tumour and endothelial cells in PCNSL. Providing
evidence for active IL-4 signaling in CNS lymphoma.
STAT 6 has been considered to provide a role in tumorigenesis
and in prognosis, because elevated expression in lymphoma cells
was associated with adverse prognosis (Seung-Ho Yang and coll.
J.Neuroncol.2009; Bruns H.A. and coll. Crit Rev Oncol Hematol
2006; Kadoch and coll. Clin Cancer Res 2009) in patients treated
with high dose MTX-based therapy.
B-cell growth factor interleukin (IL-4), that is considered STAT
6 activator, was demonstrated in PCNSL to promote lymphocyte
survival and to protect lymphoma cells from apoptosis (Kadoch
C. and coll. Clin. Cancer Res. 2009).
From 40 PCNSL patients registered in the archives of our depart-
ment, we retrieved 14 cases with known follow-up, all treated
with high doses of MTX.
The objective of this study was to investigated the immunohistochemical expression of STAT 6 and IL-4 and to compare the
results with survival.
STAT 6 was considered positive if at least 10% of the malignant
cells demonstrated nuclear staining and positivity was graduated
with + (25% of positive cells); ++ (50%), +++ (100%).
In our study, STAT 6 was positively expressed in 10 cases with
5-18 months survival; in 4 negative cases survival was longer
than positive cases with a range from 2.5 to 13 years. IL-4 resulted negative only in 3 cases (2 of these were also STAT 6
negative); in 11 case were positively expressed.
These observations suggest that STAT 6 expression is associated with short overall survival in comparison with cases that
exhibited absent STAT 6. Moreover, we observed a correlation
between intensity of STAT 6 expression and survival.
In our experience, STAT 6 expression in lymphoma cells associated with IL-4 positivity may be considered predictor of early
progression and short survival.
EBV+HHV8- Germinotropic large B cell lymphoma:
A lymphoproliferative disorder with features
intermediate between EBV+ Large B cell
lymphomas and classical Hodgkin lymphoma
L. Lorenzi*, W. Pellegrini*, C. Agostinelli**, G. Massarelli***, S.
Pileri**, F. Facchetti*
I Servizio di Anatomia Patologica, Spedali Civili di Brescia, Università
di Brescia, Italia; **Dipartimento di Ematologia e Scienze Oncologiche
«L. e A. Seràgnoli», Università di Bologna, Italia; ***Servizio di Anatomia
Patologica, Università di Sassari, Italia
*
Background. Epstein Barr Virus (EBV)-related lymphoproliferative diseases include benign and malignant lesions with a wide
array of morphologic and phenotypic features, ranging from lymphoid hyperplasia, to atypical lymphoproliferative disorders and
frank lymphoma. We report three cases of a hitherto undescribed
EBV+ lymphoproliferative process characterized by nodal proliferation of large B cells with a distinctive tropism for germinal
centers and with features intermediate between EBV+ Large B
cell lymphomas and Classical Hodgkin lymphoma.
Patients and Methods. Three patients, two women and a man,
aged 63, 77 and 65 years, with no history of immune deficiency,
presented with a high stage lymphoproliferative disease. Patients
were treated with different protocols; one died of disease after
18 months from diagnosis, another was free of disease after 28
months. No data are available on the follow up of the third case.
Tissue specimens consisted of mesenteric, cervical and inguinal
lymph node, respectively of 1.5 cm, 2.0 cm and 3.5 cm in diameter. On paraffin sections we applied immunohistochemistry for
several anti-leucocyte-associated antigens, herpes viruses type
8 and EBV (LMP1), in situ hybridization for the EBV-encoded
RNA (EBER), kappa and lambda light chain mRNA, and FISH to
detect MYC, Bcl6, Bcl2 and PAX5 gene translocations using the
break-apart probes (Dako).
Results. Lymph nodes showed an effaced architecture with
follicles colonized by large atypical cells, which displayed
heterogeneous features, from centroblasts, immunoblasts and
Reed-Sternberg-like cells. The follicles contained also variable
amounts of residual mantle B-cells and a disrupted network of
follicular dendritic cells. In two cases the follicular dendritic
cells within the follicles showed Castleman-like features. On
immunohistochemistry the large atypical cells were strongly
positive for CD20, CD30, PAX5, OCT2, BOB1 and IRF4; CD15
was expressed in one case; Bcl2 was negative in all cases, while
CD10 and Bcl6 were focally expressed in one. All atypical cells
were positive for EBV on both immunohistochemistry and in
situ hybridization, indicating a latent phase II type of infection;
169
comunicazioni orali
HHV8 was negative. The germinotropic large cells were negative for immunoglobulin light chains and corresponding mRNA;
translocations for the MYC, Bcl6, Bcl2 and PAX5 genes were not
detected by FISH.
Conclusion. Three cases with large B-cell lymphoma of the
mediastinum with a distinctive tropism for germinal centers
have been originally described by Suser [Suster S, Cancer 1992];
tumor cells were CD20+, and lacked CD15 and CD30; other 3
cases of germinotropic large B-cell lymphoma were reported by
Du [Du MQ et al, Blood 2002]. In this study tumor cells had a
plasmablastic morphology, were negative for CD20, expressed
CD30 (2/3 cases) and showed coinfection by EBV and HHV8. In
both Suster and Du series tumor cells showed monoclonal restriction for immunoglobuling light chains on immunohistochemistry.
In the present series the germinotropic large B cells were infected
by EBV; they did not express immunoglobulins and morphologically and phenotypically showed features intermediate between
large B cells and Reed-Sternberg cells. Interestingly, in age-related EBV-associated benign lymphoproliferative disorders, EBV
infected cells can predominantly occur in the germinal centers
[Dojcinov SD et al, Blood 2002], indicating that the lymphoma
here reported could represent the neoplastic counterpart of this
reactive lesion. The clinical significance of this lymphoproliferative disease is not clear, since follow up was available only in two
patients, who showed a significantly different outcome.
Uropatologia
Primary low grade sarcoma of the specialised
prostatic stroma: a case report and review of
literature
R. Zamparese, F. Corini, A. Braccischi, A. D’Angelo, L. Diamanti, M. Del Vecchio, V. Mambelli
Departments of Pathology, General Hospital C.G. Mazzoni, Ascoli Piceno
Introduction. Primary sarcoma tumors of the prostate arise from
specialized hormone-dependent mesenchymal cells and are classified, according to their histology, as stromal tumours of uncertain malignanct (STUMP) and stromal prostatic sarcoma (namely
low grade and high grade).
Case report. A 71-year-old man developed progressive urinary
obstruction symptoms and presented at our Hospital and was
performed a transurethral prostatic resection (TURP). Macroscopically, the excised tissue consist of many fragments of prostatic tissue weighing 30 grams. Microscopically, the histological
specimens showed a diffuse proliferation of epithelioid and spindle cells, with a storiform and infiltrative growth pattern. There
aren’t prostatic glands in the proliferation. No foci of necrosis, no
vascular invasion. The neoplastic cells showed rare atypical mitotic figures in addition to moderate hypercellularity and moderate nuclear atypia with pleomorphism. Immumohistochemically
the neoplastic cells characteristically express diffusely CD34 and
focally progesterone, whereas no immunoreactivity was seen
for cytocheratin (clone AE1-AE3 and Cam 5.2), desmin, S-100,
Bcl-2, chromogranin, CD117, AM,t p53 and Ki-67 showed a very
low expression. On the basis of the morphological and immunoistochemical features, a final diagnosis of low grade prostatic
stromal sarcoma was made.
Discussion. Prostatic stromal tumors arising from the specialized
prostatic stroma are rare and distinct tumours with diverse histologic pattern. In the past, these tumors have been reported under a
variety of terms including atypical stromal hyperplasia, phyllodes
type of atypical stromal hyperplasia, and cystic epithelial-stromal
tumors. Now these lesions have been classified as lesions of uncertain malignant potential (STUMP) and stromal sarcoma (low
grade and high grade).
In contrast to stromal sarcoma, the neoplastic nature of STUMP
is controversial. The predominant pattern consists of either
normocellular or midly hypercellular stroma with cytologically
degenerative atypical cells associated with benign glands. In the
past, this pattern was often reported as benign prostatic hyperplasia (BPH) with atypia and may be difficult to distinguish form
stromal sarcoma in rare case. Althought STUMP can be histologically misdiagnosed as benign prostatic hyperplasia, it is important
to recognized that these are neoplasms with unique local morbidity and malignant potential. In contrast to BPH, STUMP can recur
frequently and occur at younger man. Herawi and Epstein found
that seven of the 50 stromal tumours of the prostate they analyzed
were STUMPs associated with sarcoma (4 High grade sarcoma,
3 low grade sarcoma). Low grade sarcoma can locally invade,
despite having relatively bland cytology at times. High grade
sarcoma has the potential to metastatized.
The low grade prostatic sarcoma are a really rare neoplasm, in
the literature, at our knowledge, are described only 9 cases (age:
19 to 76, mean 51). All patients were alive and free of disease at
follow-up.
The high grade prostatic sarcoma are neoplasm rare too. In the
literature are described only 16 cases. Of these 6 were free of
disease, 3 was alive with pulmonary metastases, 2 was alive
with multiple metastases and 3 deid of disease and 2 was lost to
follow-up).
There are several difficulties with the histologic diagnosis of
STUMP, low grade prostatic sarcoma and high grade prostatic
sarcoma. It may be difficult to differentiated low-grade prostatic
sarcoma from STUMP, sometimes the tumour seemed histologically benign and only the presence of local infiltration or the presence of atypical mitotic figure was diagnostic of LG sarcoma.
Prognosis and treatment of STUMP and sarcoma are also controversial. Factors to consider in deciding wheter to proceed with
definitive resection for STUMP diagnoses on biopsy include
patient’s age, presence and size of the lesion and extent of the
lesion on tissue sampling.
Can prostatitis to be a confoundig parameter
in prostatic proteomic profile designation?
S. Bergamini*, L. Reggiani Bonetti**, E. Monari*, E. Bellei*, A.
Maiorana*, T. Ozben**, A. Tomasi*, S. Micali***, G. Bianchi***
Dipartimento Integrato di Anatomia Patologica, Medicina Legale e di
Laboratori, Sez. di Anatomia e Istologia Patologica/Università degli Studi
di Modena e Reggio Emilia, Modena, Italia; **Dipartimento di Biochimica, Facoltà di Medicina, Akdeniz University, Antalya, Turkey; ***Dipartimento di Urologia, Azienda Ospedaliera-Universitaria, Università degli
Studi di Modena e Reggio Emilia, Modena, Italia
*
Serum protein profiles were investigated in order to identify
distinctive proteins able to discriminate patients with benign prostatic hyperplasia (BPH) from those with prostate cancer (PCa).
We considered these conditions focusing on the co-existence
of inflammation. Patients with clinical suspect of PCa and candidates for trans-rectal ultrasound guided prostate biopsy were
enrolled. The analysis of protein profile of 30 patients with PCa
cancer and 30 subjects with BPH was carried out. All histological
specimens were examined in order to graduate and classify the tumor and to recognize the BPH condition and presence of inflammation, that was distincted in chronic and acute and then graduated in mild, moderate and severe. Serum was depleted of the
6 high-abundance proteins by immunoaffinity chromatography
prior to Surface Enhanced Laser Desorption/Ionization - Time of
Flight - Mass Spectrometry analysis. The comparison between
protein spectra from PCa and BPH considering the inflammation
parameter and excluding samples with moderate and/or severe
inflammation, identified 17 differentially expressed protein peaks
using H50 ProteinChip Array. The analysis of protein profile in
presence of inflammation showed different protein peaks in the
170
two groups, some of which overlapped with those found also in
the comparison between PCa and BPH in absence of inflammation. The inflammation seems to lead a crucial contribution in the
protein profile assessments of these conditions. On the basis of
our results, we believe that certain different protein peaks could
be reasonably associated to inflammation rather than to cancer.
Therefore, inflammation might be a confounding parameter in the
search of specific biomarkers to discriminate PCa from BPH.
Effectiveness Evaluation of immunofluorescence
urine test, associated with traditional urine
examination in patients followed up for bladder
cancer.
V. Nirchio*, L. Sullo**, F. Pappalettera***, L. Cormio***, G. Carrieri***
S.S.D Cytopathology, departments of Pathology, Ospedali Riuniti, Foggia, Italy; ** L.P., urologist, Foggia; ***U.O.C Urology Univ, Ospedali Riuniti, Foggia, Italy
*
Background. The sensitivity of urine cytology, in patients with
bladder cancer, especially in low grade lesions is about 20%. The
objective of this study is to evaluate the sensitivity, specificity
and efficacy of an immunofluorescence urine test associated with
a traditional examination.
Method. Patients undergoing TURB for bladder cancer in
follow-up cystoscopy and urinary cytology were also proposed to
associate the test uCyt.1 2 3
In the period from January 2010 to June 2011, 142 patients were
enrolled, for a total of 236 tests
uCyt. From three urine samples a single urine thin prep has been
prepared, while a second slide
thin-prep method was used to perform the uCyt. The test positiveness has been assessed according
to the parameters specified by the supplier company (at least 5
positive cells).
Results. 15 samples were found to be inadequate, 6 of these have
been repeated during the following 18 months. 114 cases were
negative to conventional cytology, uCyt and cystoscopy.
10 cases resulted positive in all three methods. 9 cases were false
positive. 9 cases were false negative results.
According to the previous results the test sensitivity is about 53%,
its specificity is 93%, while its
effectiveness is 87%.
Conclusions: The sensitivity of the test compared to the degree
of malignancy, predominantly lowgrade, and efficiency, in our opinion justify the technical examination cost and effort.
References
1
Mian C, Maier K, Comploj E, et al. uCyt+/ImmunoCyt in the detection of recurrent urothelial carcinoma: an update on 1991 analyses.
Cancer 2006;108:60-5.
2
Bernardi A, Berno E, Fopet F, et al. The utility of uCyt+ in the detection and surveillance of transition cell cancer of the bladder (UC)
and its application in differatiating follow-up schemes. Pathologica
2009;101:207
3
Napoli A, Napoli G, Tantimonaco L, et al. Diagnosis of differentiated urothelial carcinoma: cytology + uCyt+TM. Revision of cases
of dipartimento anatomia patologica, Policlinico Universitario Bari.
Patologica, 2009; 101:216.
Ruolo della proteina P16 quale fattore prognostico
dei carcinomi Ta G1 della vescica
da lesioni non infiltranti suddivise dal WHO/ISUP 2004, in lesioni iperplastiche, papillari e piatte.
Nell’ambito delle lesioni papillari il carcinoma uroteliale non infiltrante di basso grado, presenta delle problematiche ancora non
risolte, in quanto, nonostante l’aspetto architetturale e citologico
blando, i pazienti con tali lesioni vanno incontro non infrequentemente a recidiva o addirittura a progressione neoplastica, in
breve tempo.
La proteina p16 ha una funzione di blocco nell’ambito della proliferazione cellulare. La sua iperespressione, da tempo associata
a lesioni HPV correlate della cervice uterina, è stata di recente
valutata e studiata in lesioni neoplastiche di varia natura e sede e
molto recentemente della vescica.
Scopo del nostro studio è valutare l’espressione immunoistochimica di p16 in carcinomi uroteliali della vescica a diverso
stadio e grado, in associazione con la proteina p53, al fine di
intercettare delle differenze di espressione dei due marcatori in
maniera dipendente o indipendente, nei sottogruppi delle lesioni
esaminate.
Materiali e metodi. 100 casi di neoplasia uroteliale papillare
della vescica pervenuti presso l’U.O.C. di Anatomia Patologica
dell’ASL di Frosinone, dal gennaio 2008 al giugno 2011, suddivisi in quattro gruppi di 25 casi Ta G1, Ta G3, T1, T2-3 e classificati secondo il Consensus ISUP/WHO del 2004.
Per ogni caso la diagnosi istologica è stata effettuata da sezioni
fissate in formalina, incluse in paraffina e colorate con ematossilina-eosina. Per ogni caso sono state inoltre effettuate determinazioni immunoistochimiche per p16 (MTM laboratories) e p53
(Ventana).
Risultati. La positività immunoistochimica per p16 è stata riscontrata in 13 casi del gruppo Ta G1; 20 casi Ta G3; 23 casi T1
e tutti i casi T2-3.
La positività immunoistochimica per p53 è stata riscontrata in un
caso del gruppo Ta G1; 21 casi Ta G3; 22 casi T1 e tutti i casi
T2-3.
La positività immunoistochimica per p16+p53 è stata riscontrata
in un caso del gruppo Ta G1; 18 casi Ta G3; 20 casi T1 e tutti i
casi T2-3.
Conclusioni. I risultati dell’espressione di p16, nei vari stadi
di carcinoma uroteliale, suggeriscono un possibile ruolo di tale
marcatore quale spia di una lesione maggiormente aggressiva o
indicativa di una possibile recidiva.
P16 potrebbe essere utile nella prognosi delle lesioni papillari non
infiltranti di basso grado (pTa G1) in quanto la sua espressione
appare indipendente dal quadro morfologico.
Pertanto, la valutazione dei regolatori del ciclo cellulare, quale
p16, potrebbe essere usato come uno strumento predittivo e
fornire, informazioni utili per determinare follow-up personalizzati strategie terapeutiche.
Patologia testa collo e cavo orale
Loss of heterozigosity (LOH) as molecular
progression marker in oral squamous carcinoma
(OSCC)
A. Marsico*, M. Micheletti**, I. Rostan**, M. Pentenero***, S.
Gandolfo***, R. Navone **
U.O.C. Anatomia Patologica, Ospedale Umberto I, Frosinone
UO di Anatomia Patologica, Presidio del Policlinico di Monza, Vercelli;
Dipartimento di Scienze Biomediche e Oncologia Umana dell’Università di Torino (Sez. di Anatomia Patologica); ***Dipartimento di Scienze
Cliniche e Biologiche dell’Università di Torino (Sez. di Medicina e Oncologia Orale)
Introduzione. Il carcinoma uroteliale della vescica rappresenta la
settima causa di morte per neoplasia nei paesi industrializzati.
Dal punto di vista patogenetico la neoplasia è sempre preceduta
Objectives. Oral squamous carcinoma (OSCC) is characterised
by genetic alterations in the epithelial cells. The loss of heterozygosity (LOH) i.e. the disappearance of a more or less large
R. Reitano, S. Noto, E. Maura, C. Mirabella, P.L. Alò
*
**
comunicazioni orali
area of DNA in one or two members of a couple of homologue
chromosomes, is an event whereby the genetic loci, containing
oncosuppressor genes involved in tumoral progression, are lost.
The study of LOH in OSCC has evidenced the involvement of
oncosuppressor genes (TSGs), situated mainly in determined
chromosomal regions i.e. 3p14.2, 3p24, 3p21.3, 9p21, 17p13,
4q, 8p, 11q and 13q. The loss of specific chromosomal regions
(LOH) that contain suspected or confirmed oncosuppressor genes
represents an early predictor of potentially malignant oral lesion
(PML) progression such as leukoplakia, erithroplakia, lichen planus and proliferative verrucous leukoplakia. It has been estimated
that subjects with oral leukoplakia and dysplasia have a 36% risk
of progression versus OSCC and in the absence of dysplasia the
risk of developing carcinoma remains high at 5-10%.The presence of LOH has been observed at 3p and/or 9p in 50% of oral
leukoplakias, with a 3.8 fold increase in the risk of malignant
transformation. Additional LOH (4q, 8p, 11q, 13q and/or 17p)
lead to a 33-fold increase in the risk of tumoral progression.
Methods. We investigated the polymorphic microsatellite markers in the chromosomal loci that had a higher evidence of LOH
and a significant heterozygosity in oral PML and OSCC. Attention was focused on chromosome 3 (D3S1234 and D3S1300,
locus 3p14.2, gene FHIT i.e. the fragile histidine triad gene,
D3S1317, locus 3p26, gene VHL and chromosome 9 (IFNA,locus
9p22, gene IFNA, D9S171 and D9S1751, locus 9p21), that had
the highest number of LOH in squamous carcinoma and on cases
involving progression compared to those without progression.
We investigated a group of lesions which included 8 cases of oral
verrucous carcinoma and samples of healthy mucosa from the same
subjects as controls for LOH analysis. A microdissection protocol
and extraction and amplification of DNA for the optical microscope
was set up to examine both healthy and tumoural histological samples. The histological paraffin embedded samples were obtained
from bioptic and surgical archival material from oral verrucous carcinomas and healthy mucosa adjacent to the lesions. The mucosal
samples were obtained by means of a dermatological curette, from
surrounding areas and/or regions far from the lesion site.
Results. Four/eight cases had LOH on chromosome 3, or 9.
Herein we present a case of a 66 year-old female with oral verrucous carcinoma on the left edge of her tongue. Analysis of the
6 markers was done both on a sample of the neoplasia and healthy
mucosa of the same subject. There was a loss of heterozygosity
on the short arm of chromosome 3, evidenced by the analysis of
the D3S1300 and D3S1234 markers. Whilst there was a normal
allelic profile in the 3 heterozygote points of the markers IFNA,
D9S171, D9S1751, on the short arm of chromosome 9.
Conclusions. In our cases, 50% of the oral verrucous carcinoma
had a loss of heterozygosity; in agreement with literature data on
the study of LOH in squamous carcinoma.
Incorporating molecular data on the loss of heterozygosity at histopathologic diagnosis of PMLs may well represent a progression
marker for the evolution of these lesions, distinguishing cases with
a high probability of progression, or worsening, from those with a
lower risk. This approach may well allow for a more aggressive and
earlier therapy at an earlier stage of the disease and identify those
cases that require a stricter follow-up whilst, at the same time, avoid
invasive treatment in lesions at low risk of progression.
Beta-catenin expression and its prognostic role in
oral and oropharyngeal SCC.
G. Pannone*, A. Santoro*, M. Mattoni*, R. Franco**, G. Aquino**,
P. Bufo*
Department of Surgical Sciences, Section of Anatomic Pathology and
Cytopathology, University of Foggia, Foggia, Italy; **Istituto Nazionale
per lo studio e la cura dei tumori, Fondazione ‘G Pascale’, Napoli, Italy
*
Introduction. The beta-catenin protein is the center of the
Wnt-signaling pathway, and the disturbance of this pathway is
171
shown by abnormal expression of beta-catenin in the nucleus of
abnormal cells. Previous studies have shown that there are many
cancers with beta-catenin mutations, mainly located in the exon
3 region. Although dysregulation of the Wnt pathway via betacatenin is a frequent event in several human cancers, its potential
implications in oral cancer are largely unexplored. Aim of the
work was to define both the pathogenetic and the prognostic role
of beta-catenin in a large series of oral (OSCC) and oropharyngeal squamous cell carcinomas (OPSCCs)
Materials and methods. 374 O/OPSCCs selected from three
different geographic areas were quantitatively and qualitatively
analysed by immunohistochemistry for beta catenin and Lef/
TCF1/TCF4. All cases were stratified according to intracellular
localization (membranous, nuclear, cytoplasmic, absent) of markers. The series has been assessed in various clinicopathological
groups and the relationschips were established by statistical
analysis. Survival rates were assessed by Kaplan-Meier curves.
Beta-catenin expression was also evaluated on both normal and
neoplastic oral cell lines, by RT-PCR and fold increase definition.
A further quantitative analysis of the neoplastic DNA content was
performed on 22 O/OPSCCs by DNA-image cytometric analysis,
in order to explain the association between beta-catenin expression and the aneuploid state of the neoplastic cells.
Results. In all cancers alterations of beta-catenin levels were more
evident than in normal peritumoral epithelium (p<0.05). The protein
staining was mainly detected in the cytoplasm of the neoplastic
cells. Only focal nuclear positivities were observed. Higher expression of cytoplasmic beta-catenin correlated significantly with poor
histological differentiation, and advanced stage (p<0.05) and with
worst patient outcome, as also confirmed by Kaplan-Meier curves.
Immunohistochemistry and Western Blotting analysis for Lef/
TCF1/TCF4 shows complete absence of this marker in O/OPSCC.
The logistic regression analysis demonstrates that the tobacco and/
or alcohol consumption is associated to loss of beta-catenin expression in O/OPSCC. By RT-PCR significant fold increase was detected in neoplastic cell lines. The RT-PCR on surgical samples reveales an important increase of the beta-catenin mRNA expression
in 10/22 cases (45%). The study of the DNA ploidy demonstrates
that beta-catenin over-expressing O/OPSCCs have higher levels of
aneuploidy and numerous nodal metastases in T1.
Discussion and conclusions. Our work have underlined the keyrole of beta-catenin in oral and oropharyngeal carcinogenesis,
in chromosomal instability determination and in the prognostic
stratification of patients. To our knowledge, the present work is
the first wide study that highlights the existence of a statistical
association between beta-catenin and traditional prognostic factors in oral and oropharyngeal cancer, combining different types
of bio-molecular techniques.
Oropharynx cancer: evaluation of clinical outcome
according to HPV status and radiotherapy
technique
E. Bragantini1, S. Girlando1, V. Vanoni2, A. Bolner2, F. Valduga3,
R. Carella5, M. Barbareschi1, M. Silvestrini4, C. Grandi4, P. Dalla
Palma1
1
S. Department of Pathology, 2Departement of Radiotherapy, 3Department
of Oncology, 4Head and Neck Surgery, Chiara Hospital Trento; 5Department of Pathology, S.Maurizio Hospital Bolzano
Introduction. Epidemiologic and molecular data showed that
HPV-associated head and neck squamous cell carcinoma (HNSCC) is different from traditional carcinogen-induced HNSCC on
the levels of risk factors, tumor development, response to therapy,
prognosis and survival (1, 2). This study analyze the correlation
between HPV status, radiotherapy technique and outcome for
oropharyngeal squamous cell carcinoma (OSCC).
Matherial and methods. From September 2005 to September
2008, 62 patients with OC were treated with exclusive primary
172
radiotherapy+/-chemotherapy. Thirty-five pts (56.5%) had stage
IV disease (all M0); 33 pts (48.4%) underwent radiotherapy (RT)
combined with concomitant chemotherapy (CT) (cis-platinum).
Seventeen pts (27.4%) underwent 3D-planned simplified conventional three-field RT (3D-S) with a single isocenter, followed by
off-cord photon beam treatment with electron dose supplement
to neck nodes and finally a 3D conformal boost. Twenty-two pts
(35.5%) underwent 3D advanced RT (3D-A), with a 5- or 7-field
conformal technique (50Gy CTV1; 70Gy for CTV2). Twentythree pts (37.1%) underwent IMRT treatment with simultaneous
integrated boost (SIB) with 2 dose levels (54-66 Gy in 30 fractions) or 3 dose levels (54-60-69 Gy in 30 fractions).
HPV status was analyzed by p16-immunohistochemistry and
HPV molecular biology using the CINtec® Histology V-Kit for
qualitative detection of p16-antigen on tissue sections prepared
from formalin-fixed, paraffin-embedded biopsies. Both nuclear
and/or cytoplasmic positive staining > 5% were considered
p16-positive. DNA extraction from formalin-fixed, paraffinembedded samples was performed using a Qiagen Kit.. Molecular analysis was performed by nested PCR using MY09/11 and
GP5+/6+ primers (AB-Analitica Kit).
Results. Mean follow up was 28 months (range 5-61). 27 patients
(43.5%) were p16-positive and 34 were p16-negative (44.3%)
1 not informative. HPV PCR was positive in 29 pts (46.8%),
negative in 29 (46,8%), not informative in 4. DFS at 3 years was
76.2% for p16-positive pts and 58.4% p16-negative pts (p=.03).
OS at 3 years was 68.2% and 44.1%, respectively (p=.002). In the
p16-positive group, no statistically significant differences were
found for the three different RT techniques for DFS at 3 years
while for p16-negative patients, DFS for 3D-S, 3D-A and IMRT
was 30%, 63% and 87% (p=.05). DFS at 3 years for the HPVpositive and negative group was 74,5% and 56%, respectively
(p=.04).
Conclusions. P16 is an efficient predictive biomarker, comparable to HPV molecular analysis.
The use of IMRT with SIB seems to be more relevant for outcome of HPV/p16-negative pts than for HPV/p16-positive pts in
our series, indicating that IMRT with SIB can improve the worse
a-priori prognosis of HPV/p16-negative patients. Further studies
must be performed to confirm this hypothesis
References
1
Allen CT, Lewis JS Jr., El-Mofty SK, et al. Human papillomavirus
and oropharynx cancer: biology, detection and clinical implications.
Laryngoscope 2010;120:1756-72.
2
Mehanna H, West CML, Nutting C, et al. Head and neck cancer - Part
2: treatment and prognostic factors. BMJ 2010;341:c4690.
Oropharyngeal squamous cell primary tumor and
second primary tumors: an immunohistochemical
panel with P16, P53, MIB1 and CD44
A. Di Lorito*, S. Malatesta*, P. Viola*, E. Penitente*, S. Setta*, D.
Angelucci*, A. Croce**, A. Colasante*.
UOC Anatomia Patologica, Ospedale Clinicizzato SS Annunziata,
Chieti,Italia; ** UOC Clinica OtoRinoLaringoiatrica, Ospedale Clinicizzato SS Annunziata, Chieti,Italia.
*
Head and neck cancer (HNSCC) is the sixth leading cancer in the
world by incidence. It is likely that approximately 600,000 cases
will arise this year worldwide and only 40–50% of patients with
HNSCC will survive for 5 years. Unlike most tobacco-related
head and neck tumors, patients with oropharyngeal carcinoma
usually do not have a history of tobacco or alcohol use. Instead,
their tumors may be positive for oncogenic types of the human
papillomavirus (HPV).
The extent of the tumour, the presence of lymph-node metastases
and distant metastases determine the stage and so the treatment.
However, some patients develop a new cancer after curative treatment, named second primary tumor (SPT). These may be syn-
chronous with the index tumor or, if occurring after an interval of
longer than sixth months are described as metachronous.
Recently, some studies have identified, in HNSCC, a subpopulation of cells with a cancer stem cell property that show a CD44
positivity. Moreover, some Authors have suggested that a combined immunohistochemical panel with p16, p53 and MIB1 is
very useful in predicting the clinical outcomes of oropharingeal
cancer with more relevance than staging alone.
We evaluate in our Institution, since year 2003, the characteristics
of the SPT in 7 patients with oropharingeal squamous cell carcinoma, treated with curative intention.
Matherial and methods. We examined 2 out of 7 patients
treated by surgical and/or chemo-radiotherapy for squamous cells
carcinoma, oral (5) and tonsil (2), that developed a SPT. So, we
performed an immunohistochemical panel with p16, p53, MIB1
and CD44 in both biopsies and surgical specimens in the primary
tumor and in the SPT.
Results and conclusions. We found patient RM, p53 positive,
p16 focal and weak positive, MIB 1 ≥ 30% and CD44 moderate
membrane expression while the other patient, MV, showed p53
negative, p16 under evaluation, MIB 1 ≥ 30% and CD44 strong,
granular membrane and cytoplasmatic expression.
There were no differences, in the same patient, regarding p53,
p16, and CD44 expression on comparing biopsies and surgical
specimens both in primary tumor and in SPT; on the other hand,
we can expect differences on these markers expression in different patients. The diversity may be due to a different oncogenic
stimulus and/or to the second field theory, that is, derived from
the same genetically altered field as the index tumor.
Since additional information are needed to better understand
these preliminary data, further evaluations are in progress.
Oral microhistology: an innovative technique for
a first level diagnosis in malignant and potentially
malignant oral lesions
R. Navone*, G. D’Angelo*, I. Rostan*, A. Marsico**, M. Pentenero***, G. Tempia Valenta***, S. Gandolfo***
Dipartimento di Scienze Biomediche e Oncologia Umana dell’Università di Torino (Sez. di Anatomia Patologica); ** UO di Anatomia Patologica,
Presidio del Policlinico di Monza, Vercelli; *** Dipartimento di Scienze
Cliniche e Biologiche dell’Università di Torino (Sez. di Medicina e Oncologia Orale)
*
Objectives. Squamous cell carcinoma of the oral cavity (OSCC),
although frequent (6th cause of cancer-related mortality worldwide), still has a low survival rate as diagnosis is often late and
there is a lack of simple and reproducible diagnostic tests able to
identify early stage precancerous potentially malignant lesions
(PMLs). These are clinically subdivided into classes I and II: the
former are manifestly clinically suspicious and the latter have
an apparently innocent appearance. To date, the diagnosis of
oral cavity OSCC and PMLs has been based exclusively on the
scalpel (surgical) biopsy. This is an invasive technique, limited to
a restricted area and of difficult application with multiple lesions
and, last but not least, generally only used for class I lesions.
Oral diagnostic cytology alone, whilst providing useful information (sensitivity is higher than the Pap test, while specificity is
similar), does not suffice for the diagnosis of OSSC and PMLs
(Navone R et al: The impact of liquid-based cytology for the
diagnosis of oral dysplasia and carcinoma. Cytopathology 2007;
18: 356-60).
Methods. Recently, an original, less invasive sampling method
which does not generally require anaesthetic and uses a dermatological disposable curette, provided small epithelial fragment
from oral mucosa with results comparable to the scalpel biopsy
(Navone R et al: Oral Potentially Malignant Lesions: First Level
Microhistological Diagnosis from Tissue Fragments Sampled in
Liquid-Based Diagnostic Cytology. J Oral Pathol Med 2008, 37:
173
comunicazioni orali
358-63). However, only experts in specialised centres did this
sampling. As the territorial (private practise) dentist is the first
to observe an apparently innocent lesion i.e. class II PMLs, after
a brief training period, a clinical trial was set up in collaboration
with them. Samples were obtained according to our instructions
with the curette technique by 50 dentists and treated for histological examination (paraffin inclusion, haematoxylin-eosin staining)
as routine small biopsies.
Results. Ten of the 150 samples were inadequate (6.6%), 131/140
negative (hyperkeratosis, parakeratosis or simple hyperplasia),
6/140 (4.3%) low-grade dysplasia (OIN 1), 2/140 (1.4%) highgrade dysplasia (OIN 2-3) and 1/140 (0.7%) OSCC.
Our aforementioned study, done in a specialised centre, reported
a 3.6% rate of inadequate samples (6/164). Although inadequate
samples in this field trial are higher (6.6%, 10/150), it must be
considered that “inexpert persons” did sampling and results are
still very good as this is a first level test.
Conclusions. The sampling with the “curette technique” and the
use of “microhistology” may well be an effective first-level method to distinguish those reactive, or inflammatory lesions requiring
only follow-up, from positive lesions (dysplasia and OSCC) to be
sent to the specialised second-level centres for routine scalpel biopsy. Moreover, this method can use this material also with flow
cytometry to evaluate ploidy: the finding of aneuploidy allowed
for the identification of lesions that were at risk of evolution
(Pentenero M et al: DNA aneuploidy and dysplasia in oral potentially malignant disorders. Oral Oncol 2009; 45: 887-90) and the
selection of individuals who required a stricter follow-up regime.
Lastly, curette sampling, which covered ample surface areas and/
or multiple lesions, led to a reduction in the number of patients
that were required to return for further investigations as well as
the quantity of surgical (scalpel) biopsies. Consequently, there is
a positive cost/benefit ratio for the hospital and less discomfort
for patients. Therefore, the adoption of this technique will allow
the dentist, who is the first to see the preneoplastic and neoplastic
oral lesions, to manage even those apparently innocent lesions
(class II) of difficult definition and/or not yet considered for biopsy, in the most appropriate manner.
Bilateral synchronous pleomorphic adenoma
diagnosed by FNA cytology
D. Russo, C. Bellevicine, E. Vigliar, V. Varone, G. Troncone
Background. Pleomorphic adenoma represents the most frequent
salivary gland tumor. Its bilateral synchronous presentation is a
rarely occurrence. Case. A 28-year-old woman presented with a 2 cm firm left parotid mass; a less evident contro-lateral nodule was also present.
There was no evidence of other neck masses or lymphadenopathies. The ultrasound (US) examination showed well circumscribed hypoechoic bilateral nodules Fine-needle aspiration
(FNA) US guided was performed on both lesions. The smears
from two nodules overlapped; small, uniform, round to oval
epithelial cells arragend in cohesive sheets were evident. The
background was composed of single uniform epithelial cells and
magenta-colored metachromatic fibrillary matrix on Diff Quik
staining. These findings led to the diagnosis of bilateral synchronous pleomorphic adenoma. Histologic examination proved the
pre-operative diagnosis. Conclusion. Here we present a case of bliateral synchronous
pleomorphic adenoma. This unusual presentation of a common
salivary gland tumor generates peculiar clinical and surgical
management issues.
Ginecopatologia
Infezione da papilloma virus umano (HPV)
nelle donne HIV positive: correlazioni clinicopatologiche
F. Micheli BTD, P. Somma MD PhD, P. Micheli MD
U.O.C. Anatomia patologica e Citologia Diagnostica Azienda Ospedaliera dei Colli- Monaldi-Cotugno-CTO-Napoli
Introduzione. I papilloma virus umani (HPV) sono divisi in
ceppi ad alto e basso rischio oncogeno per la minore o maggiore
associazione displasie di alto grado e carcinomi della regione
ano-genitale.
È noto che il normale ciclo cellulare è regolato di due gruppi di
geni: gli Oncosoppressori e gli Oncogeni.
La proteina E6 degli HPV ad alto rischio lega il prodotto dell’ oncosoppressore 53 bloccandone la funzione. La proteina E7 ha una elevata affinità per la proteina p105 del retino blastoma. Nei tipi 6 e 11
(basso rischio) l’E6 non lega p53 e l’E7 ha bassa affinità per p105.
Tali interazioni, associate ad alterazioni del sistema immunitario
(HIV), determinano una proliferazione cellulare incontrollata
(immortalizzazione) con accumulo di mutazioni genetiche e trasformazione neoplastica.
Materiali e metodi. Dai Files dell’U.O.C. di Anatomia Patologica e Citologia Diagnostica dell’A.O.R.N. “Dei Colli”, Napoli
sono stati selezionati 90 casi di citologia del tratto cervico-vaginale (Thin prep). Età media di 34,6 anni (range 18-49). Popolazione controllo:96 donne HIV-negative (range 18-49).
Ricerca del DNA di HPV ad alto rischio oncogeno n tutti i casi
mediante test di ibridazione (Hybrid Capture II -Digene) anche in
quelli citologicamente negativi per HPV.
In tutti i casi positivi al DNA di HPV ad alto rischio oncogeno
studio dell’espressione di p16 (immunocitochimica).
Nei casi con citologia positiva o dubbia: esame colposcopio con
relativa biopsia
Sono state effettuate correlazioni clinico patologiche
(morfologia,risultati Hybrid Capture II ed espressione della proteina p 16).
Risultati. 90 casi HIV-positive: negativi 54 (60%), ASCUS 6
(6,6%), LSIL 21 (23,3%), HSIL 9 (10,0%). HPV- DNA ad alto
rischio (Hybrid Capture II): positivo in 11 casi HIV-positive
(15,7%) con citologia negativa o dubbia (ASCUS) e in 21
casi (70,0%) citologia positiva (LSIL e HSIL). Popolazione di
controllo:HPV-DNA ad alto rischio positivo in 9 casi (10,5%)
con citologia negativa o dubbia e in 5 casi degli 11 (45,4%) positivi (LSIL e HSIL). Proteina p16: positiva in 3 (27,2%) degli 11
casi positivi all’HPV-DNA al alto rischio (HIV-positive con citologia negativa o dubbia) ed in 7 (33,3%) con citologia positiva.
L’esame istologico dei casi positivi all’HPV- DNA ad alto rischio
ha confermato i dati precedenti.
Discussione. I nostri dati, in accordo con altri autori, confermano
la più alta prevalenza dell’infezione da HPV ad alto rischio in
donne HIV-sieropositive ed una maggiore prevalenza di HPV ad
alto rischio in donne HIV-sieropositive con citologia negativa o
dubbia (probabile persistenza subclinica dell’infezione).
In conclusione, i nostri risultati dimostrano che test virologici sensibili e specifici (Hybrid Capture II) per il DNA HPV ad alto rischio
sono importanti nello screening per prevenire neoplasie cervicali
in donne HIV-sieropositive identificando casi a rischio negativi al
semplice screening citologico. Inoltre, come è noto, lo studio della
proteina p16 identifica, tra i casi positivi per HPV ad alto rischio oncogenico, quelli con maggiore possibilità di evoluzione neoplastica.
Bibliografia essenziale
Syrjänen K. Natural history of cervical HPV infections and CIN. In: Sons
JW, ed. Papillomavirus Infections in Human Pathology. New York,
2000:142-66.
174
Ho GY, Burk RD, Fleming I, et al. Risk of genital human papillomavirus
infection in women with human immunodeficiency virus-induced immunosuppression. Int J Cancer 1994;56:788-92.
Vernon SD, Holmes KK, Reeves WC. Human papillomavirus infection
and associated disease in persons infected with human immunodeficiency virus. Clin Infect Dis 1995;21 Suppl 1(S121-4).
Risk management
Realistic technician staffing requirements in
a histopathology laboratory via an innovative
workload method
M. Bergamaschi*, G. Coccini**
Servizio Immunoematologia e Medicina Trasfusionale I.R.C.C.S. Fondazione Policlinico “San Matteo”, Pavia, Italia; ** Direzione Sanitaria
I.R.C.C.S. Fondazione Policlinico “S. Matteo”, Pavia, Italia.
*
It is well recognized that efficiency is one of the most important
objectives of clinical governance. The correct determination of
personnel required plays a central role in health economics. Inadequate staffing of clinical laboratories may compromise quality
and throughput, whereas excess staff can uselessly increase costs.
This study was undertaken to determine the most reliable and easily applicable method for determination of staffing requirements
in a histopathology laboratory.
Three published methods, namely the weighted workload model,
standard time-based and audit benchmarking methods, were compared. The strengths and weaknesses of each method is described
with the purpose of identifying the best approach.
There are only three relevant published methods, and even these
are not appropriate for current requirements. In particular, they
may be based on data that is not readily available (calculation
of standard time) or may use outdated patterns (using weighted
workload) or nonstatistical benchmarks. Although benchmarking
was widely used in U.S. even in a period of a crisis in this area
in the late 1990s, its major flaw consists of excluding the most
influential variables.
In summary, we tried to formulate a new method based on organizational activities and their categorization into sub-activities,
each of which contributes to the calculation of the total time
required to perform all activities and accurately determine the
number of technicians required.
Risk management in anatomic pathology
M. Biancalani
Dipartimento di Diagnostica, U.O.C. Anatomia Patologica, Ospedale S.
Giuseppe, Empoli, Firenze Italia
Risk Management implementation in the Anatomic Pathology
department is addressed to minimize errors that can occur during the analytical process, as wrong patient identification, wrong
sample identification and association to the right patient, biocassettes loss.
Risk Management can be effectively implemented through the
support of specific software features and hardware devices, as
bidimensional code readers, slides and cassettes label printers.
The presentation will highlight specific points of failure of the
analytical process. For each of these points of failure is necessary to act in an accurate and automated way to cut down errors
risk and to permit a continuous operations traceability during the
whole process.
The presentation will show even the most relevant software features that allow to control the process during its different steps:
from the patient identification to the sample acceptance, from the
sample processing to the reporting, the report delivery and the
slides archiving. The software solution is based on a model that
implement a deep knowledge of the specific Anatomic Pathology
process and approaches to its problems, determining the efficacy,
efficiency and quality of the offered service.
The presentation will highlight the most critical phases and activities where mistakes can occur and will identify the informational
and operating procedures through which it’s possible to avoid or
minimize errors.
Shortly, the presentation will analyse the possible support that IT
(information Technology, though software and hardware components) con provide to implement “risk management” in Anatomic
Pathology process to reach the following targets:
Improve patient and sample identification during the tissues sampling, analysis, report and communication.
Improve verification and communication of information that can
affect the patient life (critical diagnosis).
Improve identification, communication and correction of mistakes.
Put the patient safety at the centre of the healthcare organization
and of the AP process.
Pathologica 2011;103:175-267
Poster
Risk management in anatomic
pathology
Risk management: a safety lab work flow driven
by information technology and 2D BAR code
Agreement between cause of death diagnosis on
death certificate and at autopsy: a consecutive
series of 590 autopsied cases over a period of 10
years (1999-2008): a single-institute experience
Unità Operativa di Anatomia Patologica e Citologia Dipartimento di Patologia Clinica e Radiologia, Ospedale Infermi, Rimini
G. Crisman*, M. Margiotta*, V. Ciuffetelli*, G. Coletti**, P. Leocata*
Anatomia Patologica, Dipartimento di Scienze della Salute, Università
Patologica, Ospedale Civile “San Salvatore”, L’Aquila, Italia
*
Background. The term “autopsy” literally means to “see for oneself”; the terms “post- mortem”, “post-mortem examination” and
“necropsy” are use as synonymous. Autopsy rates have been declining worldwide since 1950s. The advances in medical technology (believed to provide greater diagnostic accuracy), economic
and legal reasons justify this decline.
Methods. We retrospectively analyzed the autopsy records from the
Pathology Unit of “San Salvatore” Hospital of L’Aquila (L’Aquila,
Italy) of 590 deceased people over a 10-years period (1999-2008)
and compared the clinical and post-mortem diagnoses. Furthermore, we also compared our results to two previous consecutive
series of, respectively, 1000 and 2000 autopsied cases performed in
our Department over a 10-years and a 20-years period.
Results. According to the literature, in the present study the autopsy rate (including both clinical and required by law autopsies)
shows a homogeneous decline; the only exception is represent by
a high number of autopsies performed in 2007. Fetal autopsies
represent the 20,4% of all post-mortem examination. Of 424 adult
patients, 275 were males and 149 were females, with a mean age
of 77 years old at the time of necroscopy. Among fetal autopsies,
congenital malformations and genomic aberrations represent the
most common cause of death. It is interesting to note the high
incidence of Dandy-Walker Syndrome. Among adult autopsies,
we classified findings into six groups according to the cause of
the death, namely: Cardiovascular diseases (Group1), Malignancies (Group 2), Digestive and Accessory Digestive Glands nonneoplastic diseases (Group 3), Respiratory diseases (Group 4),
Infectious diseases (Group 5) and Miscellaneous (Group 6).
As expected, in Group 1 and Group 2 are represented the most
common cause of death, with a percentage, respectively, of
54,5% and 15,5%. Group 4 collects the third most common
causes of death with a percentage of 14,5, followed by Group 3
(9,9%), Group 6 (4,2%) and Group 5 (1,8%). Comparing these results with the two previous study, we should underline a relevant
decline of Infectious and Digestive diseases as causes of death in
favour of an increasing number of Malignancies.
The diagnoses were in total agreement in 64% of cases. The most
common causes of disagreement are represented by thromboembolic events misdiagnosed as infarcts or pulmonary diseases.
Conclusions. This study underlines once again the relevant role
of post-mortem examinations, not only to confirm the cause of
death diagnosis but also as source of investigation and research,
being the frequency of misdiagnosis not decreased significantly,
despite improvements in diagnostic technology.
Thus, autopsy remains one of the most reliable methods to validate clinical diagnoses.
References
1
Ventura T, Leocata P. Principali cause di morte riscontrate su 1000
autopsie consecutive nell’Osp. San Salvatore di L’Aquila. Settimana
degli Ospedali Vol. XX N.1 Gennaio 1978.
2
Rutty GN, Duerden RM, Carter N, et al. Are coroner’s necropsy necessary? A prospective study examining whether a “view and grant”
system of death certification could be introduced into England and
Wales. J Clin Pathol 2001;54:279-84.
G. Fabbretti, A. Bagnoli, P. Bianchi, G. Giovagnini, A. Ioli, M.
Nicolini, P. Para, A. Pederzoli, D. Piccioni, R. Priano, I. Sampaoli, M. Brisigotti
The complexity of pathology lab work, a multistep process with
a lot of handoffs, is potential source of errors that can occur at
any stage. In the past five years we have approached this challenging issue in our lab. We redesigned our laboratory workflow
to standardize each step, assisted by information technology and
the 2D bar code innovation. We reviewed working procedures,
taking into account the critical elements and the weak points in
the entire process. The goal of correct patient identification has
been achieved through the elimination of easily misinterpreted
handwritten data on requests and labels and eliminating manual
data transcription. Our laboratory information system (LIS)
(Armonia, Dedalus SpA, Italy) was integrated with our hospital
information system (HIS), with an HL7 interface for: receiving
orders from physicians trough HIS order entry. All histological
and cytological requests are received via-Web. Electronic orders
with specimens labelled computer printed, resulted in a drastic
reduction of misidentification of patients, anatomic sites, laterality and misleading clinical information. At the accession the LIS
give the identification code to the patient and samples as readable
text and 2D Bar Code on printed web-request and labels. Our LIS
provides for each anatomical site and medical procedure many
parameters which are predetermined for example the topographic
snomed, number and color of cassette (white for sentinel lymph
nodes, yellow for small biopsies, blue for lymph node etc,),
number of section, routine stains or immunostains if provided (for
example for sentinel lymph node for breast cancer). Default may
modified at any time of the process.
Cassettes are directly printed (Leica Microsystems, Bannockburn, IL) easy and quickly case by case, during gross examination. Recently the LIS has been implemented with data matrix
2-dimensional bar-code and interfaced with both cassettes Leica
printer and slides printers (Leica printer for cytology lab and
Slide Mate, Thermo Fisher Scientific, printers for cutting station).
At the tissue embedding station each cassette is “read” from the
scanner before embedding tissue. LIS displays: code number,
type of tissue, number of fragments, notes if recorded during
gross examination, operator name, date, time and the ”status”:
after reading a cassette, its status change from “in processing” to
“executed” and when all the cassettes of a single case are embedded, the case change status from “ gross executed “ to “embedded”. LIS shows on the monitor, in sequence, a list of all cassettes
embedded. At each cutting station the operator reads a cassette
from the scanner and the slide printer provides all the associated slides (routine stain, special stains and immuno stains). On
the slide are printed the following data as human readable text:
code accession number, surname and name of patient, type of
stain, the name of our lab and 2D bar code which encodes also a
progressive number of printing. Only if the slide match correctly,
the LIS displays the changing status of slide from “requested” to
“checked”. Only when all slides of a single case are checked the
case change status from “embedded” to “cut”. Our LIS is also
integrated with Leica BOND-III that fully automates immunohistochemistry. In the same manner described for routine slides,
specific charged slides are directly printed with the 2D bar code.
BOND-III reads the 2D bar code on slides. At the end of whole
work process there is the final check before delivering slides to
the referring pathologist. Each slide is read from the scanner and
when all slides of the case (routine, special and immuno stains)
are “pinged” the case is ready for medical examination. The introduction of the redesigned workflow and 2D bar code has two ad-
176
vantages; the first one the correct identification of patient, specimens, blocks and slides, the second one is the traceability of each
case along the entire work flow. The knowledge of when, where
and why the misidentification defects occur is a fundamental pre
condition for their successful reduction. Our LIS allows to record
all defects in each step of work flow, quickly and easily; by a
keyboard the system open a special section where several parameter are predetermined: the type of defect, the corrective action,
date, time and operator. Cases with a defect are highlighted by a
special icon so that pathologist is alerted to control the validity of
corrective actions done, before the diagnosis. In our experience
no single technology can eliminate errors in a complex system
like a pathology work flow. Each lab have to consider their own
work flow. LIS has a leading role to drive safely the entire process but also important are standard operating procedures for each
step, accompanied by an efficient system of recording errors and
a really daily work of compliance procedures.
staining, adipocytes constriction. Using GreenFix, antibodies
used for immunohistochemical assays showed an excellent reactivity, although we needed to optimize some protocols.
GreenFix and formalin fixation can be considered equivalent
to obtain a final diagnosis and Pathologists can easily get used
to these new morphological features. Ottix and alcohol/xylene
are equivalent methods, perhaps with a better quality of Ottix
technique. In conclusion, GreenFix and Ottix guarantee a good
quality for histoprocessing, staining and specimens reactivity
evaluation assuring high standard levels of diagnosis and a safer
workplace. All these features allow a routinary use of these products in Pathology labs.
Occupational exposure to toxic agents:
a real alternative in pathology laboratories
UOS Anatomia ed Istologia Patologica /Ospedale Evangelico Fondazione
Betania, Napoli, Italia
R. Murari, E. Pica, P.L. Alò, M.T. Ramieri
Background. Advantageous preservation of histology and detaliled cellular morphology has rendered neutral buffered formalin
(NBF) the most widely fixative in clinical pathology. Despite
excellent morphology for routine diagnostics, NBF is a crosslinking agent that induces RNA chemical modifications and fragmentation, impairing predictive tests to new targeted treatments.
Furthermore formalin is a highly toxic reagent, classified carcinogenic of class I by IARC in 2004 and WHO since October 2006,
characterized by a pungent smell, much irritating to eyes as wellknow for operators, demonstrated to be at the proven beginning
respiratory pathologies, allergies and respiratory tract cancers.
Aim. Evaluation of Greenfix fixative using paraffin-embedded
human samples regarding its effects on histomorphology as well
as on immunohistochemical (IHC) properties.
Materials and methods. Human tissues (colon, liver, lung,
lymph node, pancreas, skin, small intestine, stomach, thyroid, and
uterus) were collected.
On arrival in the lab, representative samples were taken primarily
from the large surgically excised specimens.
Parallel tissue blocks were fixed in the Greenfix and NBF.
All samples were immersed in fixative within 30 minutes of
surgery. Immersion time was similar for both fixatives for each
specimen (12-24 h).
Parallel tissue blocks were processed according to the standard
protocol used for tumor diagnosis and staging in the lab.
Briefly, this protocol was performed overnight at room temperature and consisted of dehydration in absolute ethanol (2 baths for
a total of 1 hours), followed by xylene (4 baths for a total of
7hours), and paraffin-immersion (3 baths at 58°C for 5 hours).
4μm thick sections of paraffin-embedded tissue from either NBFGreenfix were prepared for routine H&E as well IHC stains.
To evaluate histomorphology, special attention was paid to the
overall pattern of tissue preservation, cellular and extra cellular
structures, and the cell and nuclear morphology, as well as to
tinctorial reactions of various tissue and cell components.
IHC was performed automatically with a autostainer (BenchMark
XT, Ventana) with antibodies to the following antigens: AML
(1A4), CA 125 (OC125), CA19.9 (121SLE), CD3 (polyclonal),
CD20 (L26), CD34 (QBend/10), CEA (TF-3H8-1), CK7 (SP52),
CK20 (SP33), desmin (DE-R-II), EGFR (3C6), EMA (E29),
LCA (RP2/18), Melanosome (HMB45), Pan CK (AE1AE3), P53
(DO-7), P63 (4A4), S100 (polyclonal), TTF1 (8G7G3/1), Vimentin (V9), KI67 (30-9).
The intensity, pattern, and specificity of the IHC reactions were
assessed and compared on all slides.
Results. Greenfix preserved tissue integrity, giving morphological information similar to that obtained using the reference fixative NBF with respect to cytoplasmic and nuclear details.
U.O.C. Anatomia Patologica; Azienda Ospedaliera Umberto I, Frosinone,
Italia
In Pathology labs there is a daily use of chemical compounds with
high environmental and human toxicity, like formalin and xylene.
Formaldehyde (FA, CH2O) is the simplest of all aldehydes. Several studies showed that chronic exposure to FA by inhalation is
associated with eyes, nose, throat and skin irritation while acute
exposure leads to irregular heartbeat, chest pain, lungs irritation,
pulmonary edema and death. FA has also carcinogenic effects:
the exposure increases the risk of nasopharyngeal cancer and
myeloid leukemia. Basing on these evidences in 2004, International Agency for Research on Cancer (IARC) classified FA as
carcinogenic to humans (Group 1). Xylene (C8H10) is an aromatic
hydrocarbon and several studies highlighted its toxicity at different levels (effects on CNS, skin and mucous membranes irritation, change in blood-pictures).
In accordance with Italian regulatory, 81stordinance of 2008 concerning health and safety protection in working place (article 15)
established that for operator’s safety an hazardous product must
be replaced by another not or less hazardous.
Basing on these evidences is strictly necessary to find new chemical products for Pathology labs. To this aim we tested GreenFix
which is formalin substitute and Ottix Shaper/ Ottix Plus instead
of xylene and alcohols, both produced by Diapath S.p.A.
GreenFix is a mixture of ethyl alcohol and ethanedial characterized by low evaporation rate and therefore lower toxicity by
inhalation. This product is classified as irritant. Ottix Shaper is
a mixture of ethanol and other not toxic compounds, Ottix Plus
is a mixture of linear hydrocarbons, aliphatic alcohols and other
not toxic components: these products substitute both alcohols and
xylene during histoprocessing and staining steps.
In this study we analyzed 27 samples from different human tissues and collected two different parts for each sample, one fixed
in formalin and processed with alcohol/xylene (traditional protocol) and the other fixed in GreenFix and processed with Ottix.
Both sections were stained with H&E, either with alcohol/xylene
or with Ottix. We chose specific antibodies for IHC assays for
each kind of tissue to test specimen reactivity.
The staining step performed with Ottix revealed better nuclear detail compared to the traditional method. We also observed a better
staining of myoepithelial cells and melanocytes. Specimens fixed
with GreenFix had excellent chromatin and nucleolar details but
they showed some morphological differences compared to formalin fixed tissues: red blood cells with transparent aspect (“ghost”like), less intense staining of eosinophilic granulocytes, partial
detachment of glands from sub-epithelial tissue and a pale mucin
Histomorphology and innumohistochemistry
of greenfix fixed tissues: our experience
M. Postiglione, L. Nugnes, M.P. Maione, A. Maglione, A. Russo,
A. Nicastro, D. Oppressore, R. Giannatiempo
177
Poster
H&E slides of Greenfix and NBF tissue showed no significant
differences in tissue architecture, cellular and nuclear morphology, or tinctorial reaction.
Some tissue retraction was observed using Greenfix fixation, but
this did not impair the global tissue architecture nor the cellular
details in the tumor analyzed.
The effects of Greenfix fixation on immunostaining were tested
on the same samples. Although Greenfix required some optimization of the immunostaining procedures including antigen
retrieval, IHC stains of Greenfix fixed tissues were comparable
to those seen in NBF fixed.
The intensity of IHC reactions for most cytoplasmic antigens was
generally equal or stronger in Greenfix tissues.
Conversely, there was a decrease in the intensity of reactions
nuclear antigens; increasing their exposure times optimized the
sensitivity of the antibodies.
A new and sensitive approach
for immunohistochemical analysis
on formalin fixed murine tissues
D. Russo*, M. Nebuloni**, F. Pasqualini***, G. Tasso*
*
LaboSpace; **Anatomia Patologica, Dip. Scienze Cliniche L. Sacco, Università degli Studi di Milano; ***Laboratorio di Ricerche in Immunologia
ed Infiammazione, Istituto Clinico Humanitas, Milano, Italia.
Immunohistochemistry is the localization of antigens in different
tissues using specific primary and secondary antibodies. Immunohistochemistry is widely used in basic research and surgical
pathology, both in human and animal models. Frozen and fixed
tissues can be used but fixation and paraffin embedding offer
the best option for preserving the specimen morphology. Unfortunately, the most common fixatives (i.e. formalin or paraformaldehyde) may alter the biochemistry of the proteins and mask
antigens. For this reason, antigen retrieval is required to allow
antigen-antibody binding; different types of digestive enzymes or
heat-induced methods can be used.
Murine models have always been a challenge for immunohistochemistry due to low sensitivity of mouse antibodies binding
mouse tissues and limited availability of immunohistochemical
reagents for FFPE tissues. In fact, most of the mouse-specific
antibodies are only functional on frozen tissues but the quality of
frozen sections is not good enough for morphological evaluation.
The aim of the study was to propose a new and sensitive approach
for immunohistochemistry on formalin fixed-paraffin embedded
murine tissues in order to provide a useful panel of antibodies for
immunology research. We used a pressure cooker specifically
designed for antigen retrieval (temperature of 125 degrees and 20
psi, unmasking buffer at pH 6.00 which turns during boiling to
7.00) and tested a panel of 28 antibodies to identify leukocytes,
endothelial and epithelial cells, cytoskeleton molecules, proliferation markers, and cytokines. These antibodies either cross-reacted
with murine antigens or were mouse-specific. Immunohistochemical staining was carried out on an automated immunostainer. We
compared several dilutions of the antibodies, and used various
detection systems.
Overall, 20 of the tested antibodies showed specific positivity,
two antibodies failed to work and 6 produced a strong background
which made it difficult to analyze the results. The morphology of
the cells and tissues was entirely preserved in all of the samples.
In conclusion, we demonstrated the useful application of an innovative method for immunohistochemical analysis on formalin
fixed murine tissues. This method will guarantee to deliver a
clear and specific staining compared to other well recognized
techniques.
Dermopatologia
KA and SCC: GLUT1, CD1a, and CD57 different
expression
Fr. Aragona, B. Belmonte, L. Schillaci, F. Aragona, D. Cabibi
Dipartimento di Scienze per la Promozione della Salute “G. D’Alessandro”, Sezione di Anatomia Patologica “Paolo Craxi”, Università degli
Studi di Palermo, Palermo, Italia
It is not easy to reach a differential diagnosis between keratoacanthoma (KA) and squamous cell carcinoma (SCC) and furthermore there is still considerable discussion about the relationship
of these 2 tumors with immunity. To facilitate such a diagnosis,
we assessed the Glut-1 antibody, reported to be strongly and diffusely expressed in SCC but never assessed in KA. We studied 43
lesions of immunocompetent patients: 17 SCCs, 13 typical KAs
(tKAs), and 13 atypical KAs (aKAs), with histologic features of
SCC in less than 30% of the lesions. In tKA, Glut-1 stained only
the basal layers of the squamous nests (basal pattern) whereas in
SCC the squamous nests were randomly and diffusely stained
(diffuse pattern). In aKA, a biphasic pattern was observed, with
the typical KA areas showing the basal pattern and the SCC-like
areas showing the diffuse pattern. Glut-1, therefore, helps to distinguish tKAs from SCCs and highlights the intermediate aKA
group, supporting the hypothesis of a progression from KA to
SCC. Finally, we used CD1a, CD57, CD4, CD8, CD3, and CD20
antibodies to assess whether or not the progression might be
related to an in situ immunologic deficit. Significant differences
were found both in CD1a+ cells, more numerous in tKA than in
SCC and in CD57+ cells, more numerous in tKA than in aKA
and in SCC. This suggests a local immunological failure in aKA
and SCC, probably related to the action of UV rays, leading us
to consider KA as a model for the study of the interaction of skin
cancer and immunity.
Metastatic basal cell carcinoma of the skin. Report
of 2 additional cases with review of the literature
M. Bisceglia*, I. Carosi*, N. De Luca*, N. Tricarico**, G. Panniello***, D. Ben-Dor****.
Unità Operativa di Anatomia Patologica / IRCCS – Ospedale “Casa
Sollievo della Sofferenza”, San Giovanni Rotondo (FG), Italia; ** Unità
Operativa di Otorinolaringoiatria / IRCCS – Ospedale “Casa Sollievo
della Sofferenza”, San Giovanni Rotondo (FG), Italia; *** Unità Operativa
di Dermatologia, Ospedali Riuniti di Foggia, Foggia, Italia; **** Department of Pathology, Barzilai Medical Center, Ashkelon, Israel
*
Background. Basal cell carcinoma (BCC) of the skin is the most
frequent malignant tumor in humans (mostly in Caucasian populations), with an approximate incidence of 400,000 to 750,000
new cases per year in USA. It usually does not give rise to metastasis. However there are exceptions to this rule. Based on a computerized literature search (PubMed) slightly more than 300 case
of ordinary (histologically-proven) cutaneous metastasizing cases
of BCC have been found on record up to 2010 (Tavin et al, 1995;
Ting et al, 2005; Saladi et al, 2004; Robinson et al, 2003; and
personal updated review). We present herein 2 personal cases,
of metastatic BBC, one with systemic metastases and 1 with regional lymph nodal metastases, from primaries arising in the head
and neck area. The former case was already previously reported
in a national dermatology journal (Bisceglia et al, 2004), and was
not captured by the above mentioned data base repository.
Case reports. Case 1. In 2004 a 43-year old lady was admitted
with diffuse bone pain and found on radiological and scintigraphic examinations to have multiple systemic skeletal metastases, involving the cranium, vertebral column, ribs, pelvis,
and left femur. Bilateral lung metastases were also documented.
The histological diagnosis of metastatic BCC, complemented
178
by extensive immunohistochemical testing, was established on
a core needle biopsy from the iliac bone. The medical history
in this patient showed three previous excisions over the last 7
years of a retroauricular locally recurring BCC, with perineurial
space invasion on the histological examination of the last tumor
recurrence. The primary tumor and recurrences, sized 0.5 to 2
cm, were reviewed, and all the diagnoses were confirmed; and no
foci of squamous or metatypical differentiation were seen. The
patient died 5 months after the metastatic disease was discovered.
Case 2. In 2009 a 70-year old man was hospitalized for a 2 cm
sized lump on the right side of the lower neck of 1 month duration, which was surgically excised and clinically suspected to be
a lymph node metastasis of unknown origin. Histologically the
nodule corresponded to a nodular metastasis of BCC with foci of
squamous cell differentiation, infiltrating adipose tissue, without
any visible lymph nodal tissue. This man had a clinical history
dating back 5 years of repeated surgical excisions of 2 cutaneous
metachronous BCC, 1 located on the nose, which locally recurred
4 times, and 1 on the left nasolabial ridge, respectively. All slides
relating to both the original cutaneous tumors were reviewed and
both primaries, as well as the recurrences, were less than 1 cm in
size and all exhibited classical cytological features of pure basal
cell carcinoma with no foci of squamous cell differentiation. Following the diagnosis of subcutaneous metastasis from basal cell
carcinoma with squamous cell differentiation, the patient underwent ipsilateral radical neck lymph node dissection which yielded
a total of 18 lymph nodes, 13 of which were massively involved
by metastatic basosquamous carcinoma, with invasion into perinodal fibroadipose tissue in 3. This patient was then given adjuvant
local external beam radiotherapy, and is currently alive with no
evidence of disease (ANED) 2 years after the metastatic disease
was discovered.
Discussion. Metastasis from cutaneous BCC is a rare occurrence,
with a frequency on average likely around 1:10,000 histologically
examined cases (Van Domarus et al, 1984; Motegi et al, 2006).
The interval until the appearance of the metastasis is between 1
and 25 years after the original diagnosis of the primary cutaneous tumor. Around 85% of all metastasizing BCC so far reported
were located in the head and neck area (Malone et al, 2000),
however metastases have been recorded also with tumors from
other other non-facial sites, including special areas, such as the
breast [nipple-areola complex], axilla, and male and female genitals (Ferguson et al, 2009; Martorell-Calatayud et al, 2011; Berlin
et al, 2002; Feakins et al, 1997; Jones et al, 2000; Ribuffo et al,
2002).The tumor has the ability to spread by both the lymphatic
and hematogenous routes, with regional lymph nodes, mostly
cervical, lung, and bones as the most frequent target metastatic
organs. Several factors, including male gender, tumor size, duration, histology, lymphatic invasion, and perineurial spread have
been postulated as markers of the aggressive basal cell carcinoma
phenotype (Walling et al, 2004). Liver, brain and kidney as well
as soft tissue (either nearby or along draining lymphatics) have
also been rarely described as the sites of metastasis. Less than
15% of cutaneous metastasizing BCC have been found to harbour foci of squamous cell carcinomas either in the primary main
tumor mass or in the metastasis (van Domarus et al, 1984). Although it has been never proved that the metastatic capability of a
cutaneous basal cell carcinoma is imparted to it by an associated
squamous tumor component, according to some the basosquamous variant of basal cell carcinomas is considered to be a more
aggressive form (Martin et al, 2000; Garcia et al, 2009). In two
cases metastatic BCC occurred in the context of a nevoid BCC
syndrome (Lamon et al, 2010). Sentinel lymph node biopsy has
been successfully used in a case in which lymphatic invasion was
seen during the histological examination of the primary excision
specimen (Harwood et al). No effective therapy has been devised
for BCC in the metastatic phase and the recorded mean survival
time from the diagnosis of systemic metastasis is 10 months.
Our former patient from 2004 with systemic metastases died in 5
months, while the latter patient who was diagnosed with regional
metastases is ANED now 2 years post surgery.
Conclusions. 1. BCC of the skin is a very low grade malignancy,
which needs to be radically treated by surgery as most of the
metastasizing cases in the literature had been repeatedly recurring
for years before they metastasized. 2. Metastasis from a cutaneous
BCC should be considered by clinicians when evaluating cervical
lymph node metastases of an uncertain head and neck primary, at
least in patients with previous surgeries for neglected BCC.
Palisaded neutrophilic and granulomatous
dermatitis in a patient with monoclonal
gammopathy of undetermined significance
C. Colato*, V. Lora**, G. Girolomoni**
Department of Pathology and Diagnostic, University of Verona, Verona,
Italy; **Section of Dermatology and Venereology, Department of Medicine, University of Verona, Verona, Italy
*
We describe the clinicopathological findings of a 81-year-old
male who presented to our dermatology clinic with erythematous
to violaceous patches and plaques, some of them with crusting,
localized bilaterally on the posterior lower extremities (on the
posterior thighs and buttoks), at metacarpophalangeal joints on
dorsum of hands, on elbows, and at proximal interphalangeal joint
on dorsum of left first toe. The lesions started ten years before.
Laboratory investigations disclosed a IgAK monoclonal gammopathy of undetermined significance. A skin biopsy showed a
superficial and deep dermal perivascular and interstitial infiltrate
characterized by neutrophils with leukocytoclasis and histiocytes.
The infiltrate appeared palisaded with focal basophilic collagen
alteration. Histochemical staining such as Alcian Blue, PAS,
Giemsa, Ziehl-Neelsen were negative. Clinicopathological correlation was thus consistent with palisaded neutrophilic and
granulomatous dermatitis (PNGD).
This entity, previously reported under several different names,
was first designated PNGD by Chu et al. in 1994. To our knowledge, only 101 cases of PNGD have been described so far. It has
a female predominance and it has been described in association
with a variety of systemic disease (autoimmune, oncological,
infective) and drugs (anti TNFalpha inhibitors).
PNGD may be a heterogeneous entity with clinical and histopathological overlapping with interstitial granulomatous dermatitis (IGD). Reviewing the literature we suggest that PNGD
represents a separate entity with its own clinical, histological
and pathogenetic features, distinct from IGD. PNGD is indeed
clinically characterized by erythematous to violaceous patches,
plaques or nodules localized in 78 % of cases on the extremities
while IGD is typified by erythematous plaques or linear cords
mainly on the trunck (bilaterally at lateral chest, axilla, flank).
Moreover, PNGD histologically begins with a dense dermal neutrophilic infiltrate with nuclear dust and sterile abscess formation
(early lesion) followed by a basophilic degenerated collagen and
palisaded granuloma formation (fully developed lesion). Otherwise, IGD shows interstitial and palisaded granulomatous patterns around tiny foci of degenerated collagen with occasionally
rare neutrophils. In addition, to the best of our knowledge, IGD
never shows modification of the histological features over time.
Although the etiology of both entities remain unknown, some
suggestions about their pathogenesis are proposed. According to
the Gell and Coombs classification established for the hypersensitivity reactions, both entities are mediated by a T-Cell-Delayed
immune reaction of type IV. What appears different is the initial
phenomenon that seems to be related to a hypersensitivity reaction involving neuthophils only in PNGD. If it represents a type
III-immune complex reaction or a type VI d reaction in which
both neuthophils and T cells are involved remains to be clarified.
179
Poster
Finally, from a clinical perspective, recognition of the association
of PNGD with underlying systemic disease processes is important
as it may lead to an early diagnosis of the underlying systemic
conditions.
Atypical histopathological findings in cutaneous
gvhd after cord blood transplantation
C. Fondi , I. Donnini , C. Nozzoli , C. Delfino , N. Pimpinelli***, A. Bacigalupo****, A. Bosi**, S. Guidi**, R. Saccardi**,
D. Massi*
*
**
**
***
Division of Pathological Anatomy, AOU Careggi, Florence, Italy; ** BMT
Unit, Dept. of Haematology, AOU Careggi, Florence, Italy; ***Division of
Dermatology, Department of Critical Care Medicine and Surgery, AOU
Careggi, Florence, Italy; ****BMT Unit, Dept of Haematology, San Martino Hospital, Genua, Italy
*
Objectives. Cord blood (CB) transplant has been increasingly
used in the last 10 years as an alternative source of Haematopoietic Stem Cell Transplantation. This type of transplant presents
the advantage of easy procurement, the absence of risk to donors,
low risks of transmitting infections, greater tolerance of human
leukocyte antigen disparity. Although CB transplantation is often associated with a reduction in the incidence and severity of
graft-versus-host disease (GVHD), recent experience has shown
that GvHD remains a challenging problem. Cutaneous rash is the
most frequent clinical manifestation of GvHD, the skin is easily
accessible, therefore cutaneous biopsies are frequently carried
out. We report an analysis of clinical and histological features of
cutaneous GVHD in adult recipients of unrelated CB transplants
in two BMT Units.
Materials. Twenty-eight skin biopsies were collected from 22
CB recipients for a clinical suspect of cutaneous GvHD at a
median of 183 (range 14-1280) days from transplant. Median
age at HSCT was 44 yrs (range 26-60), M/F distribution was
13/9; HLA matching was 4/6 (23), 5/6 (4), 6/6 (0). Eighteen
patients received one CB Unit, 4 a double Unit. Myeloablative/
non-myeloablative conditioning regimen ratio was 21/1. CB was
infused intra-bone (11) and intravenously (11). GvHD was classified as aGvHD (14), de novo cGvHD (6) and 2 patients developed
a cGvHD following an acute onset. Skin biopsies were cut and
stained with E&E. Sections were immunohistochemically stained
with antibodies anti- Elafin (FL-117, Santa Cruz), also known as
elastase-specific inhibitor or skin-derived antileukoproteinase, a
recently described GvHD biomarker.
Results. Our study shows that the clinical picture of cutaneous
GvHD in CB recipients is protean, displaying a variable constellation of histopathological alterations: 9/22 (41%) patients showed
an atypical skin rash characterized by erythematous-squamous
patches suggestive of an eczema-like or atopic dermatitis. Such
patients showed itchy, erythematous-squamous patches mostly
located in the trunk and extremities, more rarely on the head &
neck region. Histopathology in these cases revealed epidermal
hyperplasia, spongiosis and a dermal mixed inflammatory infiltrate with lymphocytes and eosinophils, in some cases associated
with folliculitis. There was no evidence of the typical apoptotic
or fibrotic tissue damage commonly associated to GvHD. Elafin
was variably expressed in skin tissues but high expression (defined as significant staining extending to more than 50% of the
epidermis) was demonstrated only in 2/22 (9%) cases, with no
correlation with clinical presentation. All patients were treated
with steroids and diagnosis of GVHD was made on the basis of
clinical evolution.
Conclusions. Improving diagnosis and treatment of GvHD in CB
recipients is crucial for the outcome of this increasingly used procedure. We report unusual eczema-like eruptions in CB recipients.
Although the significance of such observation is still unclear, we
suggest that they represent atypical, eczema-like GvHD rashes.
Histopathology of skin biopsies in such patients does not show
the typical apoptotic or fibrotic GvHD-related tissue change, possibly leading to a false negative assessment, resulting in relevant
therapeutic implications. Prospective clinical and immunological
studies are needed for a better understanding of these unusual
features, which might lead to GVHD under diagnosis.
TRPV4 is downregulated in keratynocytes
in different human skin tumors
V. Maio*, R. Nassini**, S. Materazzi**, T. Oranges*, P. Pedretti**,
C. Fusi**, D. Massi*
*
Sezione di Anatomia Patologica, Dipartimento di Area Critica MedicoChirurgica; **Dipartimento di Farmacologia Preclinica e Clinica, Università degli Studi di Firenze
The transient receptor potential (TRP) family of channels encompasses 28 proteins expressed in a variety of cell types where they
mediate a large series of physiological functions and play major
pathophysiological roles. The TRP vanilloid 4 (TRPV4) has been
found highly expressed in a subset of somatosensory neurons
which also express the capsaicin receptor, TRPV1, and by releasing neuropeptides mediate neurogenic inflammation. It is gated
by small reductions in tonicity and by temperatures >27°C 1. The
expression of TRPV4 by neurosensory structures, including circumventricular organs, which detect changes in systemic osmolality, inner ear hair cells, Merkel cells and sensory neurons, and
its activation by hypotonic stimuli, suggests that it functions to
detect osmotic and mechanical stimuli. TRPV4 immunoreactivity
was differentially identified on basal and suprabasal keratinocytes
of healthy human skin 2-3 and their functions have been related to
cell survival after skin exposure to noxious heat. However, the
expression and function of TRPV4 in skin cancer is poorly understood. The TRP family of proteins exhibits differential expression
in cancer tissues. Rather than mutations, changes in expression
of TRP proteins seem to be related to alterations in wild type
protein level, which might be associated with specific stages of
cancer. Here, TRPV4 protein expression has been investigated by
immunohistochemistry in human normal skin and in a series of
premalignant and invasive cutaneous carcinomas, including solar
keratoses, Bowen’s disease, squamous cell carcinomas (SCC) on
sun-exposed skin, SCC on sun-protected genital skin and BCC of
different histotypes. Formalin-fixed, paraffin-embedded sections
(4 μm) were dewaxed and hydrated with graded ethanol. Antigen
retrieval was performed in citrate buffer at 97°C for 15 min followed by cooling at room temperature. Endogenous peroxidase
activity was blocked with 3% hydrogen peroxide. After blocking with normal horse serum, sections were incubated with an
anti-rabbit TRPV4 antibody (dilution 1:500) overnight at 4 °C.
Staining was achieved using Avidin-Biotin-Peroxidase method.
Signal was detected using aminoethylcarbazol as chromogen. In
normal skin, TRPV4 was diffusely expressed in basal and suprabasal epidermal keratinocytes, and was consistently observed in
adnexal structures. Intense immunostaining was detectable in the
epidermal (i.e. the acrosyringia) and dermal part of the eccrine
sweat gland ducts. The secretory portion of sweat glands showed
staining of single secretory and myoepithelial cells. Endothelial
cells decorating dermal blood vessels were also TRPV4 positive.
In solar keratoses and Bowen’s disease, atypical keratinocytes
showed a partial to complete loss of TRPV4 expression. In UVinduced SCC on sun-exposed skin and in SCC on protected sites
TRPV4 was strongly downregulated while BCC, irrespective of
different histotypes, were TRPV4 negative. In addition, the ability of TRPV4 agonists to promote a calcium response was studied
in vitro in human cultured keratinocyte cell lines. TRPV4 agonist,
4αPDD evoked a calcium response in immortalized cultured
keratinocytes (NVTC cells) and in cells obtained from a basal
cell carcinoma. These responses were inhibited by the TRPA1
antagonist, HC-030031. Present results suggest that TRPV4 is
substantially downregulation in skin cancer tissues compared
180
with normal skin tissues. Whether downregulation of TRPV4 in
skin cancer is required for or is a consequence of cancer progression remains to be investigated.
References
1
Liedtke, W, Choe Y, Martí-Renom MA, et al. Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate
osmoreceptor. Cell 2000;103:525-35.
2
Chung, MK, Lee H, Caterina MJ. Warm temperatures activate TRPV4
in mouse 308 keratinocytes. J Biol Chem 2003;278:32037-46.
3
Radtke, C, Sinis N, Sauter M, et al. TRPV channel expression in human skin and possible role in thermally induced cell death. J Burn
Care Res 2011;32: 150-9.
FKBP51 and CAF-1 look as promising proliferation
and prognostic markers for skin melanoma
M. Mascolo*, G. Ilardi*, M. Siano*, M.F. Romano**, S. Romano**,
G. De Rosa* ***, S. Staibano*
Dipartimento di Scienze Biomorfologiche e Funzionali, Sezione di Anatomia Patologica, Università di Napoli, Napoli, Italia; **Dipartimento
di Biochimica e Biotecnologia Medica, Università di Napoli “Federico
II”, Napoli, Italia; ***Centro di Ricerche Oncologiche della Basilicata
(C.R.O.B.), Rionero in Vulture, Potenza, Italy.
*
Skin melanoma (SM) is one of the most lethal malignancies, with
a poor response to conventional anticancer treatment. Currently,
a subgroup of patients is still diagnosed with advanced stage SM,
having a very little chance to survive for a long time. It is then
mandatory to better understand the molecular pathways involved
in melanoma progression, with the aim to identify specific target proteins for new molecular therapy protocols. Recently, we
focused our efforts to clarify the role played by FK506 binding
protein 51 (FKBP51), on melanoma biology. This protein is a
cochaperone belonging to the immunophilin family, which exert
a pivotal role in the regulation of several fundamental biological processes in normal and neoplastic cells. We demonstrated
that its expression correlates with melanoma aggressiveness,
being maximal in metastatic lesions; furthermore, it regulates
the Rx-induced melanoma cell apoptosis. These data have led
us to consider FKBP51 as either a promising prognostic marker
for melanoma patients, and a possible target for radiosensitizing
melanoma metastases. We explored also the role in melanoma
progression of the Chromatin Assembly Factor-1 (CAF-1), a
heterotrimeric protein complex which exherts a major role in the
regulation of cell proliferation and has been proposed as a novel
sensible proliferation marker for several malignant tumors. We
found that the co-hyperexpression of CAF-1 and FKBP51 identifies a more aggressive subgroup of melanomas, highlighting their
synergistic promoting effect in determining the metastasizing
behaviour of SM.
methyl-guanine-methyl-transferase (MGMT) gene promoter has
favorable prognostic significance in cancer patients by enhancing cancer cells chemosensitivity to alkylanting drugs. Human
CD133 (PROM1, prominin-1, AC133 antigen) has been proposed
as a potential cancer stem cell marker in several human cancers,
including melanoma, and CD133+ cells have been suggested to
be implied in the transformation from nevus to melanoma and in
the progression to metastatic disease.
Aim and methods. In the present study we analyzed the immunohistochemical expression of the CD133 protein in a subset
of primary melanomas and corresponding recurrences and/or
metastases. The results obtained were correlated with MGMT
gene promoter methylation status in the same cases and with the
clinical outcome of the patients.
Results. A total of 37 primitive cutaneous melanomas and 74
subsequent recurrences (36 visceral, 21 lymph node and 17 cutaneous) diagnosed in the Department of Anatomia Patologica of
Modena from 1988 to 2008 were analyzed. Patients included 21
males and 16 females. The mean age at diagnosis was 62.43±13.93
years (range 34-88). Histologically 1 case was Clark’s 2 level, 6
were Clark’s 3, 24 were Clark’s 4 and 6 were Clark’s 5. In 13
cases Breslow’s thickness was > 4 mm, in 12 cases was over
2 to 4mm, in 11 was over 1 to 2 and in 1 case was until 1mm.
Ulceration of the tumor was observed in 14 cases and more than
4 mitoses x10 HP were counted in 18 primitive tumors. Overall
survival (OS) was50.51±33.55 months (range 6 - 132) whereas
the disease free survival (DFS) was 27.24+21.6 months (range 3
- 72). Immunohistochemical staining for CD133 showed positive
immunoreactivity in 16/37 (43.24%) primitive malenoma, and in
26/74 (35.13%) recurrences. Of the 37 primitive tumors, only 1
case (2.7%) had MGMT promoter methylation; differently in the
recurrences group the frequency of methylation was detected in
22 malignancies (29.7%). Cross between CD133 expression and
MGMT status identified 4 groups: MGMT methylated/CD133+,
MGMT un-methylated/CD133+, MGMT methylated/CD133-,
and MGMT un-methylated/CD133-.
Respect to the clinical pathological data as well as OS and DFS,
no statistical significant differences were found related to CD133
expression. A statistically significant difference was detected
with MGMT status and recurrence (p< 0.04). The distribution
of methylation status by localization of recurrences showed a
predominantly frequency in visceral recurrences respect other
sites (p<0.007). No statistical significant differences have been
found respect to the clinical and pathological data. Kaplan-Meyer
curves OS showed better outcome in metastases MGMT positive
respect to the negative cases. Similar trend was observed for
the DFS. The longest mean OS and DFS were recorded among
patients with MGMT positive/CD133+. No patients with unmethylated MGMT promoter/CD133- were alive.
Correlation between CD133 expression and mgmt
status in recurrences melanoma
Ossyfying trichilemmal cyst
M. Migaldi*, L. Reggiani Bonetti*, A.M. Cesinaro*, A. Maiorana*,
A. Farinetti**, S. Bettelli*, A. Sgambato***
Laboratori, Sez. di Anatomia e Istologia Patologica, Università degli Studi di Modena e Reggio Emilia; **Dipartimento di Chirurgia Generale e
Specialità Chirurgiche, Università degli Studi di Modena e Reggio Emilia,
Italia; ***Centro di Ricerche Oncologiche “Giovanni XXIII”, Istituto di
Patologia Generale, Università Cattolica del Sacro Cuore, Roma.
*
Introduction. Melanoma is one of the most life-threatening skin
tumor because of its metastatic potential, with a survival rate estimated about 60% and 45% for patients stage II, and about 15%
and 10% for patients in stage IV, respectively at 5- and 10-years.
Among the new therapies for patients with advanced melanoma,
the alkylating drugs have shown promising results in a subset
of patients. Recent studies reported that metylation of the O6-
D. Morichetti, T. Pusiol, M.G. Zorzi
Introduction. Trichilemmal cysts (TCs) occur as solitary or multiple intradermal or subcutaneous lesions, mainly on the scalp.
The TCs are lined by stratified squamous epithelium, showing
trichilemmal keratinisation. Sebaceous and apocrine differentiations have been reported in the wall. In the present report we
describe two cases of metaplastic bone formation within a TC.
Case reports. Case 1: A previously healthy 46-year old woman
presented with a 8 month history of a tender dome shaped 2.5x2
cm. cutaneous lesion on the scalp. The lesion was locally excised
with free margins. No evidence of recurrence nor metastases has
been observed 1 year and 4 months after resection. The entire
biopsy specimen measured 3x2x2 cm. and was routinely fixed,
processed and stained with hematoxylin-eosin. Microscopically
181
Poster
the nodule was a well circumscribed a cystic unilocular dermal
lesion. The wall was composed of an outer layer of basaloid cells,
surmounted by several layers of pale, eosinophilic squamous
cells. Rupture of the cyst wall was found. Intraluminal dystrophic
calcification and membranous ossification were evident as well as
adjacent to parietal rupture.
Case 2: A 64 year-old man presented with irregularly pigmented
asymmetric plaque with irregular borders. The lesion had been
present on the palm of the right hand for more than a year and
measured cm 1.2 x 1. The lesion was locally excised with free
margins for histologic examination with conclusive diagnosis of
invasive malanoma. Histologically the vertical growth phase was
composed by epithelioid atypical melanoma cells that fills and
expands the papillary dermis (Clark Level III, Breslow thickness:
1.8 mm). The dermal mitotic rate was seven mitosis per square
millimetre. The radial growth phase showed the features of superficial spreading melanoma. The clinical examination showed
cutaneous nodule of 1.2 x 1 cm localized on the back. The nodule was a well circumscribed a cystic unilocular dermal lesion.
Rupture of the cyst wall was found. Intraluminal dystrophic
calcification and membranous ossification were evident as well
as adjacent to parietal rupture.
Discussion. TC is a nodular cystic lesion derived from the isthmus of the hair follicle. Cholesterol clefts are common and foci
of calcification occur within the cyst lumen in about 25% of
cases. Occasionally rupture of the cyst wall is observed, associated with a granulomatous response to the cyst contents. Repair
of the rupture defect may produce the entry of inflammatory cells
and fibroblasts into the cysts with subsequent organization. Irregular hyperplasia of the epithelial lining may be a consequence
of this fact. The latter change may account for the proliferating
trichilemmal cyst. Cutaneous ossification has traditionally been
classified into primary event without a demonstrable preceding
skin lesion and a secondary type (metaplastic ossification). In the
latter possibility ossification develops in association to local conditions such as trauma, scarring, inflammatory processes or, most
commonly, benign/malignant cutaneous tumours. The majority of
these neoplasms tend to be epithelial or melanocytic lesions. Several bone-forming growth-regulating factors have been identified
that may also participate in secondary ossification. Cutaneous
bone usually develops by membranous (mesenchymal) ossification without the presence of a cartilage precursor. In our cases,
no areas of mature cartilaginous were observed near the focus of
ossification. The cutaneous bone may have been directly formed
from osteogenic stromal elements without a cartilaginous precursor (membranous or mesenchymal ossification). However the
dystrophic calcification secondary to cyst wall rupture might also
contribute to the bone-forming process.
References
Civatte J, et al. Ann Dermatol Syphiligr (Paris).1974;75:402-3.
Eccrine siringofibroadenoma of the eyelid
T. Pusiol, D. Morichetti, M.G. Zorzi
Institute of Anatomic Pathology, Rovereto Hospital, Italy.
Introduction. Eccrine siringofibroadenoma (ES) was first described by Mascaro in 1963 1 and is an uncommon tumour of the
acrosyringium. Several types of eccrine and apocrine tumours are
observed in the eyelids, but the ES has not been reported previously in this site.
Materials and methods. A 84-year-old man attended the eye
clinic with a soft ovoid mass (8 mm diameter) in the right upper
eyelid. The lesion has been present for several years. No recent
change had been noticed. The tumour was excides. The lesion appears to be arising at the eyelid margin, close to the mucocutaneous junction. The lesion is characterised by multiple downgrowths
of squamoid or cuboidal epithelial cells which anastomose around
a bland fibroblastic stroma. In many areas, one can appreciate
formation of multiple small ductal structures by these epithelial
downgrowths, as well as multifocally scattered mucinous cells
(Figure 1). No evidence of malignancy was found. The diagnosis
of EC was performed. ES usually affects the extremities of elderly individuals either as solitary or multiple tumours.
Discussion. It has been suggested that ES, as describe by Mascaro, is identical to the acrosyringeal nevus of Weedon and
Lewis, but there do appear to be some clinicopathological. The
terms “acrosyringeal adenomatosis” and “eccrine syringofibroadenomatosis” have been suggested as appropriate designations for
the more diffuse cases. Multiple lesions have been reported in
association with Schopf syndrome 2 and Clouston’s syndrome
3
. In our patients no features of these syndrome were found.
The overall appearance of ES incorporates aspects of mammary
fibroadenoma as well as fibroepithelioma of Pinkus (fibroepitheliomatous basal cell carcinoma). The stroma is likely “induced”
by the epithelial proliferation. It has been suggested that ES is
identical to the acrosyringeal nevus of Weedon and Lewis 4, but
there do appear to be some clinicopathological differences.
References
1
Mascaro JM. Considerations sur les tumeurs fibroepitheliales. Le
syringofibroadenome eccrine. Ann Derm Syphiligr 1963;90:146.
2
Starink TM. Eccrine syringofibroadenoma: multiple lesions representing a new cutaneous marker of the Schöpf syndrome, and solitary
nonhereditary tumors. J Am Acad Dermatol 1997;36:569-76.
3
Clouston HR. A Hereditary Ectodermal Dystrophy. Can Med Assoc J
1929;21:18-31.
4
Weedon D, Lewis J. Acrosyringeal nevus. J Cutan Pathol 1977;4:1668.
On a stengel-wolbach sclerosis: a century
after the last case report
B.J. Rocca, M.R. Ambrosio, M. Onorati, R. Santopietro, V.
Mourmouras, C. Cardone, S. Lazzi S
Department of Human Pathology and Oncology, Anatomic Pathology Section - University of Siena, Italy
Background. Stengel-Wolbach sclerosis (Kettle’s disease, RobbSmith reticulosis, Boeck’s sarcoidosis) is a rare form of granulomatosis. It has been observed mainly in adults, without significant
differences of gender. Its cause is unknown and it has been interpreted as a histological variant of sarcoidosis. Firstly described
by Wolbach in 1910, only few cases have been published as case
reports, all at the beginning of the last century.
Methods. We describe the case of a 29- year-old female, who
entered the Hospital complaining of increasing weakness and
anorexia. The abdomen was soft but presented a large, smooth,
flat, tender, hard mass in the left costal margin. Laboratory findings showed: red blood cell count 3.5x106/mm3; hemoglobin 11g/
dL; total platelet count 90.2x109/L, calcium 11 mg/dl. The clinical diagnosis was “systemic amyloidosis of unknown origin”. In
the attempt to find the etiology, abdomen ultrasound, whole body
computed tomography and umbilical adipose tissue biopsy were
performed, however with negative results. Therapeutic splenectomy, together with removal of an accessory spleen and of some
perisplenic lymph nodes, was performed and representative
samples of the spleen, accessory spleen and lymph nodes were
stained with haematoxylin and eosin. Picromallory and Congo
red stains were also carried out as well as polymerase chain reaction (PCR) for mycobacterium.
Results. The surgical specimen consisted of enlarged spleen
measuring 19x12x10 cm and weighing 1100 g. The capsule was
bluish red and slightly nodular. Small, nodules, ranging from 2 to
5 mm were observed. The nodules were gray, translucent, quite
discrete and sharply circumscribed. On cut section, the splenic
pulp was reddish blue and bulging and showed moderately elevated, papular lesions with a tendency towards aggregations into
182
groups. The nodular lesions were firm and whitish and sometimes
presented a depressed, white, scarred area in their central portion.
The intervening splenic parenchyma was moderately congested
and homogeneously dark reddish. Microscopic examination
of the spleen showed that the pulp was filled with, and almost
completely replaced by, epithelioid cell granulomas, with multinucleated giant cells, encircled by connective tissue. Giants cells
were both of Langhans and foreign-body type, with star-shaped
asteroid bodies and lipidic vacuoles in their cytoplasm. No
Schaumann bodies were detected. The accessory spleen and perisplenic lymph nodes showed similar lesions. Necrosis was not
observed (Picromallory stain was negative). Gram and PCR for
mycobacterium were negative. The final diagnosis was StengelWolbach sclerosis involving the spleen and perisplenic lymph
nodes. Nine months after the diagnosis, a thoracic TC revealed
multiple nodules in the lungs and mediastinal lymphadenopathies
consistent with sarcoidosis.
Conclusions. The cause of sarcoidosis is unknown. A variety of
infectious and noninfectious agents have been implicated, but
there is no proof that any specific agent is responsible. However,
all available evidence is consistent with the concept that the
disease results from an exaggerated cellular immune response to
a limited class of antigens or self-antigens. Sarcoidosis usually
begins with thoracic (hilar) lymph nodes and pulmonary involvement; on the contrary, in our case, the first sites involved were
the spleen and perisplenic lymph nodes. We conclude that, after
a century, a new case of Stengel-Wolbach sclerosis is herein illustrated.
nerve sheath lesions, in order to assess if it is differently expressed in sporadic, NF1 associated and malignant lesions. We
studied 18 typical, solitary, sporadic neurofibromas (Group A)
and 21 cases (Group B) consisting of 11 NF1 associated cases
(10 neurofibromas and 1 malignant peripheral nerve sheat tumor)
and 10 cases without known history of NF1 at the time of the
histological diagnosis, consisting of 2 malignant peripheral nerve
sheath tumors and 8 atypical neurofibromas.
We found that CD10 immunopositivity was absent or very weak
and focal in Group A. On the contrary, CD10 was strongly expressed in Group B, including all the MPNST and their metastases, with 95% sensitivity and 72% specificity in distinguishing
between the two groups.
Granulomatous reactions from silicone:
a diagnostic trap for the dermatopathologist
Unità Operativa, Ospedale “Casa Sollievo della Sofferenza”, San Giovanni Rotondo, Italia; 2Department of Pathology, Cook Children’s Medical Center, Fort Worth, TX, USA; 3Unità Operativa di Anatomia Patologica, Ospedale Policlinico, Bari, Italia; 4Unità Operativa di Patologica,
Istituto “Gaetano Pini”, Milano, Italia; 5Unità Operativa di Anatomia
Patologica, Ospedale Generale “S. Maria della Misericordia”, Udine,
Italia; 6Diagnostico Italiano, International Center for Oncologic Pathology Consultations, Milan, Italy
M. Trombatore*, D. Giallombardo*, M. Castiglia**, B. Belmonte*,
D. Cabibi*
Studi di Palermo, Palermo, Italia; **Dipartimento di Oncologia, Università degli Studi di Palermo, Palermo, Italia.
*
We present a case of late granulomatous reaction from silicone
that first appeared in a site different from that of the injection, The
histological picture was a cystic-macrophagic granuloma in both
the injection site (upper lip) and the migrating site (paranasal regions). The case is interesting, due to the long time (8 years) that
elapsed between the cosmetic operation and the appearance of the
clinical symptoms (which led initially to a misleading diagnostic
picture), for the site of onset of the dermopathy (which was different from the injection site) and for the unusual histologic features
(all these aspects, in the absence of the correct clinical picture and
of immunohistochemical support, led to an uncorrect diagnosis of
liposarcoma in the beginning.) We think that the foreign body has
undergone an antigravity migration from the upper lip to the right
paranasal region. The antigravitary migration hypothesis, to our
knowledge, has not been yet reported in literature.
Patologia dei tessuti molli
Immunohistochemistry of peripheral nerve sheath
tumors: usefulness of cd10 antibody
B. Belmonte, O. Schillaci, Fr. Aragona, V. Rodolico, D. Cabibi D.
Dipartimento di Scienze per la tutela della Salute “G. D’Alessandro”,
Sezione di Anatomia Patologica “Paolo Craxi”, Università degli Studi di
Palermo, Italia.
Neurofibromas are sporadic or associated with type 1 Neurofibromatosis (NF1), with a higher risk of malignant progression
123
. We investigated CD10 immunoexpression in 39 peripheral
References
1
National Institutes of Health. Neurofibromatosos: National Institutes
of Health Consensus Development Conference Statement. Vol 6, July
13-15, 1987, pp. 1-9
2
Weiss SW, Goldblum JR. Enzinger & Weiss’s Soft Tissue Tumors.
Fifth Edition. St. Louis, Missouri: Mosby-Elsevier 2008.
3
Zhou H, Coffin CM, Perkins SL. Malignant peripheral nerve sheath
tumor: a comparison of grade, immunophenotype, and cell cycle/
growth activation marker expression in sporadic and neurofibromatosis 1-related lesions. Am J Surg Pathol 2003;27:1337-45.
Immunohistochemical investigation of WT1
expression in 117 embryonal tumors
M. Bisceglia1, M. Vairo1, C. Galliani2, G. Lastilla3, A. Parafioriti4,
G. DeMaglio5, J. Rosai6
1
Background. Wilms tumor transcription factor-1 (WT1), a
449-aminoacid, 52-62 kDa molecular weight protein, is encoded
by the Wilms tumor supressor gene (located on chromosome
11p13). WT1 controls the expression of growth factors that
regulate glomerular capillary development and is involved in the
phenotypic plasticity of cells during the mesenchymal epithelial
transition process, exerts a cooperative effect on p53, activates
the bcl-2 gene, and is normally expressed in mesothelium and
podocytes of the kidneys. WT1 is expressed also in neoplasms.
Sporadic nephroblastoma is the prototypical neoplasm expressing
WT1 protein in tumor cell nuclei with a frequency of around 8085% of cases (Ghanem et al, 2000; Bisceglia et al, 2009). Other
tumors which have been described as exhibiting nuclear immunohistochemical staining for nuclear WT1 protein are ovarian
and mesothelial malignancies, Sertoli cell tumors, thyroid carcinoma, and DSRCT, and occasionally also congenital mesoblastic
nephroma and renal and extrarenal malignant rhabdoid tumors.
However WT1 protein or mRNA has been documented with variable frequency even in the cytoplasm of the tumor cells of other
neoplasms from various anatomical sites, including vascular and
uterine tumors, breast and thyroid carcinomas, melanoma, high
grade astrocytomas, acute leukemias, salivary gland pleomorphic
adenomas, and even in gastrointestinal, pancreatobiliary, urothelial and bone malignancies.
Aims of the study. We investigated 117 embryonal tumors, other
than nephroblastomas, mostly of the small round cell type, for
WT1 immunohistochemical expression.
Materials and methods. The study included (i) 14 peripheral
neuroblastomas; (ii) 62 embryonal soft tissue and bone tumors
(28 Ewing sarcoma/peripheral primitive neuroectodermal tumors,
18 embryonal rhabdomyosarcomas, and 16 alveolar rhabdomy-
183
Poster
osarcomas); (iii) 11 visceral embryonal tumors [4 hepatoblastomas, 4 pleuropulmonary blastomas (PPBs; 2 type I, 1 type II, and
1 type III), 1 pancreatoblastoma, 1 paraganglioblastoma, and 1
undifferentiated embryonal liver sarcoma from an adult]; (iv) 5
desmoplastic small round cell tumors (DSRCTs), of which 3 were
intra-abdominal and 2 extra-abdominal; and (v) 25 embryonal
tumors of the central nervous system (CNS) [10 supratentorial
central primitive neuroectodermal tumors (cPNETs), 14 infratentorial (medulloblastomas), and 1 intraspinal]. Of the 117 patients
in this study, 75 were males and 42 were females; 76 were in the
pediatric age group (<21 y), 15 were young adults (>21 y and ≤30
y), and 26 were adults (≥31 y). Eligible cases were retrieved from
the pathology files of the participating institutions, a few of which
had been referred as consultations or as a courtesy. Sections of
formalin fixed, paraffin-embedded specimens were exposed to
two 15 minute cycles of heat-induced antigen retrieval in 10mM citrate buffer (pH=6.0) using a 360-W microwave oven.
The monoclonal antibody used was WT1 (1:50 dilution; clone
6F-H2; DakoCytomation). Immunohistochemical staining was
performed using the labeled Envision detection system according
to the manufacturer’s recommendations in a DAKO Autostainer
(Dako).
Results. No embryonal tumor in this study expressed nuclear
WT1 expression. WT1 cytoplasmic positivity was seen in 17 of
18 cases of ERMS (diffuse and strong in 15 and focal in 2, one
with staining in less than 30% and the other in less than 10% of
tumor cells, respectively), and in 15 of 16 ARMSs (mostly diffuse and of moderate to strong intensity in 6, widespread with less
than 50% in 6, and focal with 10% or less positive tumor cells in
3). Furthermore, moderate cytoplasmic staining with WT1 was
observed in the following: 3 of 5 DSRCTs (focal reactivity); 7
of 13 peripheral neuroblastomas (focal and weak); 2 out of 4
PPBs (positivity in diffuse, spindle cell component in 1 PPB type
II with anaplasia and in 1 PPB type III with rhabdomyoblasts,
respectively); 4 of 10 supratentorial (diffuse in 2, focal in 2)
and 3 of 14 infratentorial PNETs (diffuse in 1, focal in 2); 2 of
5 hepatoblastomas (1 diffuse, 1 focal); 7 of 28 EWSs/pPNETs
(diffuse in 3, focal in 2, rare cells only in 2); and in the single
paraganglioblastoma (focal, in small round tumor cells).
Discussion. WT1 cytoplasmic immunohistochemical staining
should be no longer considered as non-specific since post-translational phosphorylation of WT1 results in cytoplasmic retention
of the protein which may play a role in translational regulation
in tumor cells (Hohenstein and Hastie, 2006) and there is also
some evidence that the rate of immunohistochemical detection of
cytoplasmic WT1 expression correlates with that of the nuclear
expression in some tumors (e.g. thyroid cancer) in which it likely
functions as a differentiation promoter (Tanaka et al, 2007). In
our study the consistent cytoplasmic WT1 immunostaining in
almost all cases of ERMS and in the majority of ARMS make
us consider cytoplasmic staining with WT1 as a reliable and
sensitive adjunctive immunomarker for rhabdomyoblastic differentiation. This finding is in agreement with previous studies
by other authors (Carpentieri et al, 2003; Mentzel et al 2006) as
well as with our own previous experience with 7 cases of rhabdomyomatous nephroblastomas (Bisceglia et al, 2009), 1 case of
rhabdomyosarcoma of the prostate in an adult (Bisceglia et al,
2011), and 1 case of spindle cell rhabdomyosarcoma of the heart
(Fraternali et a, 2010l). The significance of the cytoplasmic WT1
immunostaining noticed in 3 of 5 DSRCTs, in 2 PPBs, and in a
single case of paraganglioblastoma might reflect the polyphenotypic nature of these tumors.
Additionally the WT1 cytoplasmic positivity in 2 cases of central
PNETs (1 supratentorial PNET and 1 anaplastic medulloblastoma) in our study is of note, because of the previous immunohistochemical evidence of rhabdomyoblastic differentiation (nuclear
immunopositivity with myogenin) we demonstrated in the course
of diagnostic routine work in both. In contrast, of more dubious
significance is the WT1 cytoplasmic immunopositivity seen in
the rest of the embryonal tumors of neural lineage (which is,
however, usually focal and weak and seen in a minority of cases)
of the CNS and soft tissue, and in 2 hepatoblastomas. Finally,
nuclear negativity for WT1 in the DSRCTs in our cases is not
surprising, given that the clone we used is directed to the amino
terminus of the protein (in DSRCT – because of the EWS-WT1
gene fusion – only the carboxy terminus portion is made which is
recognized only with the (WT)C-19 antibody).
Conclusions. Embryonal tumors in this study did not express immunohistochemical nuclear WT1 expression. Cytoplasmic WT1
expression in embryonal tumors can be regarded as an helpful
and consistent immunomarker for tumors with skeletal muscle
differentiation. The cytoplasmic WT1 expression, which is rarely,
mostly focal, seen also in embryonal tumors of neural lineage and
hepatoblastomas, still needs to be elucidated.
Soft tissue hemangioblastoma-like tumor:
a new tumor or a soft tissue variant of peripheral
sporadic hemangioblastoma?
M. Bisceglia*, L. Muscarella**, L. D’Agruma***, G. Pasquinelli****
Unità Operativa di Anatomia Patologica; **Laboratorio di Oncologia;
Unità Operativa di Genetica Medica, IRCCS, Ospedale “Casa Sollievo
della Sofferenza”, San Giovanni Rotondo (FG), Italia; ****Unità Operativa
di Surgical Pathology, Dipartimento di Ematologia, Oncologia e Patologia Clinica, Policlinico S. Orsola, Università di Bologna, Bologna, Italy
*
***
Background and aims of the study. Two forms of capillary
hemangioblastomas (HGB) have been nosologically delineated: the familial form, which is the most frequent manifestation of von Hippel-Lindau (VHL) disease, a genetic autosomal
dominant condition, and the non-familial form. The majority of
cases of HGB (70%) are of the non-familial type. VHL-associated HGB as well as sporadic HGB mostly can occur in any
part of the CNS (central or neuraxial HGB), including the optic
nerve and retina. However, rarely, HGB of either form may
also occur outside the CNS (extraneuraxial HGB). Sporadic
HGB outside the CNS has been described in the following anatomical locations: i. spinal nerve roots or pia, filum terminale,
and cauda equine (perineuraxial HGB); ii. internal organs, such
as the kidney (visceral HGB); iii. soft tissues of body cavities,
such as the retroperitoneum and pelvis, and somatic soft tissues, either related or unrelated to peripheral nerves; iv. skin;
and v. even in bone (peripheral HGB). We report on a case of
a “richly vascularised, benign mesenchymal hemangioblastoma-like tumor of soft tissue”, posing significant difficulty in
categorical diagnosis, which was analyzed histologically, immunohistochemically, and with electron microscopy (EM) and
molecular testing.
Case report. A 34-year old unmarried female underwent surgical
excision of a 2.5 cm subcutaneous lump from her left thigh. The
lesion was present for 1 year. The clinical diagnosis was fibroma.
Grossly this lesion was rubbery in consistency and solid on sectioning. Histologically the most characteristic features were a
tumor cell population consisting of round to polygonal clear cells
with microvacuolated cytoplasm and a well developed arborizing
network of capillary-sized vessels surrounding the tumor cells.
Immunohistochemically the tumor cells were diffusely immunopositive with vimentin, S-100 protein, NSE, CD57, BCL-2,
CD-99, and alpha-inhibin; and immunonegative with EMA,
cytokeratins, muscle specific actin, alpha-SMA, desmin, Hcaldesmon, and calponin. Scattered tumor cells also showed focal
dot-like immunoreactivity for neurofilaments. On the assumption
that the tumor cells were the same “stromal cells” one sees in
hemangioblastomas of the CNS and taking everything into account a diagnosis of peripheral extraneuraxial HGB was rendered
and the suggestion was given to look for other (possible) clinical
184
manifestations of VHL syndrome in the patient and her family,
which were not found. Following that EM investigation on a
piece of tumor from the paraffin block and molecular analysis of
both a peripheral blood sample from this patient and tumor tissue
from the paraffin block were also performed. EM documented the
tumor cells as being of mesenchymal nature with tracts of basal
lamina on the external cell surface, and abundant glycogen particles, intermediate filaments, and rough endoplasmic reticulum
in the cytoplasm. Notably occasional electron-dense granules of
secretory type were noticed in some cells; lipid droplets were not
demonstrated. Molecular characterization of the VHL gene was
performed by mutation analysis, fluorescent loss of heterozygosity (LOH) with microsatellites, and methylation analysis, but no
germline or somatic mutation was revealed in this patient either
in the peripheral blood examined or in the tumor by means of any
of these techniques employed.
Discussion. The case presented herein showed histological
and immunohistochemical features consistent with HGB. EM
could not document lipid droplets which are the most frequent
ultrastructural findings usually seen in this type of tumor and, in
addition, demonstrated neurosecretory granules which have not
been previously reported in this context. Molecular analysis did
not reveal any DNA alteration of the VHL gene (gene mapped
to chromosome 3p25-26), which would support our initial diagnosis, however it did not exclude such a possibility. In fact,
although the VHL gene is presumed to be involved also in the
development of sporadic HGB, either central (neuraxial) HGB
or peripheral types, molecular analyses remain elusive in most
of these cases. In regard to the sporadic HGB of CNS (cerebellar
HGB) there are a few reports of molecular analyses, documenting either somatic mutations of the VHL gene in a proportion
of these cases (around 50%) studying microdissected tumor
stromal cells (Lee et al, 1998), or ineffective germline mutations, not at risk of developing classical VHL disease (5-15%),
or mosaic germline mutation (4%) studying DNA from the
peripheral blood of these patients (Olschwang et al, 1998;
Catapano et al, 2005; Woodward et al, 2007). In regard to sporadic extra-axial HGB, only a few cases have been molecularly
analyzed: of the first 2 so studied one was a multifocal, recurrent lesion, arising from different spinal nerve roots of the mid
cervical medullary segments in a 57 year-old male (Raghavan
et al, 2000) and the other was a soft tissue tumor of the ankle in
a 74 year-old woman (Michal et al, 2004); in both patients no
genetic alterations of the VHL gene were documented by both
complete sequence analysis and LOH analysis in the former
and mutation analysis only in the latter, respectively. In another
study (using mutation, microsatellite, and methylation analyses)
presented both at the last SIAPEC-IAP Congress of 2010 in
Italy (Muscarella et al, 2010) and the 2011 USCAP in the US
(Muscarella et al, 2010), we reported the results from 5 cases
of sporadic extraneuraxial HGB, in which one single somatic hit
in 2 cases (one perineuraxial HGB and another peripheral) and
two somatic hits in another (perineuraxial) one were found, thus
confirming the hypothesis that the VHL gene is involved also in
the development of sporadic extra-axial HGB. However in the
remaining 2 cases no mutation was found. In the case herein
presented, not included in the previous study, using the same
multimodal analyses, no mutation was found.
Conclusion. This tumor is intriguing and difficult to classify
completely because: i. light microscopy and immunohistochemistry would suggest the diagnosis of HGB; ii. EM seems to
militate against HGB, still it may just indicate a more evident
neural differentiation; and iii. molecular testing in this case was
indefinite.
Intranodal palisaded myofibroblastoma:
a case report
M. Del Vecchio, R. Zamparese, F. Corini, A. D’Angelo, L. Diamanti, A. Braccischi, R. Taborro, V. Mambelli
Department of Pathology, Division of Anatomic Pathology, General Hospital C.G. Mazzoni, Ascoli Piceno, Italia
Introduction. Intranodal palisaded myofibroblastoma (IPM),
also known as intranodal hemorragic spinde cell tumor with amianthoid fibers, is a rare bening tumor originating from differentiated smooth muscle cells and myofibroblasts of the lymph node.
To date, approximately 56 cases have been reported in the literature.
Case report. A 57-year-old woman presented with a mass in
the right inguinal region; grossly, the round lesion, 4x4x3 cm in
size, had a tan, solid cut surface, with patchy red-brown areas.
Microscopic examination revealed a lymph node almost replaced
by a spindle cell proliferation with nuclear palisading alongside
homogeneus eosinophilic accumulation. The lesion demonstrated
diffuse hemorragic findings; no atypia, mitosis or necrosis was
identified in the cells forming the lesion, which was surrounded
by compressed lymphoid tissue. Immunohistochemical analysis
showed that the neoplastic cells were positive for vimentin and
smooth muscle actin, whereas they were negative for S-100 protein, CD34, desmin and cytokeratin. In this clinical setting, the
morphology of the tumor along with its immunohistochemical
findings were characteristic for an intranodal palisaded myofibroblastoma.
Discussion. Intranodal palisaded myofibroblastoma is a rare
neoplasm arising from the stromal component of the limph
node. Almost all cases develop in lymph nodes of the inguinal
region; submandibolar and cervical lymph nodes have also
been reported as rare originating sites. It was first well characterized in 1989, when three group of investigators describe this
tumor giving it three different names: “palisaded myofibroblastoma” (Weis et al.), “intranodal hemorragic spindle-cell tumor
with amianthoid fibers” (Suster et al.) and “solitary spindle-cell
tumor with myoid differentiation of the limph node” (Lee et
al.). Its predominant morphologic features include the bland
appereance of its constituent spindle cell population, often with
nuclear palisading, the presence of acellular eosinophilic stellate areas (so called “amianthoid fibers”) and intraparenchymal
hemorrhage with erythrocyte extravasation. In most cases the
tumor is observed with lymphoid tissue compressed to the
periphery of the lesion. The immunohistochemical profile of
the neoplastic cells is indicative of myofibroblastic or smooth
muscle differentiation; the spindle cells are in fact positive
for vimentin and smooth muscle actin, whereas no reaction
is observed with many other antibodies, such as neural or endothelial markers. The tumor cells exhibit a low proliferative
index. Pathologic differential diagnosis should comprise the
large spectrum in wich spindle cells are involved. Malignant
spindle-cell tumors and non-IPM benign mesenchimal lesions
of the limph node must be taken into account. Schwannoma
and sarcoma di Kaposi are the most important tumors in the
differential diagnosis, but hemangioendothelioma, dendritic
reticulum cell tumor and inflammatory myofibroblastic tumor
should also be considered. It is also important to distuguish
IBM from metastatic malignant lesions of the limph node with
spindle cell features. Although a wide range of tumors can
be included in the differential diagnosis, intranodal palisaded
myofibroblastoma has a quite distinctive morphological feature
and an immunohistochemical profile. Excellent prognosis has
been reported after surgical treatment with a 6% recurrence rate
and no malignant transformation in all the cases described.
185
Poster
EGFR, MMP9 and steroid receptors reltionship
in human sarcomas
Synovial sarcoma: two cases reports
, M. Mattoni*, S. Cagiano*, M. Di
M. Postiglione*, L. Nugnes*, M.P. Maione*, A. Maglione*, A.
Russo*, A. Nicastro*, D. Oppressore*, R. Franco**, L. Marra**, A.
Monticelli***, R. Giannatiempo*
*
Department of Surgical Sciences, Surgical Pathology Section of the University of Foggia, Foggia, Italy; **Department of Anatomic Pathology,
University of Bari, Bari, Italy; ***Dipartimento di Patologia Generale,
Seconda Università degli Studi di Napoli
UOS Anatomia ed Istologia Patologica, Ospedale Evangelico Fondazione Betania, Napoli, Italia; **AF Anatomia Patologica, INT Fondazione G.
Pascale, Napoli, Italia; *** UOS Citopatologia, ASL NA2 nord, Napoli,
Italia
Recently, Authors have proved that mesenchimal cells express
steroid receptor hormones at low levels. In the human fibrosarcoma, an androgen-dipendent increased expression of MMPs,
correlating with the neoplastic progression and with EGFR
expression, has been shown. EGF stimulates Estrogen Receptor
(ER) phosphorylation on tyrosine, in MCF-7, thereby promoting
the association of a complex among EGFR Androgen receptor
(AR)/ER, Src that activates EGF-dependent signaling pathway.
Moreover, the metastatic progression of malignant tumors, such
as fibrosarcoma, requires the association between ER /AR and
EGFR. In the present work we analyzed, by immunohistochemistry, various sarcomas with different grade of malignancy in order
to correlate MMP9 expression with prognosis as well as with
overexpression of EGFR and AR. We observed a positivity for
MMP9 in dermatofibrosarcomas and undifferentiated pleomorphic sarcomas and we show EGFR overexpression in these cancers. Our study population was characterized by different types
of soft tissue tumors, placed in different anatomical sites (skin,
subcutaneous, parotid gland, uterus, vagina and kidney) and
with different locoregional (Dermato fibrosarcoma protuberansDFPS) and distant invasion potential (Adult Fibrosarcoma-AFS,
Infantile Fibrosarcoma-IFS, Anaplastic Sarcoma-AS, Myxoid
Liposarcoma-MLS, Leyomiosarcoma-LMS, Rhabdomyosarcoma-RMS and Undifferentiated Pleomorphic Sarcoma/Malignant
Fibro-Histiocytoma-UPS/MFH). Therefore, we analyzed 6 UPSs
(undifferentiated pleomorphic sarcomas), 3 DFSPs (dermatofibrosarcoma protuberans), 3 AFSs (adult fibrosarcomas), one
developed on a DFSPs and 1 IFS (infantile fibrosarcoma), and
single cases of LMS, RMS, MLS, and AS. We showed by immunohistochemistry that AR expression was up-regulated with
a heterogeneous percentage of nuclear positivity, according to
different hystotypes. This analysis revealed high degree of expression in a case of pleomorphic MFH, in LMS, in RMS and in
all AFSs, whereas it was moderately expressed in the remaning
MFHs, in MLS, in AS, in IFS, and in DFSPs. Particularly, a case,
diagnosed at first as a well differentiated neoplasia (DFSP) and
then (after 4 years) clinically relapsed as an AFS, showed 55% of
AR immunostaining. We observed diffusely strong membranous
and/or cytoplasmic positivity for EGFR in all AFSs, in IFS and in
1 DFSP. In two MFHs we noted moderate or low levels of protein
immunostaining. In particular, in DFSP characterized by progression to anaplastic sarcoma EGFR was negative, in the DFSP
precursor subsequently arised anaplastic sarcoma, with high proliferative index, high degree of EGFR expression was observed
with a membranous pattern. Evaluated soft tissue neoplasias
showed only scattered and focal positivity for MMP9 in the majority of cases. Particularly, differences in immunohistochemical
expression were noted by comparing the case of DFSP clinically
relapsed (5% of immunostained tumoral cells) and the anaplastic
sarcoma (90% of positive neoplastic cells) subsequently arisen
from it. High levels of MMP9 expression have been observed
also in IFS and in 1 MFH. All these relationships with all their
important implications for therapy have been the subject of our
study. From all these concepts, EGFR merges as key-molecule in
mesenchimal tumorigenesis. These results, along with the determination of EGFR/AR/MMP expression in mesenchimal tumors,
may open the door to clinical trials of currently available specific
EGFR inhibitors.
Background. The diagnosis of Synovial sarcoma can be challenging even in histopathology and requires ancillary techniques
for confirmation of the morphological diagnosis. We presented 2
cases occurring during 2010
Case presentation 1. A 49-year-old woman was admitted with a
complaint of swelling and pain in her left ankle.
She was asymptomatic until one year ago when she sustained
injury to the left ankle due to a trivial fall.
Physical examination had revealed only a swelling in the left ankle and otherwise, no mass had been palpated at that time.
Six months later the swelling gradually increased until 3 mounths
later at a follow-up examination, a soft tissue mass had become
palpable in her ankle.
It measured about 3 cm in diameter and was located on the anterolateral aspect of the ankle.
Plain radiographs of the ankle were normal. Magnetic resonance
imaging revealed a soft tissue mass attached to the ankle joint.
She subsequently underwent a total excision of the mass. At
surgery, the mass was found to originate from the ankle joint
capsule.
A part of the joint capsule was excised along with the mass.
Grossly, a cross section of the mass, measuring 2.5 cm in diameter, was tan to yellow in color, soft and fragile.
H&E stained sections showed a cellular tumor comprising oval to
spindly cells, displaying ‘stag-horn’ arrangement of vasculature,
reminiscent of a hemangiopericytoma-like pattern.
Focal areas showed myxoid change, necrosis and apoptosis.
Occasional mitotic figures were observed: immunohistochemically, the tumor cells were positive for SMA, EMA, and CD34
but negative for desmin and CD99;AE1/AE3, CK 19, CK7,
BCL2 and S100 showed weak positivity.
The molecular analysis of FFPE samples by FISH revealed a
SYT-SSX fusion transcript.
Based on these findings, a primary malignant synovial sarcoma
was diagnosed.
The patient was postoperatively given 50 Gy of local irradiation.
Since she rejected adjuvant chemotherapy, the patient did not
receive further treatment. There was no evidence of local recurrence or metastasis 8 months postoperatively.
Case presentation 2. A 23year-old woman presented to orthopedic outpatient department with pain, limp and swelling of the
left knee. She had felt a progressive swelling and pain for more
than two months without any obvious cause. The pain was dull
aching in nature, poorly localized in the knee.
There was loss of terminal extension of the knee with 15-20 degrees of fixed flexion deformity. Radiographs of the knee were
normal.
The diagnostic arthroscopy was done under general anesthesia.
Intercondylar area revealed a polypoidal lesion in the synovial tissue which was excised and sent for histopathology examination.
The gross specimen consisted of multiple gray white and gray
brown bits measuring about 2cms.
Sections revealed tissue lined by synovial cells with a cellular
lesion in the subsynovial area. Cells are spindle shaped arranged
in interlacing bundles and fascicles. Nuclei are elongated with
fine chromatin clumping, mitoses were sparse. Interspersed in
these spindle cells are cleft like and gland like structures lined
by cuboidal to low columnar epithelium. Nuclei revealed fine
chromatin clumping
G. Pannone*, A. Santoro
Domenico***, P. Bufo*
* **
*
186
IHC revealed strong expression of EMA, weak positivity for
AE1/AE3, CK20, CK 7. However tumor cells were negative for
S100, Desmin, SMA, CK8 and CK18.
Also this case was proven as SS by the demonstration of the SYTSSX fusion transcript by FISH and PCR assays.
Our patient was treated with hinged knee replacement which
includes excision of the lower end of femur up to supracondylar
fossa - excision of intra articular synovium with a safe margin and
one cm of the proximal tibial end. The patient has been on regular
follow-up since the last 6 months with no radiological evidence
of metastasis.
Conclusions. Our experience distinctly highlights the great value
of a molecular analysis of an SYT associated genetic alteration in
the diagnosis of SS especially when IHC results are equivocal.
A comparative immunohistochemical study
of oncofetal cytoplasmic WT1 expression in
human fetal, adult and neoplastic skeletal muscle
cells was seen. The extent of cytoplasmic staining was extremely
variable in the different muscle cells, ranging from focal to diffuse. Notably, WT1 expression was completely lacking in the
two cases of soft tissue rhabdomyomas, while it was strongly and
diffusely expressed in the cytoplasm of the neoplastic cells of all
cases of rhabdomayosarcomas, including embryonal, alveolar
and pleomorphic types. No nuclear WT1 staining was obtained
in any of the tissues studied.
Discussion. The present study shows that WT1 is developmentally expressed in the cytoplasm of human skeletal muscle tissue
from the 7th week of gestational age. The comparative evaluation
of the immunohistochemical findings in the different tissues
reveals that the cytoplasmic expression of WT1 in rhabdomyosarcomas may represent an ontogenetic reversal, and this nuclear
transcription factor can also be considered an oncofetal protein.
A rare case of paravertebral myelolipoma
L. Salvatorelli*, R. Caltabiano *, M. Migliore**, S. Lanzafame*
L. Salvatorelli1, M. Bisceglia2, G. Vecchio1, R. Parenti3, C.
Galliani4, R. Alaggio5, A. Gurrera1, E. Giurato1, A. Torrisi6, G.
Magro1
*
1
Dipartimento G.F. Ingrassia, Università di Catania, Catania, Italia; 2
Dipartimento di Patologia Clinica, Ospedale Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italia; 3Dipartimento Scienze Bio-Mediche,
Università di Catania, Catania, Italia; 4 Department of Pathology, Cook
Children’s Medical Center, Fort Worth, TX, USA; 5 Dipartimento di Patologia, Università di Padova, Padova, Italia; 6 Registro Tumori Integrato
CT-ME-SR, Catania, Italia
Introduction. Myelolipoma is a tumor that tipically arises in the
adrenal glands with double components, fat and erythropoietic
tissues. It can rarely occur in the extra-adrenal site (chest, retroperitoneum, presacral region, mediastinum, liver, spleen, testis,
lung). Only four cases of paravertebral myelolipoma are reported
in the literature. The myelolipoma is more common after the
fortieth year of age. Tumor size usually varies between 3 and 7
cm. Small tumors tend to be asymptomatic and often are detected
as incidental findings during radiologic studies or surgery for
some unrelated disease or at autopsy. Radiologically, it is a wellcircumscribed radiolucent mass. At gross examination, myelolipoma resembles a lipoma, but may appear grayish when myeloid
component prevails. Microscopically it is composed of bone
marrow elements mixed with fat tissue. The histogenesis of these
lesions may be the metaplasia of undifferenziated adrenal stromal
cell or from choristomatous hematopoietic stem cell rests.
Method. We report a case of a woman of 57 years old with a mass
localized in the Th8-Th9. Magnetic Resonance imaging showed
a paravertebral, well circumscribed mass of 2 cm in maximum
diameter. Grossly, tumour appared yellowish-white with a soft
consistency. Histologically, the lesion consisted of erythropoietic
bone marrow mixed with adipose tissue, fibrovascular tissue and
dense connective tissue type ligamentous. Considering clinical,
radiological and morphological features the diagnosis of extraadrenal myelolipoma was performed.
Conclusion. This is a case of myelolipoma in extra-adrenal rare
site. The site of the lesion suggests the importance of surgical
removal to prevent the onset of compressive symptoms.
Wilms tumor transcription factor-1 (WT1) is encoded by Wilms
tumor suppressor gene located on chromosome 11p13. Although
WT1 was originally identified as a tumour suppressor, there is
increasing evidence indicating its role as a potential oncogene
involved in proliferation and apoptosis, depending on the cellular
context. While it is widely known that many malignant tumors
(nephroblastoma, ovarian and mesothelial neoplasms, Sertoli
cell tumor and desmoplastic small round cell tumor) exhibit nuclear expression of WT1, many pathologists are not aware of the
possibility that this tumor transcription factor may be detected
concurrently in the cytoplasm of the above mentioned tumors,
or exclusively in the cytoplasm of other tumors, such as vascular
neoplasms. Although the cytoplasmic immunohistochemical expression of WT1 was originally interpreted as a cross-reactivity
with an epitope unrelated to WT1, accumulating data from in
vitro studies and Western blot analyses confirm the specificity of
the cytoplasmic staining. In this regard, there is some evidence
that WT1 is also expressed in the cytoplasm of human rhabdomyosarcomas suggesting its potential diagnostic use in the context of
pediatric small round blue cell tumors. The significance of WT1
expression in rhabdomyosarcoma cells is still to be established.
The aim of the present study was to immunohistochemically
investigate the expression and distribution of WT1 in developing, adult and neoplastic human skeletal muscle tissues, using
antibody clone 6F-H2 directed to the. WT1 N-terminus of the
protein.
Materials and methods. Tissues samples were collected from: i)
15 human fetuses obtained from legal interruptions, ranging from
the 7th to the 24th week of gestational age; ii) 10 samples of adult
normal skeletal muscle tissue included in specimens from surgical resection of soft tissue tumors; iii) 2 cases of adult soft tissue
rhabdomyomas; iv) 20 cases of pediatric soft tissue rhabdomyosarcomas, including both embryonal and alveolar variants; v) 4
cases of adult soft tissue pleomorphic rhabdomyosarcomas.
Results. During all the different phases of development studied,
skeletal muscle cells of the trunk, head and neck, and extremities
showed a strong and diffuse cytoplasmic expression of WT1. In
adults a mosaic pattern of expression, consisting of WT1-negative
skeletal muscle cells alternating with a minor number of positive
Dipartimento G.F. Ingrassia, Anatomia Patologica, A.O.U. PoliclinicoVittorio Emanuele, Catania; ** Dipartimento di Chirurgia, Chirurgia Toracica, A.O.U. Policlinico-Vittorio Emanuele, Catania
Post-traumatic intraosseous hemangioma
of the parietal bone
L. Ventura1, S. Marzi 2, F. Marampon3, A. Catalucci4, M. Capulli5,
A. Ricci2
U. O. C. di Anatomia Patologica, Ospedale San Salvatore, L’Aquila, Italia; 2U. O. C. di Neurochirurgia, Ospedale San Salvatore, L’Aquila, Italia;
3
Divisione di Radioterapia e Radiobiologia, Dipartimento di Medicina
Sperimentale, Università, L’Aquila, Italia; 4U. O. C. di Neuroradiologia,
Ospedale San Salvatore, L’Aquila, Italia; 5Dipartimento di Medicina Sperimentale, Università L’Aquila, Italia
1
Bone cavernous hemangiomas are rare lesions, accounting for
0.7% of all osseous neoplasms. Calvarial hemangiomas are even
less frequent, representing about 0.2% of all benign skull tumors.
Frontal localization is the most frequent one, followed by temporal and parietal regions. Most skull hemangiomas are congenital
tumors, with rare post-traumatic lesions. We report a case of in-
187
Poster
traosseous hemangioma of the parietal bone, occurred in a young
woman 3 years after an accidental head injury.
The patient was a 31-year-old female, who had noted the appearance of a painful, slowly growing swelling in the right parietal region of the head. She had experienced an accidental indoor head
injury 3 years earlier, in the same site of the tumor. No other complaints or neurological deficits were noted. Radiography showed
a radiolucent, osteolytic lesion with a sunburst pattern of bony
spicules radiating mainly to the outer table, without sclerotic margins. Computed tomography (CT) confirmed a diploic mass, 3 cm
in largest diameter, with outer and inner tables thin and partially
defective. At magnetic resonance imaging (MRI) the mass was
hyperintense at T1-weighted images and revealed heterogeneous
hyperintensity of the lesion, with hypointense borders.
The patient underwent craniectomy with en bloc removal of the
mass and the subsequent reconstruction of the resulting calvarial
defect was performed with an acrylic resin prosthesis. She recovered from surgery without complications.
Gross examination of the formalin-fixed specimen revealed a
fragment of flat bone measuring 3,7 x 3,3 x 1 cm, occupied by a
thicker area (1,6 cm) with ill-defined margins and porotic external
surface. The cut surfaces showed the red-brownish mass mainly
located in the diploe, with radiated spicules and disruption of the
outer and inner table. Cross sections obtained from the specimen
were decalcified with nitric acid 7% for 3 days, routinely processed and embedded in paraffin to obtain histological sections
stained with hematoxylin-eosin.
Microscopy highlighted large, blood-filled vessels, arranged in
an haphazardly diffuse manner and lined by a single layer of
endothelial cells, with rarefaction of diploic bony spicules. Focal
proliferation of capillary network was also observed within the
lesion, with inner and outer tables perforated by vascular spaces.
Calvarial hemangiomas are benign lesions with a distinct female
predilection, that affecting any age group and commonly occuring in middle-aged women.
They are asymptomatic and usually represent incidental findings
during imaging evaluation performed for other reasons, but may
also grow and manifest as palpable masses. Trauma may be an
antecedent factor, although not always elicited in the patient’s
history. Post-traumatic lesions may differ from the majority of
skull intraosseous hemangiomas as they are characterized by the
presence of a painful lesion, without sclerotic borders.
Calvarial intraosseous hemangiomas can be missed or misinterpreted as more ominous lesions like multiple myeloma, osteosarcoma, metastatic carcinoma, eosinophilic granuloma and meningioma, which represent the main differential diagnoses. Radiographic appearance of skull hemangiomas can be diagnostic, but
histology is almost always needed to yield the final diagnosis.
Patologia tiroidea
Neutrophil gelatinase-associated lipocalin (NGAL):
a new diagnostic marker in follicular cell-derived
thyroid tumors?
V. Barresi, A. Ieni, G. Tuccari, G. Barresi
Dipartimento di Patologia Umana, Università di Messina
The discrimination between benign and malignant thyroid nodules represents one of the major problems encountered in diagnostic surgical pathology. In the World Health Organization
(WHO) Classification, the follicular carcinoma of thyroid gland
is defined as a malignant epithelial tumour showing follicular
cell differentiation and lacking the diagnostic nuclear features of
papillary thyroid carcinoma. The diagnosis is based on the presence of invasion through the capsule and ⁄ or invasion into the
blood vessels. Thus, the histological criteria, though posing little
problem in cases of widely invasive carcinoma, may lead to interobserver variation among pathologists, in discriminating minimally invasive carcinoma from adenoma. Also, the differential
diagnosis of the follicular variant of papillary thyroid carcinoma
from follicular adenoma or carcinoma may be challenging for the
pathologist when the nuclear features of papillary carcinoma are
not well developed or are only focally expressed.
In the light of this, there is the need of more objective markers
able to discriminate benign from malignant thyroid nodules.
Neutrophil gelatinase-associated lipocalin (NGAL) is a 25 KDa
protein which seems to favor cancer progression through the
(positive) modulation of matrix metallo-proteinase-9 (MMP-9),
which degrades the basement membranes and extracellular matrix enabling the invasion of neoplastic cells. In the present study
we analyzed whether a different NGAL expression is present in
follicular cell-derived thyroid tumors on the basis of their infiltrative potential. Thus our goal was to determine whether the
evaluation of NGAL immuno-expression may be of use in the
differential diagnosis between benign and malignant follicular
cell-derived thyroid neoplasias. 40 surgical specimens of primary thyroid tumors were retrieved from our files and tested for
NGAL immunohistochemical expression. The cohort included
eight follicular adenomas (FA), two Hurthle cell adenomas (HA),
two atypical adenomas (AA), eight minimally invasive follicular
carcinomas (MIFC), nine widely invasive follicular carcinomas
(WIFC), three Hurthle cell follicular carcinomas (HC) and eight
papillary carcinomas (PC) with five follicular-variant PC (FVPC)
and three PC not otherwise specified (PC-NOS). For each case,
an intensity distribution (ID) score was calculated by taking into
account the intensity of staining and the area of staining positivity for NGAL. Cases displaying an ID score 0 were considered
as negative for NGAL. No NGAL staining was observed in the
follicular cells of the thyroid parenchyma adjacent to the tumors.
92% of benign tumors (specificity) were negative for NGAL,
whereby NGAL immuno-expression was found in 82% (sensitivity) of malignant tumors, and, specifically, in 100% of MIFC, in
87% of WIFC, in 100% of HC, in 80% of FVPC. None of the
PC-NOS displayed NGAL staining. When only the tumors with
a follicular architecture were considered, NGAL specificity for
malignant lesions was 92%; sensitivity, positive predictive value
and negative predictive value were 92%, 96% and 85%. Diagnostic accuracy of NGAL expression in the differential diagnosis
between benign and malignant follicular tumors was 92%.
In conclusion, according to our preliminary findings, NGAL
protein, which is involved in the acquirement of cancer cell
invasive potential, seems to represent a marker of malignant follicular cell-derived thyroid tumors, and especially of those with
follicular architecture. Hence we suggest that the assessment of
its expression may be of use with respect of differential diagnosis
of the latter from benign neoplasias.
Trop-2 expression in thyroid lesions
M. Centrone*, T. Addati*, C. Quero*, G. Giannone*, F. Palma*, G.
Achille**, S. Russo**, L. Grammatica**, G. Simone*
Anatomic Pathology Unit, **Otorhinolaryngology Unit, National Cancer
Institute “Giovanni Paolo II”, Bari, Italy
*
Background. Difference between benign and malignant thyroid
tumors is critical for the management of patients with thyroid
nodules.
HBME-1 and TROP-2 expression could be significantly associated with differentiated thyroid carcinomas. HBME-1 is a
biologic marker of the microvillous surface of mesothelioma cell
and recent study (1) had shown that it was positive expressed in
thyroid malignancies. TROP-2 is a cell surface protein found to
be highly expressed in various epithelial cancers and its expression correlates with aggressive tumor behaviour. Notably, in
contrast to tumor cells, somatic adult tissues show little or no
188
Trop-2 expression. Being a stimulator of human cancer growth
and a marker of invasion, TROP-2 could be a target of diagnostic
and therapeutic procedures. The aim of this study was to evaluate
TROP-2 protein expression in benign and malignant thyroid lesions, in order to analyze its role as a marker of neoplastic lesions
and to correlate it with HBME-1 status.
Materials. 39 thyroid nodules, from 2007 to 2009, with corresponding cytological specimens, entered this study. In cytology, 1 out of
39 cases was classified as TIR 2, 25 as TIR 3 and 13 as TIR 4. In
histology, 13 out of 39 cases were diagnosed as carcinoma (of this
2 cases was TIR 3 in cytology and 11 were TIR 4 in cytology) 13
were classified as adenomas (of this 12 cases were TIR 3 and 1 was
TIR 4 in cytology) and 13 were negative to neoplastic lesions (of
this 1 was TIR 2; 11 were TIR 3 e 1 was TIR 4 in cytology).
HBME-1 and TROP-2 immunoreaction (IR) were assessed, using
immunohistochemistry (IHC), on all histological cases, whereas
cytological HBME-1 IR value were avaible. HBME-1 immunostain was carry out with an anti-human Mesothelial Cell (Clone
HBME-1, DAKO). HBME-1 immunoreactivity was evaluated by
a scoring method which took into account intensity of staining.
TROP-2 reaction was performed with an anti–human TROP-2
antibody (R&D System) on all histological cases. The proportion TROP-2 score described the estimated fraction of positive
stained tumor cells (0=none; 1=<10%; 2=10–50%; 3=51–80%;
4=>80%). The intensity score represented the estimated staining
intensity (0, no staining; 1, weak; 2, moderate; 3, strong).
Results. HBME-1 immunoreaction, on cytological samples,
was present in 12 out of 13 carcinomas, in 6/13 ademomas,
whereas none of benign nodules had HBME-1 staining, although
2 case were focal positive and 2 were positive. Also, HBME-1
and TROP-2 IR in histological samples showed this distribution pattern in malignant lesions: 11 out of 13 carcinomas were
HBME-1(+)/TROP-2(+), 1 was HBME-(-)/TROP-2(+) and 1
case showed only a scant TROP-2 IR. In no malignant lesions, 1
out of 13 adenomas was HBME-1(+)/TROP-2(+), 7 were HBME(-)/TROP-2(-), 4 were HBME-1(+)/TROP-2(-), and 1 was
HBME-1(-)/TROP-2(+). Finally, 1 out of 13 iperplastic lesions
was HBME-1/(+)/TROP-2(+), 10 were HBME-1(-)/TROP-2(-), 1
was HBME-1(+)/TROP-2(-) and 1 was HBME-1(-)/TROP-2(+).
Conclusions. This study evidenced that there was a distinct and
clear difference of immunoexpression for all two markers with a
progressive increase in their staining rate from non-neoplastic to
benign to malignant lesions. Our data showed weak or no stain
in benign/negative nodules respect to a high positivity of both
antigens, HBME-1 and TROP-2, in thyroid carcinomas, this evidenced that these protein could be used as biological markers of
malignancies. However, in hystological samples TROP-2 showed
to be more sensitive and specific than HBME-1.
Combination of the two markers could be proposed in early cytological diagnosis to find a better management of patient with
thyroid nodules.
Reference
1
Saleh HA, Feng J, Tabassum F, et al. Differential expression of galectin-3, CK19, HBME1, and Ret oncoprotein in the diagnosis of thyroid
neoplasms by fine needle aspiration biopsy. Cytojournal 2009;6:18.
Rose (rapid on site examination) reduces
the number of non diagnostic cases in thyroid
nodules
R. Murari*, G. Beretta Anguissola**, A. Palermo**, P.L. Alò*, P.
Pozzilli**, M.T. Ramieri*
U.O.C. Anatomia Patologica, Azienda Ospedaliera Umberto I, Frosinone, Italia; ** Dipartimento di Endocrinologia, Università Campus BioMedico, Roma, Italia
*
Introduction. Thyroid nodular pathology is endemic in the Frosinone area mainly depending on genetic and environmental factors.
Fine needle aspiration cytology (FNC) is the gold standard technique
to detect nodules eligible for surgery. However FNC sensitivity can
be very low due to the large number of non-diagnostic samples.
A back to back cooperation between endocrinologists and pathologists as a rapid on site examination (ROSE), where the pathologist,
after the aspiration performed by the endocrinologist, screens the
slides immediately to check their adequacy, could be useful to avoid
cell re-picking from diagnostic nodules or to re-invite patients for an
inadequate sampling therefore enhancing compliance.
Material and methods. From June 2008 to September 2010, 583
ROSE was performed on 457 patients with a progressive increasing
performance from the first semester (62 cases) to the fifth (131 cases). All Fine Needle Cytology (FNC) was performed with 25-23G
needles in cooperation between endocrinologist and pathologist.
On all patients we acquired detailed medical history and ultrasound nodules characteristics: we consider these data really useful for a correct definition of a diagnosis.
Results and discussion. Our results reveled 23% of THY1, 66%
of THY2, 7% of THY 3 and 4% of THY4 and THY5.
Data from literature suggest that, for a good practice, the number
of non-diagnostic cases (THY1) may not exceed 20%. Our data
reported 23% of THY1s. We believe that this depended on our
learning pathway since we observed a progressive decrease of
inadequate cases from the first (40%) to the last semester (18%)
(p<0,001); moreover 18% of THY1 are on colloid nodules; at
last repetition of FNC on 44% of the THY1 group enabled a
conclusive diagnosis in 80% of the cases. Finally a not conclusive
diagnosis could be assessed in 8% of our patients, percentage
comparable with thin-layer technique.
We collect the histological diagnosis of the patients gone to the
surgical intervention: all the THY4 and THY5 diagnosis was
confirmed as malignant tumors; about THY3 nodules we collect histological data in 78% of the patients, cause 22% of them
didn’t turn back to the ambulatory after surgical intervention; the
histological diagnosis of carcinomatous nodule was confirmed in
13% of the patient we are following-up.
Conclusion. In our experience ROSE reduces non diagnostic cases,
favors a better characterization of suspicious or malignant nodules,
enhances experience when handling problematic cases and is time
and resource sparing; moreover, pathologist with a complete clinical knowledge of the case should define smears’ adequacy with a
lower number of cells. At last repeated FNC may be immediately
decided on the patient’s bed-side. Finally ROSE, stimulating a
multi-disciplinary approach, may be the first step for a thyroid
unit where endocrinologists, pathologists and, when necessary
surgeons, can assure, for the patient, a conclusive and accurate
diagnosis and suggest the best subsequent clinical approach.
p-NFkB expression in thyroid cancer
G. Pannone, M. Mattoni, A. Santoro, F. Sanguedolce, P. Bufo
Department of Surgical Sciences, Section of Anatomic Pathology, University of Foggia, Foggia, Italy.
Background. NFκB is a protein complex that controls the
transcription of DNA. NFkB has been recently shown to play
an important role in thyroid cancer for its ability to control the
proliferative and the anti-apoptotic signaling pathways of thyroid
tumor cells.
Methods. The aim of this study was to investigate the expression
of NFkB and its phophorylated form in a series of human thyroid
cancer and to evaluate its clinical and prognostic significance.
Protein expression was analyzed by immunohistochemistry in
70 patients with thyroid cancer and normal tissue. The series has
been assessed in various clinicopathological groups and the relationships were established by statistical analysis.
Results. Alterations of p-NFkB levels were evident in neoplastic
cells but not evident in normal peritumoral tissues. In our study
protein staining was mainly detected in the cytoplasm of the
189
Poster
neoplastic cells, and only focal nuclear signals were observed.
By comparing tumors in different stages, cytoplasmic p-NFkB
expression was prevalently revealed in T3/stage group of thyroid
cancers. Our work also highlights that the immunohistochemical detection of sporadic nuclear staining for p-NFkB was more
observable in early stage (St I/ T1), without nodal metastases
(N0). The same result has been observed for cytoplasmic p-NFkB
expression in normal peritumoral tissues. Our preliminary results
indicate that assessment of p-NFkB may be useful as prognostic
factor in patients with thyroid cancer.
Microrna profiles in familial and sporadic
medullary thyroid carcinoma: preliminary
relationships with ret status and outcome
G. Pennelli1, C. Mian2, M. Fassan1 3, M. Balistreri1, S. Barollo4, E.
Cavedon2, F. Galuppini1, M. Pizzi1, F. Vianello4, M.R. Pelizzo5,
M.E. Girelli2, G. Opocher4, M. Rugge1 4
The first two Authors contributed equally to this work.
Department of Medical Diagnostic Sciences and Special Therapies, Surgical Pathology & Cytopathology Unit, University of Padova, via Gabelli
61, 35128 Padova, Italy; 2Department of Medical and Surgical Sciences,
Endocrinology Unit, University of Padova, Via Ospedale 105, 35128 Padova, Italy; 3Department of Oncological and Surgical Sciences, General
Oncology Unit, University of Padova, via Giustiniani 2, 35128 Padova,
Italy, 4Veneto Institute of Oncology – IRCCS, via Gattamelata 64, 35128
Padova, Italy; 5Department of Medical and Surgical Sciences, Special Surgery Unit, University of Padova, via Giustiniani 2, 35128 Padova, Italy.
1
Background. microRNAs (miRNAs) are involved in the pathogenesis of human cancers, including thyroid carcinomas. Here,
we investigated miRNA dysregulation in medullary thyroid carcinoma (MTC), correlating miRNA expression with RET status
and patients’ prognosis.
Methods. We analyzed the expression of six miRNAs (miR-21,
miR-127, miR-154, miR-224, miR-323 and miR-370) by quantitative RT-PCR in 30 MTCs and 3 cases of C cell hyperplasia
(CCH). Sporadic MTCs were genotyped for somatic RET mutations. Disease status was defined on the basis of the concentration
of serum calcitonin at the latest follow-up.
Results. MTC and CCH are both characterized by a significant
overexpression of the whole set of miRNAs (the increase being
4.2-fold for miR-21, 6.7-fold for miR-127, 8.8-fold for miR-154,
6.7-fold for miR-224, 6-fold for miR-323 and 6.3-fold for miR370, p<0.001). In sporadic MTCs carrying somatic RET mutations,
the upregulation of miR-127 and miR-323 is significant less pronounced than in cases without mutations (p=0.02 and p=0.03, respectively). The upregulation of miR-323 and miR-224 correlated
with biochemical cure (p=0.01 and p=0.008, respectively).
Conclusions. miRNAs are significantly dysregulated in MTCs,
and this dysregulation is probably an early event in C cell carcinogenesis. Our preliminary findings suggest that miR-323 and
miR-224 upregulation could represent a favorable prognostic
indicator.
Ectopic thyroid tissue misdiagnosed as metastatic
carcinoma: case report with immunohistochemical
study
E.D. Rossi MD PhD, A. Santoro MD, V.G. Vellone MD, G.F.
Zannoni MD, M. Raffaelli MD, G. Chiarello MD, G. Fadda MD,
MIAC
Division of Anatomic Pathology and Histology Chair prof Rindi G. and
*
Division of Endocrine Surgery, Chiar prof Bellantone R. Università Cattolica del Sacro Cuore, Rome, Italy
Ectopic thyroid tissue represents a frequent finding associated,
from a pathogenetic point of view, with an abnormal embryologic
development and migration of the thyroid gland.
This ectopia is the result of an abnormal migration along the path
of descent of the thyroid gland with a possible final displacement
of the gland tissue in different sites of the anterior neck midline.
With lower frequency the ectopic thyroid tissue has also been
described in the lateral portions of the head and neck district.
The histologic identification of these microscopic residues may
cause a differential diagnostic dilemma, especially in patients
undergoing surgery for thyroid cancer.
The present report is the evaluation of the correct diagnostic iter
in presence of incidental microscopic findings of contralateral
thyroid microfollicular residuals in a 36-year-old female patient
who underwent a left thyroid lobectomy for a 1.7 cm nodule.
A previous right lobectomy for a benign goiter was performed
in another institution. The histological diagnosis of the thyroid
neoplasm was papillary thyroid carcinoma tall cell variant with
evidence of residual microfollicular thyroid tissue in the anterior
right neck tissue. The immunohistochemical pattern made up of
HBME-1 and Galectin-3 carried out on both the thyroid lesion
and the ectopic microfollicular residuals has lead to an opposite
concordant expression of the antibodies. In fact, the tumor yielded positive for both HBME-1 and Galectin-3 whereas the ectopic
foci resulted completely negative, ruling out a metastatic tumor
with all its clinical and prognostic implications.
In conclusion, in presence of ectopic thyroid tissue, a possible
metastatic spread must be accurately set out not only on conventional morphology but, if convenient, with the application of
immunohistochemical techniques. A future help could be represented by the molecular techniques by identifying the different
genetic patterns of the lesions.
References
Mace AD et al. ISRN Surgery, 2011.
Kumar R et al. Thyroid 2000;10:363-5
Thyroid fine-needle aspirates (FNA) diagnosed as
THYR-3 can be typed into a lower and a higher risk
subgroups
A. Somma, E. Guadagno, G. Troncone
Dipartimento di Scienze Biomorfologiche e Funzionali, Università di Napoli Federico II, Naples, Italy.
Background. Several thyroid FNA classifications have been
proposed; both classifications issued by the SIAPEC-IAP Italian
Consensus Working Group and by the British Tryroid Association (BTA) feature a THYR-3 group. This latter corresponds to
the NCI Bethesda follicular neoplasms / suspicious for follicular
neoplasm class. Very recently, the British Royal College of Pathologists subclassified BTA THYR-3 into two further groups.
However, there is little concordance whether morphological criteria underlining this THYR-3 subtyping are consistent. The aim
of this ongoing study is to assess whether retrospective THYR-3
subtyping may better refine the outcome of malignancy associated to histology following uncertain FNAs.
Methods. Special care was taken to select from a larger pool of
cases a subset of FNAs (n=44) in which the THYR-3 diagnosis
was unquestionably agreed by the three authors and whose histological follow-up was available. According to the British Royal
College of Pathologists guidelines THYR-3 were reclassified into
THYR-3 (f) and THYR-3 (a). The former (n= 25) featured a predominant microfollicular pattern; the latter (n= 19) showed either
focal nuclear or architectural atypias, whose degree was milder
than that associated with a THYR-4 diagnosis.
Results. Our data show that THYR-3 subtyping may be effective. In fact taken as a whole, THYR-3 malignancy rate (MR)
was 20%; however, the THYR-3 (a) subgroup was associated
with a higher MR (26%) than THYR-3 (f) (12%). In fact, only
3/25 THYR-3 (f) were malignant (2 papillary carcinoma [PTC];
1 follicular carcinoma), whereas 5/19 THYR-3 (a) were PTC,
190
including 2 follicular variants. Thus, the MR associated to PTC
was significantly (P < 0.0001) higher (26%) in THYR-3 (a) than
in THYR-3 (f) (8%).
Conclusion. This ongoing study shows that morphological
criteria may be identified to consistently subtype THYR-3 into
a lower (f) and a higher risk (a) malignancy associated groups.
These findings warrant evaluation in larger prospective series.
Reference
1
Guidance on the reporting of thyroid cytology specimens. Royal College of Pathologists http://www.rcpath.org/resources/pdf/g089guidanc
eonthereportingofthyroidcytologyfinal.pdf (accessed 25.07.11)
Usefulness of immunohistochemistry
in the differential diagnosis between papillary
carcinoma in the ectopic laterocervical location
and node metastases
M. Trombatore*, D. Giallombardo*, M. Castiglia**, C. Guarnotta*,
D. Cabibi*
Studi di Palermo, Palermo, Italia; ** Dipartimento di Oncologia, Università degli Studi di Palermo, Palermo, Italia.
*
The distinction between a true laterocervical metastasis of an
undetected thyroid carcinoma and a primary tumor outside the
gland is challenging. Aim of this study was to verify whether
immunohistochemistry might be useful. Galectin-3, cytokeratin
19, and HBME-1 were assessed in six cases (group A) of laterocervical masses harboring papillary thyroid carcinoma (PTC)
without a thyroid tumor, and in eight cases (group B) showing
PTC both in the thyroid and in the laterocervical masses. In both
groups, normal-looking follicles adjacent to the laterocervical
neoplasia were present. We found that the apparently normal
follicles in group A were negative for all the antibodies, while
group B showed strong and diffuse positive immunostaining.
The neoplastic areas were always positive for all the antibodies
in both groups. In conclusion, even if residual follicles of group
B are morphologically very well differentiated that they resemble
normal thyroid parenchyma, the immunohistochemical pattern
suggest that they could be metastatic tissue. On the contrary,
the presence of morphologically and immunohistochemically
normal-looking follicles in group A, with no intrathyroid tumor,
suggests that they consists of ectopic normal tissue, from which
the primary PTC might possibly develop. So, in cases showing
morphologically and immunohistochemically normal looking
follicles in laterocervical masses, these findings might lead to a
reduction of the overdiagnosis of metastatic disease of an undetected carcinoma.
Patologia pancreatica
Pancreatic gastrointestinal stromal tumour (GIST)
simulating a cystoadenocarcinoma. Report of two
cases
M.R. Ambrosio, B.J. Rocca, M. Onorati, F. Scaramuzzino, P.
Arcuri, L. Barbagli, L. Pacenti, M.T. del Vecchio, S. Tripodi
Department of Human Pathology and Oncology, Anatomic Pathology Section, University of Siena, Italy
Background. Gastrointestinal stromal tumors (GIST) are the
most common mesenchymal tumors in the stomach and small
intestine, characterized by spindle or epithelioid cells and by
the immunohistochemically expression of c-kit (CD117). Extragastrointestinal stromal tumors (EGIST) are rare and found in the
omentum, mesentery, retroperitoneum and other intra-abdominal
sites. Pancreatic EGISTs are extremely rare and, until now, only
seven cases have been reported in the literature and only one presented as a cystic lesion. We report two cases of an EGIST arising
in the pancreas and radiologically mimicking a cystic neoplasia
of the pancreas.
Patients and methods. Case 1: a 70-year-old man presented with
upper abdominal discomfort, pain and dyspnea. No jaundice was
found. From a computed tomography (CT) scan, a 18-cm cystic
tumor was found between the posterior wall of the stomach and
pancreas, suggesting the diagnosis of pancreatic cystoadenocarcinoma. Case 2: a 53-year-old woman presented with a palpable abdominal mass on physical examination. The CT-scan documented
a 7-cm cystic neoplasm of the pancreas. Both patients underwent
surgery and the specimens were processed for routine histological examination. The following antibodies were checked: CD117,
CD34, smooth muscle actin (SMA), desmin, S100, vimentin and
Ki-67. Molecular analyses for c-kit and PDGFR-a were also performed. Two months after surgery the patient 1 developed hepatic
metastases and was treated with imatinib mesylate (Gleevec). At
the moment, remission has been obtained in both patients.
Results. Case 1: the cystic lesion was firm and whitish with
brown papillary projections into the lumen and well circumscribed. The lumen contained a dirty tan liquid. On histology,
it showed a mixed epithelioid and spindle cell morphology; the
neoplastic cells were embedded in a thin reticular stroma, focally
mixoid. Epithelioid cells formed thick digitations in the lumen
simulating a papillary pattern. Spindle cells were arranged in
a fascicular or storiform pattern. No skenoid fibres were seen.
There were necrotic areas. The tumor cells showed mild nuclear
atypia and mitotic index >5/10HPF. On immunophenotyping,
the tumor was CD117 and CD34 positive and desmin and S100
negative; vimentin and SMA were weakly and focally expressed.
These findings indicated a high risk of aggressive behaviour according to Fletcher. Case 2: the specimen consisted of a necrotic
and hemorrhagic cystic lesion which, on histology, presented a
spindle cell pattern of growth. The neoplastic cells showed moderately pleomorphic nuclei, irregularly distributed chromatin, inconspicuous nucleoli and moderate to abundant eosinophilic cytoplasm. Immunohistochemically the tumor cells were CD117+,
CD34+, SMA+, desmin-, S100-. The mitotic rate was <5/10HPF
and the proliferative index (Ki-67) was <5%. The findings indicated an intermediate risk of aggressive behaviour according to
Fletcher. In both cases, sequence analysis of c-kit and PDGFR-a
genes revealed a point mutation in c-kit exon 11.
Discussion. The two cases here presented have been clinically
misdiagnosed as cystoadenocarcinoma of the pancreas; in fact,
the gross morphology of a GIST is that of a solid or partially cystic lesion. Only the histopathological and immunohistochemical
findings established their true origin. Clinicians must be aware
that the differential diagnosis of pancreatic cystic lesions should
include GISTs with pseudocystic changes, due to their rapid
growth and necrotic areas.
CK19 and KIT immunostaining is an useful adjunct
to Ki67 based grading of pancreatic NET: a study
on matched cytological and histological samples
C. Bellevicine, A. Iaccarino, G. De Rosa, G. Troncone
Dipartimento di Scienze Biomorfologiche e Funzionali, Università Federico II, Naples, Italy.
Background. Pancreatic neuroendocrine tumors (NET) are rare neoplasms. Recent evidences suggest that Ki-67 staining predicts their
behaviour. Even more recently a prognostic role has been proposed
also for CK19 and KIT. This panel might guide patient management.
CK19 and KIT have not been evaluated on NET FNAs yet.
Methods. Nine patients underwent EUS-FNA examination before surgery for primary (n=7) and metastatic (n=2) pancreatic
NET. On site evaluation by an experienced cytopathologist en-
191
Poster
sured proper tissue handling for the prepration of a representative
cell block for immunostainings. These latter were compared to
those performed on the matched resected samples.
Results. Cytological diagnosis of G1 (n=6), G2 (n=2) and G3
NETs (n=1) were histologically confirmed. Ki-67 expression
was concordant (cut-off 2%). Agreement was also found for the
CK19-/KIT- (n= 6), CK19+/KIT- (n=2) and for CK19+/KIT+ (n=
1) assessments.
Conclusions. CK19 and KIT can be reliably assessed together
with Kì-67 to stain cells aspirated from pancreatic NET.
Reference
Zhang L, Smyrk TC, Oliveira AM, et al. KIT is an independent prognostic marker for pancreatic endocrine tumors: a finding derived
from analysis of islet cell differentiation markers. Am J Surg Pathol
2009;33:1562-9.
Inflammatory myofibroblastic tumour
of the pancreas in a 6-month-old infant
R. Liotta, I. Potortì, R. Gentile
Pathology Service, Department of Diagnostic and Therapeutic Services,
ISMETT, Palermo, Italy
Introduction. Inflammatory myofibroblastic tumors (IMT) are
rare benign solid lesions of unclear etiology, very rarely founded
in the pancreas. Clinically and radiologically they can be confused with malignancy.
Clinical case. A 6-month-old caucasian infant presented with
jaundice and hypocholic stools. The upper abdomen ultrasound
showed a biliary dilatation. The CT and the cholangio-MRI
showed a cephalopancreatic mass and dilatated intra- and extrahepatic biliary ducts and Wirsung. A EUS-FNA of the lesion
didn’t lead to a definitive diagnosis due to shortage of material,
so an exploratory laparoscopy with biopsy followed. Based on
histological findings and following surgical-pathological discussion, patient underwent Whipple’s resection.
Materials and methods. Needle biopsy and surgical specimen
samples were H&E stained and immunohistochemical stained
with CD31, CD34, CD45, CD68, CD99, CD117, desmin, SMA,
muscle-specific actin, myogenin, MNF116, pCEA, EMA, synaptophysin, chromogranin, S-100, NSE, neurofilaments, HMB-45,
WT-1, cyclin D1, calretinin, ALK-1, CD14, fascin, F13a, CD163,
Mib-1. Electron microscopy was made on surgical material.
Results. The needle biopsy showed a fibrohistiocytic lesion.
Macroscopy of Whipple’s specimen showed a yellowish-white
nodule of the pancreatic head (max diameter of 2.8 cm), protruding into the duodenum and pressing on the papilla. Histologically
the lesion was composed of spindle cells in vague storiform pattern, as well as elements with foamy or vacuolated cytoplasm
and peripherally displaced nucleus, with some areas showing
multinucleated giant cells and scattered lymphocytes and granulocytes. The proliferation included and occasionally infiltrated
vascular and nervous structures, sleeve-like folding the ducts,
infiltrated the pancreatic parenchyma and the duodenal wall up
to the submucosa, and was much close to the excision margins.
Since from a merely morphological perspective the lesion was
difficult to classify, several differential diagnosis were considered. IHC stains showed high and diffuse expression of vimentin,
CD68 and CD34, weak positive SMA, muscle-specific actin and
CD99, weak and focal positive ALK, focal positive CD45; doubtful and weak myogenin; negative S-100, desmin, cytokeratins and
all the other stains. The growth fraction amounted to 10-12% of
neoplastic cells. Immunohistochemical results allowed ruling out
Langerhans’ cell lesion, granular cell tumor, PEComas, favoring an inflammatory myofibroblastic tumor but, due to positive
CD14, fascin, F13a and CD163, a visceral form of xanthogranuloma was also considered. Electron microscopy showed evidence
of spindle cells containing thin filaments and dense bodies, with
extracellular collagen bands, with no complex interdigitation of
cell membranes and myeloid bodies, thus substantiating the final
diagnosis of inflammatory myofibroblastic tumor.
Conclusions. In our case, only an accurate immunohistochemical
and electron microscopy examination on the surgical specimen
lead to the final diagnosis. Seven months after surgery the patient
is free of disease.
Intrapancreatic accessory spleen
C. Luchini, S. Gobbo, F. Pedica, P. Capelli
Italia
Intrapancreatic accessory spleen (IPAS) is a congenital anomaly
due to the fusion failure of primordial mesenchymal tissues of the
spleen which can mimic a pancreatic neoplasm.
We report three cases of IPAS that were resected because misdiagnosed by radiology as pancreatic neoplasms.
Two lesions were detected by routine ultrasonography (US)
showing respectively a nodular lesion of 3,5 cm and 1,5 cm in
diameter. In the third case there was a 1,9 cm nodule at first
observed by US and than studied by magnetic resonance. All the
three lesions resulted as well-circumscribed nodules localized
within the pancreatic tail characterized by hypervascularization
and radiologically diagnosed as pancreatic endocrine neoplasms.
After resection the gross examination revealed brick-colored
capsulated nodules within the pancreatic parenchyma. Microscopically these lesions resulted to be composed by typical
splenic tissue with reticular sinusoidal structures filled with red
blood cells and lymphoid follicles. The surrounding pancreatic
parenchyma did not show any fibrotic or inflammatory reaction
and the principal pancreatic duct were impinged but not occluded
by the nodules. The final diagnosis was intrapancreatic accessory
spleen.
Curiously two of the three patients underwent splenectomy for
abdominal trauma some years before the finding of the IPAS
raising the idea that a compensatory hyperplasia of the accessory
splenic tissue can develop these tumor-like lesions.
For this reason, in the case of a hypervascularized well-circumscribed nodule within the pancreatic tail in a patient that previously underwent splenectomy is very important to distinguish a
possible IPAS from a pancreatic endocrine neoplasm and avoid
an unnecessary surgery. Fine needle aspiration cytology in IPAS
provides a typical smear, showing polymorphous population of
lymphocytes and other inflammatory cells, with red blood cells
and thin-walled blood vessels with endothelial cells making possible this differential diagnosis.
Solid pancreatic hamartoma: report of two cases
and review of the literature
L. Marcolini*, A. Parisi*, F. Pedica*, G. Zamboni**, M. Chilosi*,
P. Capelli*
Italia; ** Dipartimento di Patologia, Ospedale Sacro Cuore Don Calabria
di Negrar, Verona, Italia
*
We report two cases of solid hamartoma of the pancreas in adult
patients and we reviewed the literature about these extremely rare
tumor-like lesions.
Non neoplastic pancreatic tumour-like lesions give rise to detectable solid or solid and cystic masses and may be either of inflammatory or non inflammatory origin. Since these lesions may
mimic pancreatic cancer, in particular ductal adenocarcinoma,
the preoperative diagnosis often refer to a malignancy. Surgical
resection and histopathological examination are usually necessary to determine the benign nature of the lesion and define the
diagnosis. Only a small number of these non neoplastic lesions
192
would be classified as true pancreatic hamartomas if the entity is
defined strictly.
The prevalence of pancreatic hamartoma is difficult to establish
and a certain amount of cases are likely to be asymptomatic and
remain undetected. Our hospital is a high-volume centre for pancreatic pathology and we examined almost 1500 cases of pancreatic surgical specimens in the last 5 years, but from 1994 to 2011
only two cases of pancreatic hamartoma have been collected.
In both cases, the lesions presented as solid, well circumscribed,
whitish-grey masses, with a homogeneous appearance on cut
surface and a maximum diameter of 1.5 cm.
In the first case, a 57-year-old woman presented with a pancreatic
mass, incidentally found by abdominal ultrasonography. The patient underwent a intermediate resection of the pancreatic body.
The second case was a 50-year-old-men with a suspected solid
lesion located in the pancreatic body. Radiologic findings suggested the presumptive diagnosis of ductal adenocarcinoma.
Intraoperative fine needle aspiration was performed for 3 times
and each time only normal acinar cells were found out. For this
reason the patient underwent a minimal intermediate resection
of the pancreatic body with the aim of achieve an intraoperative
histological diagnosis.
Microscopically both lesions were composed of well-differentiated acinar and ductal cells, without atypia, disposed in a radial
trabecular arrangement. A wide sclerotic paucicellular area was
present in the centre of the lesions. Acini and small intralobular and interlobular ducts showed atrophic aspects without any
evidence of dysplasia. Rare vascular structures were interspersed
between acini and ducts.
Discrete islets of Langerhans were lacking while scattered small
endocrine cell clusters, detected with immunohistochemistry,
were distributed between peripheral acini and ducts. In both cases
the surrounding pancreatic parenchyma was regularly lobulated
and without evidence of pancreatitis.
In both lesions the acini, stained positively for trypsin, and the
ductal cells, for cytokeratin 7, showed a trabecular radial arrangement. Masson’s trichrome stain underlined connective tissue,
stained blue, in the central fibrotic area and the surrounding red/
purple acini and ducts. Immunohistochemical staining for chromogranin A and synaptophysin showed the lacking of islets and
the presence of individual scattered endocrine cells distributed
between acini and ducts.
In both cases the preoperative diagnosis was that of solid tumour of
the pancreas suspicious for malignancy, but at histology we demonstrated the benign and non neoplastic nature of the lesions.
In particular in the second case, repetitive fine needle aspiration
shown normal acinar cells and the intraoperative histological
diagnosis of a non neoplastic lesion led the surgeon to perform a
minimal resection of the pancreatic body.
Fluorescence in situ hybridisation in the
cytological diagnosis of pancreatobiliary tumours
A. Paganotti*, S. Allegrini**, U. Miglio**, C. Veggiani*, M. Sartori***, J. Antona**, R. Mezzapelle**, R. Boldorini* **
SCDU Anatomia Patologica, Ospedale Maggiore della Carità, Novara, Italia; ** Dipartimento di Scienze Mediche, Università del Piemonte
Orientale, Novara, Italia; *** SCDU Gastroenterologia, Ospedale Maggiore della Carità, Novara, Italia
tion of the tumor. However, imaging techniques are not always
able to discriminate neoplastic stenosis from inflammatory obstruction and to define the neoplastic histotype. The cytological
diagnosis is currently based on the removal of cells by brushing
the biliary tract in the course of endoscopic retrograde cholangiopancreatography (ERCP). This method, although showing a high
specificity (almost 100%), is characterized by low sensitivity,
due to the difficult distinction between tissue cells and reactive
cells of well-differentiated CCA, at the tumor site - tract cancers
proximal bile show a high rate of false negatives related to the
difficulty of implementation of the levy - and the concomitant
presence of inflammatory processes, stenotic and sclerotic. The
high number of false negative makes this method unreliable and
in case of equivocal or negative with persistent clinical suspicion
can not exclude with certainty the nature of the neoplastic lesions
(false negatives). Recent study, including one conducted by our
group, proposed the introduction of methods for fluorescent in
situ hybridization (FISH) on cytological preparations to increase
diagnostic sensitivity.
Materials and methods. Bile duct brush samples for routine cytology and FISH were collected from 70 patients who underwent
ERCP because of a clinical suspicion of pancreatobiliary cancer
between June 2007 and September 2009. The inclusion criteria
were a pancreatobiliary stricture revealed by ERCP and a definite
histological diagnosis (benign or malignant) based on bioptic or
surgical specimens. When the patients’clinical condition did not
justify surgical procedures, a clinical follow-up of at least 12
months and nuclear magnetic resonance or computed tomography
imaging studies were used to establish a benign or malignant
disease course. Brushing samples collected from 64 patients were
evaluated cytologically and by means of a multi-probe FISH set
(Urovysion). The cytological diagnoses, in relation to cellularity,
the presence and type of cellular atypia, according to the criteria
proposed by Koss, were: “inappropriate” for lack/poor cellularity, improper fixation, etc., “negative” for the absence of cancer
cells, “suspect” is not sufficient atypia for a diagnosis of cancer
and “positive” to secure the presence of atypia of neoplastic nature, whereas criteria, proposed by Moreno Luna, for FISH were:
“negative” due to lack of polyploidy and “positive” for the presence of more than five polysomic cells or more than 10 trisomic
cells for chromosomes 3 or 7.
Results. Forty-eight of the 64 patients showed histological or
clinical signs of malignancy. The sensitivity of cytology was
high (77%) if suspicious cases were considered positive, but was
significantly lower than that of FISH if suspicious cases were
considered negative (58% versus 90%). The specificity of cytology was 81% (positive and suspicious) or 100% (negative and
suspicious), and the specificity of FISH was 94%. FISH yielded
one false negative result (isolated chromosome 7 trisomy). FISH
allowed a definite diagnosis of 9/12 cytologically inconclusive
cases.
Conclusions. Our findings suggest using FISH in the case of bile
duct strictures cytologically negative or inconclusive; a FISH
diagnosis of malignancy should only be made in the presence of
polysomic pattern.
*
Background. Cholangiocarcinoma (CCA) represents 3% of all
cancers of the gastrointestinal tract and a poor prognosis. CCA
are also characterized by high chromosomal instability with
resultant aneuploidy (reported in 80% of cases), sometimes
preceding the morphological manifestations of cancer (irregular
nuclear contours, chromatin compaction, prominent nucleoli). In
the case of clinical suspicion, the first approach to the diagnosis
of cancer of the bile ducts involves the use of image analysis
techniques, used to locate the site of occlusion and thus the loca-
Adenosquamous carcinoma of the pancreas:
a series of 12 cases
F. Pedica, I. Cataldo, L. Marcolini, C. Luchini, S. Gobbo, A.
Parisi, P. Capelli
Dipartimento di Patologia e Diagnostica, Policlinico G.B. Rossi, Università di Verona, Verona, Italia
Ductal adenocarcinoma and its variants are the most frequent
neoplasms of the pancreas (approximately 85-90% of all neoplasms).
The incidence varies from 1 to 10 on 100000 people in developed
countries.
Poster
Given the very poor survival, mortality rates closely parallel
incidence rates, with a survival of 10-20 months for patient who
underwent surgical resection and 3-5 months for unoperable
patients.
The poor prognosis depends on the advanced stage of disease
at the time of clinical presentation and the lack of effective
therapies, also because of the refractory nature of pancreatic
adenocarcinoma to conventional chemotherapy and radiotherapy
regimens.
One variant is adenosquamous carcinoma (ASC), a rare aggressive subtype, with higher potential for metastasis and an even
worse prognosis than conventional ductal adenocarcinoma.
Clinical, radiological and macroscopical aspects overlap those of
common ductal pancreatic cancer (DPC).
ASC is defined as a neoplasm with 30% or more malignant squamous cell carcinoma admixed with ductal adenocarcinoma.
We collected 12 cases of ASC of the pancreas from our archive,
surgically resected between 2005 and 2010. We reviewed the
slides and we look for the percentage of the squamous component
(also applying 2 immunohistochemical markers such as CK5 and
p63), the possible presence of preneoplastic alterations and the
type of metastasis occurring in the lymph nodes.
The maximum diameter of tumor varied from 2 to 6 cm and they
were located mainly in the head of the pancreas (80% of cases).
All cases showed massive perineural and lymphatic invasion and
where associated to a severe chronic pancreatitis.
The percentage of squamous component varied from 30 to 95%
of the tumor and 8 of 12 cases (66,67%) had more than 50% of
squamous component.
Eight cases had lymph node metastasis with different morphological aspect: 2
cases were completely squamous (16,67%), 3 were totally adenocarcinomatous (25%) and 3 cases has both components. These
last cases of mixed metastasis presented 10%, 30% and 50%
of squamous component in the lymph node. This characteristic
didn’t reflect the percentage of the two component in the primitive lesion, as in the primitive tumor the squamous component
was respectively 90%, 40% and 85%.
Preneoplastic lesions were found in all cases and involved either
the main duct than the peripheral ducts. We observed mucinous
metaplasia, intraductal pancreatic neoplasm (PanIn, from low
grade to high grade), squamous metaplasia and the combinations
of them.
Mucinous metaplasia alone was present only in one case (8,33%),
but it was more often combined with squamous metaplasia (6
cases, 50%) and in an other cases was mixed with low gradePanIn (8,33%).
For what concern squamous metaplasia, it was never present
alone but always together with mucinous metaplasia (as just illustrated above) and with low grade-PanIn (16,67%). Moreover
PanIn was present alone in only one case (8,33%).
Several theories have been proposed about ASC etiopathogenesis.
In the first one, the “squamous metaplasia theory”, squamous
metaplasia occurs as a result of ductal inflammation due to chronic
pancreatitis or obstruction by an adenomatous tumor and it ultimately transforms into a malignant adenosquamous pancreatic
tumor.; the second is the “collision theory”, suggesting that the
two histologically distinct tumors, adenocarcinoma and squamous
cell carcinoma, arise independently from different sites and then
melt; the third one, the “differentiation theory”, suggesting that
ASC arises as a arises as a result of malignant differentiation from
a pluripotential duct cell into the two distinct histologic types.
In our cases squamous-cell component seemed to be deeply intermingled in the body of the predominant adenocarcinoma type and
the main and peripheral ducts often presented squamous metaplasia. These data seem to support the third theory and the possibile
origin of both components from a pluripotent stem cell.
193
Impact of fnac on diagnosis and treatment of
solid pancreatic lesions in current clinical practice
G. Perrone*, C. Brunelli*, C. Rabitti*, F.M. Di Matteo**, D. Borzomati***, R. Coppola***, A. Onetti Muda*
*
Anatomia Patologica, ** Unità di Endoscopia Digestiva, *** Chirurgia
Generale/Università Campus Bio-Medico di Roma, Roma, Italia
Early and accurate diagnosis of pancreatic lesions is critical for
efficient pancreatic cancer management. Endoscopic ultrasoundguided / fine needle aspiration cytology (EUS/FNAC) has a significantly higher yield over percutaneous biopsy and is currently
the principal diagnostic procedure for pancreatic mass lesions.
Aim of this study was to assess the accuracy rate of EUS/FNAC
sampling of solid pancreatic mass.
Methods. A total of 205 pancreatic FNAC samples were obtained by EUS/FNAC. The cytologic material was evaluated
by using 5 clinically relevant pathologic categories: 1) non
diagnostic\inadequate; 2) negative for neoplasm: normal acinar
and/or ductal epithelium; 3) atypical/inconclusive: reflected
mild to moderate cell atypia, with a low suspicion of malignancy, often in an inflammatory background; 4) suspicious for
carcinoma: strongly suggesting malignancy but cytological
features are not sufficient in quantity and/or quality for a definitive diagnosis; 5) diagnostic of carcinoma: adenocarcinoma,
metastatic disease, neuroendocrine tumours. Clinical follow-up
information were obtained from 102 of the 205 pancreatic FNA
cases performed.
Results. In the total of 205 pancreatic FNACs, 94 (45,8%) resulted positive for carcinoma, 29 (14,1%) as suspicious, 21 (10,2%)
atypical\inconclusive, 27 (13,2%) negative for neoplasm and 34
(16,6%) non diagnostic\inadequate.
In order to assess the accuracy of EUS-FNAC, follow-up information were reviewed from 102/205 patients. 94/102 [positive
predictive value (PPV) = 90,4%] patients with pancreatic solid
mass at EUS were found affected by carcinoma. When using
FNAC, 49/49 (PPV= 100%) patients with cytological diagnosis
of “diagnostic of carcinoma” were affected by adenocarcinoma (43/49), or neuroendocrine tumors (6/49); 22/22 (PPV=
100%) patients with cytological diagnosis of “suspicious for
carcinoma” were affected by adenocarcinoma (21/22), or
neuroendocrine tumours (1/22); 9/13 (PPV= 69,23%) patients
with diagnosis of “atypical/inconclusive” were affected by
adenocarcinoma. In 5/9 (55,5%) patients with diagnosis of
“negative for neoplasm” a ductal carcinoma was found. Sensibility and specificity of EUS-FNAC was 84,5% and 100%
respectively. The positive and negative predictive value was
100% and 43,5%.
Conclusions. EUS-FNA cytological examination is an accurate
procedure for the diagnosis and planning of therapy for focal
pancreatic lesions. Atypical/inconclusive diagnosis creates
a difficult management dilemma. One possible approach to
enhance the EUS-FNA diagnostic yield is to combine routine
cytology with auxiliary diagnostic techniques, such as, tumour
marker analysis. Moreover, benign FNA findings do not necessarily exclude the presence of pancreatic malignancy, and
repeated biopsies should be considered whenever the presence
of malignancy is suspected clinically. In our experience, the use
of standard diagnostic categories may facilitate communication
among pathologists, endoscopists, surgeons and radiologists
and may facilitate cytological–histological correlation for pancreatic diseases.
194
Quantitative analysis of K-RAS mutation
in pancreatic speciments obtained
by EUS guided–FNA
G. Perrone*, L.M. Gaeta*, C. Brunelli*, D. Righi*, D. Borzomati**,
F.M. Di Matteo***, R. Coppola**, A. Onetti Muda*
Anatomia Patologica, ** Chirurgia Generale, *** Unità di Endoscopia
Digestiva, Università Campus Bio-Medico di Roma, Roma, Italia
*
Endosonography-guided fine needle aspiration (EUS-FNA) is a
well established, safe and effective technique in the diagnosis and
staging of pancreatic cancer (Wiersema MJ, et al. Gastroenterology
1997). EUS alone usually displays high sensitivity rates in detecting pancreatic cancer (Kadish SL et al. Am J Gastroenterology
1995), but specificity rates are low. Therefore, the introduction
of EUS-FNA in the study of pancreatic lesions has improved the
specificity of EUS in the diagnosis of pancreatic cancer (Erickson
RA et al. Acta Cytol 1997), although differential diagnosis between
pancreatic ductal adenocarcinoma (PADC) and pseudotumoral
forms (e.g. chronic pancreatitis) remains difficult. KRAS gene mutations (point mutations of codon 12) have been found in 75-90%
of infiltrating PADC (Koorstra JBM, et al. Pancreatology 2008)
and such a frequency seems sufficiently high to deserve diagnostic
application. However, KRAS mutations have been apparently
reported also in non-neoplastic pancreatic diseases such as chronic
pancreatitis (Tada M et al, American Journal of Gastroenterology
2002), therefore limiting the clinical relevance of the data.
Aim of the present study was to investigate the feasibility and
reproducibility of combined cytopathological examination and
KRAS analysis in improving the diagnostic accuracy on atypicalinconclusive pancreatic FNA samples obtained by endoscopic
ultrasonography.
KRAS mutation analysis in codon 12 was performed in 27 EUSFNA specimens from patients with an indeterminate cytopathological diagnosis, which were subsequently diagnosed as PADC (n = 12)
or chronic pancreatitis (n =15). As controls, KRAS mutation analysis
was also performed in surgical samples from patients with chronic
pancreatitis (n = 10) or pancreatic adenocarcinoma (n = 10). In the
latter group mutation analysis was performed also in areas of chronic
pancreatitis collected at a distance from the tumour.
In surgical samples, significant differences were found between
carcinoma vs. non neoplastic tissue (p= 0.0001). In particular, allele
frequency of KRAS mutation in cancer samples ranged from 7%
to 36% (median 27,2%; IQR 14,7% - 30,9%), in pancreatitis from
0% to 5% (median 0,65%; IQR 0,07%-1,9%) and in pancreatitis
associated with carcinoma from 2% to 4,5% (median 3,45%; IQR
2,9% - 4,0%). On this basis, 6% of allele frequency of KRAS mutation was defined as an adequate cut-off to distinguish neoplastic
from non-neoplastic lesions. In FNA specimens, KRAS mutation
analysis was successful in all samples in terms of amount and quality of extracted DNA and sequencing process. The frequency of the
mutated allele ranged from 0% to 31,6%; in particular, all cancer
patients except one displayed a mutation rate higher than 6%.
In conclusion, our preliminary data suggest that in cases of FNA
of pancreatic solid lesions with an atypical-inconclusive cytopathological diagnosis, KRAS mutation analysis may be useful
in strongly suggesting a diagnosis of PADC.
Mucinous cystadenoma of the pancreas: a case
report
V. Tralongo, G. Becchina, G. Ottoveggio, F. Genovese, R. Canciglia, C. Nagar.
Unità Operativa Complessa di Anatomia Patologica, Presidio Ospedaliero
“G.F. Ingrassia”, Azienda Sanitaria Provinciale di Palermo, Palermo, Italia
Introduction. Mucinous cystadenomas of the pancreas are
benign mucin-producing cystic tumours with an ovarian-like
stroma, lack of communication with the pancreatic duct system,
occur mostly in middle-aged females and are most frequent located in tail and the body of the pancreas 1-4.
Case report. A 27-year-old woman presented with a complaining
of a vague epigastric pain; on examination, a palpable epigastric
mass was noted. Upper computed tomography (CT) scanning
revealed a large multilocular cystic mass in the body to tail of the.
The patient underwent a distal pancreatectomy with splenectomy
to remove the tumour
The neoplasm was composed of large multilocular cysts and
showed a thick fibrous wall; the neoplasm do not visibly communicate with the pancreatic ductal system. Microscopically internal
surface of the wall was lined by columnar, mucin-producing
epithelium with underlying ovarian-like stroma. Immunohistochemical studies showed positive staining for estrogen and
progesterone receptors in the stromal cell; mucin-producing
epithelium expressed Citokeratin 7 and EMA, but not expressed
estrogen and progesterone reactivity.
Discussion. Mucinous cystadenoma of the pancreas are similar
to mucinous cystadenoma of the ovary; this resemblance includes
the presence of a hormone sensitive stroma ovarian-like around
the cysts that is now considered a prerequisite for diagnosis. This
tumor is most frequent located in tail and the body of the pancreas
and has not communication with the duct system 5-6.
The predominantly female occurrence and the expression of estrogen and progesterone receptors in ovarian-like stroma has been
the subject of two hypotheses on the origin of this tumor.
The first hypothesis is that these lesions arise from rests of
embryologic ovarian tissue which are incorporated into the
embryonic pancreas when the left primordial gonad is in close
proximity to the dorsal pancreatic anlage during embryogenesis;
the dorsal pancreatic anlage gives rise to the body and tail of the
pancreas and this hypothesis could explain the predilection of
mucinous cystic tumors for the distal pancreas 3 7 8. The second
hypothesis is that the stroma represents a recapitulation of periductal fetal mesenchyme and this concept is based on the fact
that the stroma in the fetal pancreas is similar to that of mucinous
cystic neoplasms 9.
References
1
Ng DZW, Goh BKP, Tham EHW, et al. Cystic neoplasm of the pancreas: current diagnostic modalities and management. Ann Acad Med
Singapore 2009;38:251-9.
2
Freeman HJ. Intraductal papillary mucinous neoplasm and other pancreatic cystic lesions. World J Gastroenterol 2008;14:2977-9.
3
Ikuta S, Aihara T, Yasui C, et al. Large mucinous cystic neoplasma
of the pancreas associated with pregnancy. World J Gastroenterol
2008;14:7252-5.
4
Patrinou V, Skroubis G, Zolota V, et al. Unusual presentation of
pancreatic mucinous cystadenocarcinoma by spontaneous splenic
rupture. Dig Surg 2000;17:645-7.
5
Klöppel G, Solcia E, Longnecker DS, et al. Histological typing of
tumours of the exocrine pancreas. World Health Organization - International Histological Classification of Tumours. 2nd ed. SpringerVerlag, Berlin 1996.
6
Solcia E, Capella C, Klöppel G. Tumors of the pancreas. Armed
Forces Institute of Pathology, Atlas of Tumor pathology. 3nd series,
fascicle 20, Washington DC 1995.
7
Zamboni G, Scarpa A, Bogina G et al. Mucinous cystic tumors of the
pancreas: clinicopathological features, prognosis and relationship to
other mucinous cystic tumors. Am J Surg Pathol 1999;23:410-22.
8
Goh BK, Tan YM, Chung YF, et al. A review of mucinous cystic neoplasms of the pancreas defined by ovarian-tipe stroma: clinicopathological features of 344 patients. World J Surg 2006;30:2236-45.
9
Volkan Adsay N. Cystic lesions of the pancreas. Mod Pathol 2007;20
(Suppl 1):71-93.
195
Poster
Emolinfopatologia
Lymphomatoid granulomatosis with gastric
and pulmonary localization
MR Ambrosio, M Onorati, BJ Rocca, C Bellan, A Barone, D
Spina, C Vindigni, T Amato, S Lazzi, L Leoncini
Background. Lymphomatoid granulomatosis (LG) is an extranodal B-cell lymphoproliferative disorder with a background of
reactive T lymphocytes. It is characterized by Epstein-Barr virus
(EBV) association. It has an angioinvasive behaviour, and grade
3 LG is considered as a diffuse large B cell lymphoma (DLBCL).
It can originate at any age, but 80% of the cases occur between 4th
and 6th decades, with a male prevalence. As with other EBV-associated lymphoproliferative disorder, LG occurs with increased
frequency in immunosuppressed patients. The lung is the most
common involved organ; skin and central nervous system may
be also affected. Gastrointestinal involvement is uncommon and
represents a very poor prognostic factor.
Methods. A 45 year-old female presented with persistently
dyspeptic symptoms. Gastroscopy showed mild hyperemia of
the gastric body mucosa. Gastric biopsy samples were processed
for routine histological examination, immunohistochemistry for
CD20 and CD3 was performed. The patient underwent therapy
for helicobacter-pylori and after two months, she was submitted
to another gastroscopy for control. The gastroscopy showed the
worsening of endoscopic findings. Another biopsy was performed
and a panel of antibodies were checked (CD20, CD3, CD4, CD8).
Due to the onset of severe dyspnea, a thoracic computed tomography was made. In the suspicion of diffuse interstitial lung disease,
right lower lobectomy was carried out. Serial section of the surgical specimen were stained with H&E and immunohistochemistry
for CD20, CD79a, CD3, CD4, CD8 was performed. Both gastric
and pulmonary specimen were evaluated for EBV-encoded RNA
(EBER). The rearrangement of the immunoglobulin heavy chain
genes and of the T cell receptor genes was done.
Results: The first gastric biopsy showed a marked infiltration of
gastric surface and foveolar epithelium by T cells (CD4+) (>25
IELs/100 epithelial cells) and a lymphoplasmacytic infiltrate in
the lamina propria; a diagnosis of lymphocytic gastritis HP-related
was made. The second gastric biopsy showed an angiocentric proliferation of small T lymphocytes (CD4+, CD8+) admixed with
a polymorphous atypical lymphoid infiltrate of CD20 positive
cells with blastic appearance. Scattered pleomorphic, enlarged,
multinucleated “Hodgkin-like” cells were also observed. The infiltrate extended deeper into the muscolaris mucosae causing focal
ulceration of the mucosa and lymphoepithelial lesions. Necrosis
was absent. Atypical cells were shown to express EBER positivity. No classical Reed-Stenberg cells were observed and atypical
B lymphocytes were CD30- and CD15-. Pulmonary parenchyma
showed a complete effacement of the architecture because of the
presence of nodular infiltrates of T cell lymphocytes (CD4+),
intermingled with scattered large, pleomorphic, multinucleated
B cells (EBER+), actively proliferating. Blood vessels showed
obliteration of the lumen and transmural infiltration of tumor cells.
Necrotic foci were also observed. In both the lung and second
gastric biopsies, a diagnosis of LG grade 2 was made. Considering
these findings, the first gastric biopsy was reevaluated and EBER
was performed with positive result in B cells, so a diagnosis of
LG grade 1 was made. Molecular studies showed monoclonal IgH
genes rearrangement on polyclonal background in both the gastric
biopsies and in the nodular lesions of the lung.
Conclusions. Differential diagnosis of LG include inflammatory
process (Wegener’s granulomatosis, Churg-Strauss vasculitis,
inflammatory pseudotumor), infectious diseases (histoplasmosis,
tuberculosis, interstitial pneumonia, abscesses) and neoplasms
(nasal type T/NK lymphoma, Hodgkin lymphoma, DLBCL). Although the evolution of LG from grade 1 to grade 3 has not been
unequivocally demonstrated, several studies showed that an early
diagnosis followed by a specific treatment with corticosteroid,
interferon, monoclonal antibodies and sometimes chemotherapy,
may avoid the development of a DLBCL.
Rosai-Dorfman disease: report of an aggressive
case in an adult patient, successfully treated
with bone marrow transplantation
M. Bisceglia1, G. Cimino2, A. Chiaramonte3, G. Giannatempo4,
M. Greco5.
1
Unità Operativa di Anatomia Patologica; 2 Unità Operativa di Otorinolaringoiatria; 3 Unità Operativa di Chirurgia Toracica; 4 Unità Operativa
di Radiologia; 5 Unità Operativa di Ematologia, IRCCS, Ospedale “Casa
Sollievo della Sofferenza”, San Giovanni Rotondo (FG), Italia
Background. Rosai-Dorfman disease (RDD) or sinus histiocytosis with massive lymphadenopathy (SHML) is a polyclonal
proliferative histiocytic disorder, of unknown cause, affecting
mainly children or young adults. RDD is more often a selflimiting disease with a protracted clinical course consisting of
alternating remissions and flare-ups, but it may also undergo
spontaneous resolution and occasionally may show a poor prognosis. RDD is generally treated with corticosteroids, usually with
a good response, but the therapeutic strategy can vary according
to individual disease presentation and extent. We present a new
case in an adult patient with an aggressive presentation and massive nodal involvement, who was for the first time successfully
treated with autologous bone marrow transplant.
Case report. A 57-year old male was admitted with a diagnosis
of suspected lymphoma due to constitutional symptoms (such as
malaise, cough, mild serotinous fever and a significant weight loss
of 7 kilos) and the appearance of superficial neck and supraclavian
lymphadenopathy lasting for 1 to 2 months, with lymph node size
ranging from 1.5 to 2.5 cm. Hematology and blood chemistry tests
were all normal, except for a direct Coombs test which was positive.
In addition CT scan of the thorax also showed enlarged mediastinal
lymphadenopathy. A left neck lymph node biopsy was performed and
the histological examination showed classical morphological features
of SHML, which was immunohistochemically confirmed (all intrasinusoidal histiocytes were positive for CD68/KP-1, CD68/PGM-1,
and S100 protein; and CD1-alpha negative). Trephine bone marrow
biopsy revealed a normal bone marrow haematopoietic population
with no evidence of RDD infiltration. Follow-up. The patient was
given 6 courses of chemotherapy with cyclophosphamide, vincristin,
and prednisone, with a partial clinical response. 16 months later due
to the persistence of symptoms and multiple enlarged lymph nodes a
second left neck lymph node was excised and histologically examined with confirmation of SHML involvement. Mediastinal lymph
node enlargement was again confirmed by means of a new CT scan.
4 courses of a new chemotherapy protocol were administered, using
ifosfamide, epirubicin, and etoposide along with rituximab, and, with
the aim of curing this patient by means of bone marrow transplantation, CD34-positive peripheral blood stem cells were collected during
the phase of reconstitution of the hematopoietic system and cryopreserved. 6 months later myeloablation was accomplished using the
FEAM regimen, including fotemustine, arabinoside-C, vepesid, and
melphalan followed by autologous peripheral blood stem cells infusion. Currently 5 months after bone marrow transplant the patient is
alive and well and is being followed-up.
Discussion. In around 60% of cases RDD is an exclusively nodal
disease. Nodal involvement may be limited to a single node or
may involve numerous lymph nodes, usually bilaterally in the
neck area, but also in other anatomical locations (mediastinal,
intrabdominal, and inguinal). In the remaining 40% of cases
RDD manifests itself as extranodal disease either in association
196
with nodal involvement (more often) or in an exclusively extranodal localization. Among extranodal sites almost every organ has
been described, including skin, soft tissue, bone, central nervous
system (both brain and meninges), upper respiratory tract (nasal
cavity and paranasal sinuses), breast, eye and orbit, and rarely the
gastrointestinal tract, including the liver, and serosal membranes
(mostly pleura). Some patients are asymptomatic at presentation,
but many manifest constitutional symptoms, and around 10%
also exhibit autoimmune phenomena (autoantiboides against red
blood cells, neutrophils, and platelets). Patients with extranodal
localizations may complain of local symptoms related to the specific organ involvement. Furthermore, RDD has been reported
also in association with other diseases or in other clinical settings,
that is: i. in patients with Hodgkin’s and non-Hodgkin’s lymphomas, either coincidentally in the same organ or simultaneously in
a different one (Di Tommaso et al, 2010); ii. in patients affected
by autoimmune lymphoproliferative disease (ALPS) (Maric et
al, 2005); iii. following intensive treatment for T-cell acute lymphoblastic leukemia (Allen et al, 2001); and also iv. coincidental
with Langerhans’ cell histiocytosis (Sachev et al, 2008; Wang
et al, 2007; O’Malley et al, 2010), this latter condition maybe
representing a monoclonal subset of the disease (O’Malley et al,
2010). Intrathoracic manifestations have only occasionally been
reported. Of 21 patients with RDD diagnosed over a period of
30 years in the Mayo Clinic records (1975-2005), only 9 were
found to have intrathoracic manifestations, 6 of which consisted
of lymphadenopathy, with the remaining cases involving the lung
parenchyma and appearing as cystic changes or interstitial lung
diseases (Cartin-Ceba et al, 2010). In 1 case RDD presented as
a solitary, most likely soft tissue mediastinal mass (Hida et al,
2009). The heart has been reported as being involved in 7 cases,
in which the (right) atrium was more often the main site of involvement (Ajise et al, 2011). Etiologic hypotheses in regard to
RDD include immune dysregulation and infectious agents, with
polyoma virus (SV40) as the last among multiple pathogens, to be
documented, in 3 of 18 soft tissue cases (Al-Darraji et al, 2011).
Therapy is variable and is mainly based on corticosteroids, however
chemotherapy using methotrexate and mercaptopurine either singly
or combined with corticosteroids are used as well (Komp et al, 1990;
Horneff, 1996; Jabali, 2005). Based on a computerized literature
search, no case of RDD treated with bone marrow transplantation
has been recorded to date.
Conclusions. The case herein described is a classical case with
classical histology, location, constitutional symptoms, haematological and chemical laboratory abnormalities, and intrathoracic
manifestations, who was treated (maybe the first case worldwide)
with autologous bone marrow transplant by means of peripheral
CD34-positive blood stem cells.
Dysregulation of global microrna expression
in splenic marginal zone lymphomas and impact
of chronic hepatitis c virus infection
G. Crisman1* , J. Peveling-Oberhag2*, A. Schmidt3, C. Doring4,
M. Lucioni5, L. Arcaini6, S. Rattotti6, S. Hartmann4, A. Piiper2,
W.P. Hofmann2, M. Paulli5, R. Kuppers3, S. Zeuzem2, P. Leocata1, M.L. Hansmann4
U.O. Anatomia Patologica, Dipartimento di Scienze della Salute, Università degli Studi dell’Aquila, L’Aquila, Italia; 2 Department of Internal Medicine, J.W. Goethe- University Hospital, Frankfurt am Main, Germany;
3
Institute of Cell Biology, University of Duisburg-Essen, Medical School
Essen, Essen; 4 Senckenberg Institute of Pathology, J.W. Goethe- University Hospital, Frankfurt am Main, Germany; 5 Anatomia Patologica, Fondazione IRCSS Policlinico San Matteo, Università di Pavia, Pavia, Italia; 6
Unità Operativa di Ematologia, Dipartimento di Oncoematologia, Fondazione IRCSS Policlinico San Matteo, Università di Pavia, Pavia, Italia
1
According to the World Health Organization (WHO 2008),
Splenic Marginal Zone Lymphoma (SMZL) represents a rare
indolent B-cell neoplasm, accounting for less than 2% of nonHodgkin Lymphomas, that primarily affects the white and red
pulps of the spleen. The relationship between tumour cells and
normal spleen as well as the aetiopathogenesis of this lymphoma
are still unknown, even though chronic hepatitis C virus (HCV)
infection seems to be involved in a subset of patients, especially
in endemic areas. As a matter of fact, it is proved that HCV infection frequently leads to chronic hepatitis and eventually to liver
cirrhosis and to hepatocellular carcinoma (HCC), but an association with the development of some lymphoproliferative diseases,
such as mixed-cryoglobulinemia and B-cell non-Hodgkin lymphoma (B-NHL), has been demonstrated as well.
MicroRNAs (miRNAs) are members of a class of small, noncoding RNAs that modulate gene expression at the post-transcriptional level in a sequence specific manner. They play a role in
controlling a variety of biological functions, including developmental patterning, cell differentiation, cell proliferation, genome
rearrangements and transcriptional regulation. Dysregulation of
miRNA expression is thought to play a pivotal role in carcinogenesis and also viral infection, e.g. hepatitis C virus (HCV)
infection, has been shown to distinctively influence miRNA
expression in vivo and in vitro.
Formalin fixed paraffine embedded (FFPE) splenic tissue from
15 patients with SMZL and 11 matched controls from healthy
patients (splenectomy was performed for blunt abdominal
trauma), respectively, was selected for multiplex miRNA expression analysis from the Dept. of Pathology, Pavia, Italy,
Dept. of Pathology, L’Aquila, Italy and the Dept. of Pathology, Frankfurt, Germany. Additional 20 cases of SMZL and
5 splenic controls were selected from the Dept. of Pathology,
Pavia and the Dept. of Pathology, Frankfurt, Germany for
validation. Diagnosis of SMZL was established by standard
morphological, cytochemical and immunophenotypic methods
according to 2008 WHO classification and the diagnostic criteria proposed by Matutes et al.
A large-scale miRNA expression profiling analysis of 381 miRNAs has been performed by quantitative reverse-transcription
PCR (Q-RT-PCR) on 26 microdissected splenic tissue samples
(7 SMZL, HCV+; 8 SMZL, HCV- and 11 non-tumorous splenic
controls). Single assay Q-RT-PCR and miRNA in situ hybridisation were used to confirm findings. Unsupervised hierarchical clustering of miRNA expression profiles demonstrated a
distinct signature of SMZL compared to the healthy splenic
marginal zone. A supervised analysis revealed differentially
expressed miRNAs, including ones previously recognized for
their tumour suppressive or oncogenic potential. Five miRNAs
were found significantly overexpressed in SMZL, including miR-21, miR-155, and miR-146a, while seven miRNAs
showed significantly lower expression, including miR-139,
miR-345, miR-125a, and miR-126. Low expression of miR-29c
was associated with worse overall survival (p-value: 0.028).
Furthermore, we identified miR-26b, a miRNA with known
tumor suppressive properties, as significantly down-regulated
in SMZL arising in HCV positive patients (p-value: 0.0016).
In conclusion, our results revealed a specific dysregulation of
miRNA expression in SMZL and it should be considered to
play almost a role in the molecular pathway and in the prognosis of the SMZL itself.
*
Authorship contributions: J. Peveling-Oberhag: designed and
performed research, revised and analyzed clinical data; G. Crisman performed research and collected and characterized histological samples.
197
Poster
DNA hypermethylation in myelodysplastic
syndromes: bone marrow cell immunoreactivity
for 5-methylcytosine in single and double
immunostainings
G. Goteri1, A. Poloni2, A. Zizzi1, F. Giantomassi1, D. Stramazzotti1, B. Costantini2, S. Trappolini2, M. Mariani2, R. Re3, F.
Alesiani4, M. Catarini5, P. Leoni2
Anatomia Patologica, Dipartimento di Scienze Mediche e Sanità Pubblica; 2Clinica di Ematologia, Dipartimento di Scienze Mediche e Chirurgiche, Università Politecnica delle Marche, Ospedali Riuniti di Ancona,
Ancona, Italia; 3Divisione di Medicina, Ospedale Civitanova Marche;
4
Divisione di Oncologia, Ospedale di Camerino; 5Divisione di Medicina,
Ospedale di Macerata.
1
Myelodysplastic syndromes (MDS) are a heterogeneous group of
clonal haematopoietic disorders with ineffective haematopoiesis
leading to cytopenia and an increased risk of transformation to
acute myeloid leukaemia (AML). Clonal cytogenetic abnormalities have prognostic significance. The importance of epigenetic
events which regulate gene expression at post-translational level
has been highlighted. The response of MDS to drugs that reverse
aberrant hypermethylation suggest that aberrant hypermethylation
might play a causative role in MDS. DNA methylation occurs at
the cytosine residues of CpG dinucleotides by an enzymatic reaction producing 5-methyl-cytosine (5-mc) and 5-mc expression
considered in several neoplasms as a reliable immunohistochemical marker of DNA methylation status.
Methods. We retrieved from our archives the original slides and
the complete clinical history of 130 MDS and 40 AML cases
diagnosed at our institutions. As healthy controls we included
46 normal bone marrows resulted negative at lymphoma staging.
The histological categories were uni- and multi-lineage without
excess of blasts (UL/45 cases and ML/29 cases), multilineage
with excess of blasts (EB-ML/49 cases), based on the number
and type of hematopoietic lineages involved by dysplasia, and the
percentage of CD34/CD117 positive precursors. Chronic Myelomonocytic leukemias (CMMoL/7 cases) were separately considered. Cases were stratified also for the FAB and WHO classifications, the cytogenetic classes, and the International Prognostic
Scoring System (IPSS) risk scores (low, intermediate-1 and -2,
high). Immunohistochemistry was performed on formalin-fixed,
EDTA-decalcified bone marrow sections; 5-mc immunoreactivity was expressed with a “H-score” obtained by multiplying the
percentage of positive cells and the intensity score, as previously
described by Elsheikh et al. (2009).
Double immunostainings were performed for 5mc and one of four
cytoplasm/cell membrane markers (CD34, MPO, Glycophorin-C,
Factor VIII) by using EnVision™ G|2 Doublestain System, Rabbit/Mouse (Dakocytomation).
Results. A “H-score” increase was observed in MDS (mean,
62.54) compared to normal bone marrows (mean, 19.80) and in
AML (mean, 168.62) compared to MDS (ANOVA test, p=0.001;
pairwise comparisons p<0.05). Among MDS cases, a significant
increase was observed in EB-ML MDS (mean, 81.48) and in
CMMoL (mean, 110,00) compared to UL MDS (mean, 36.00;
ANOVA test, p<0.001; pairwise comparisons p<0.05). The
“H-score” correlated significantly with FAB and WHO classifications (Kruskal-Wallis test, p=0.0102 and p=0.0016), the
cytogenetic abnormalities (p=0.04), the IPSS risk score (p=0.04).
With double staining, in normal bone marrows the intermediate
myeloid MPO-reactive and early erytroid glycophorin-C-reactive
precursors (less than 10%) were 5mc-positive. Segmented granulocytes, neutrophilic polymorphonucleated cells and orthochromatic erythroblasts were negative. CD34+ precursors with
double stainings were not visualized. Compared to normal bone
marrows, de novo and secondary AML were characterized by the
highest percentage of 5mc+/CD34+ (>60% and 30%) and 5mc+/
MPO+ cells (>90% and 70%), including also more mature cells
like segmented granulocytes and band cells; 5mc+/glycophorin+
cells were few in de novo and numerous in secondary AML
(30%). UL and ML MDS showed a mild increase of 5mc+/MPO+
(>20%) and 5mc+/glycophorin+ precursors (>30%) compared to
normal bone marrows. EB-ML MDS exhibited a slight increase
of 5mc+/CD34+ precursors (>5%) compared to MDS without
blasts and normal bone marrows. Factor VIII+ megakaryocytes
were frequently reactive for 5mc in all conditions.
Comclusions. Our data suggest that global DNA hypermethylation correlate with MDS aggressiveness and provide molecular
explanation for the therapeutic success of hypomethylationinducing agents in MDS. Future studies have to analyze whether
these parameters may serve as a new predictive marker for
therapy response.
Bone marrow stroma cd40 expression in splenic
marginal zone lymphoma is associated with
prominent mast cell infiltration and correlates
with shorter time to progression
C. Guarnotta1, S. Sangaletti2, G. Franco3, B. Frossi4, M. Cacciatore1, M. Calvaruso1, P.P. Piccaluga4, C. Pucillo5, E. Boveri6, L.
Arcaini7, A.M. Florena1, M.P. Colombo2, C. Tripodo1
Dipartimento di Scienze per la Promozione della Salute, Università di
Palermo, Palermo, Italia; 2 Fondazione IRCCS, Istituto Nazionale dei
Tumori, Milano, Italia; 3 Unità Operativa di Ematologia, University of
Palermo, Palermo, Italia; 4 Instituto di Ematologia ed Oncologia Medica, Università di Bologna, Bologna, Italia; 5 Dipartimento di Scienze e
Tecnologie Biomediche, Università degli Studi di Udine, Udine, Italia; 6
Dipartimento di Patologia, Università di Pavia Italia; 7 Dipartimento di
Oncoematologia, Università di Pavia, Italia.
1
Splenic marginal zone lymphoma (SMZL) is an indolent mature
B-cell malignancy. However, nearly one-third of patients display
a rapidly progressive disease and a dismal outcome. Risk stratification has been recently proposed based on the assessment of
clinical and laboratory parameters on diagnosis. Biological prognostic factors are still lacking and their identification might prove
of great value for identifying patients at high risk of unfavorable
disease. In SMZL, bone marrow (BM) infiltration is almost invariably observed on diagnosis and the BM microenvironment
may play an important role in the disease progression. Aim of this
study was to characterize the BM microenvironment associated
with SMZL infiltrates in order to identify potential influences of
the stroma on the biology and natural history of this lymphoma.
Routinely processed Bm biopsies (BMB) of fifty-six consecutive
cases of SMZl diagnosed between
2001 and 2010 were collected. All patients had a BMB at the time
of diagnosis. The density and distribution of stromal elements
were evaluated in the context of SMZL BM lymphoid infiltrates
by semiquantitative immunohistochemical analysis using the following markers: CD10 (adventitial reticular cells), CD31 (blood
vessels), and CD40 (activated stromal cells and endothelia).
Moreover, the amount of immune cells infiltrating the SMZL
lymphoid aggregates was estimated by counting the number of
CD68+ macrophages, DC-Sign+ myeloid DCs, CD2+ T-cells,
and tryptase+ mast cells. Time to progression (TTP) was used as
clinical endpoint. TTP was calculated as the time interval between
diagnosis and progressive disease (PD). PD was defined either as
an increase in size of previously documented disease greater
than 25%, or as the appearance of disease at any new site or even
the shift to a more aggressive histotype. On multivariate analysis,
we found a significant correlation between the density of the
CD40+ stromal cell meshwork of neoplastic BM infiltrates and a
shorter TTP (p=0,02). Notably, a shorter TTP was also associated
with a conspicuous amount of infiltrating tryptase+ mast cells in
the lymphoid aggregates (p=0,01). Consistently, the presence of
CD40 in the stromal network of SMZL BM infiltrates proved
associated with the amount of mast cells (p=0.0001) while no
198
significant associations were detected with that of other bystander
immune cells such as CD68+ macrophages, DC-Sign+ dendritic
cells, or CD2+ T-cells. On these bases we hypothesized the existence of a cross-talk between mast cells and BM mesenchymal
cells (BM-MSC), which could be functional to the engendering
of a permissive environment for SMZL clone progression. Thus,
we performed co-culture experiments using human mast cells
(LAD2 MC line) and BM-MSC obtained from marrow aspirates,
aiming at detecting variations in CD40 expression on BM-MSC
upon mast cell co-culture. Strikingly, we observed that co-culture
with mast cells resulted in CD40 upregulation on BM-MSC and
in IL-6 release by mast cells. Accordingly, mast cells detected in
SMZL BM infiltrates rich in CD40+ stromal cells, were found to
express IL-6. Here we demonstrated that BM microenvironmentrelated features, namely the presence of a CD40-expressing
stromal meshwork and the prominent mast cell infiltration could
have a role in influencing the clinical course of SMZL. Our preliminary results point out a possible functional link between mast
cells and BM stromal cells towards SMZL clone stimulation via
CD40-CD40L and IL-6 production.
References
1
Arcaini L, Lazzarino M, Colombo N, et al. Splenic marginal zone lymphoma: a prognostic model for clinical use. Blood 2006;107:4643-9.
2
Iannitto E, Tripodo C. How I diagnose and treat splenic lymphomas.
Blood 2011;117:2825-95.
3
Tripodo C, Sangaletti S, Piccaluga PP, et al. The bone marrow stroma
in hematological neoplasms-a guilty bystander. Nat Rev Clin Oncol
2011;8:456-66.
Nodal extramedullary hematopoiesis in a patient
with idiopathic myelofibrosis: differential
diagnostic issues
S. Malatesta*, P. Viola*, N. Vazzana**, A. Di Lorito*, G. Lattanzio*
UOC Anatomia Patologica, Ospedale Clinicizzato “SS. Annunziata”,
Chieti, Italia; ** U.O. Ematologia Clinica, Ospedale Civile “S. Spirito”
di Pescara, Pescara, Italia
*
Background. Proliferation of bone marrow elements in extramedullary sites, known as extramedullary hematopoiesis
(EMH), is an infrequently finding in routine pathology practice.
Sometimes it presents differential diagnostic difficulties, especially when seen in unusual sites, with infections, lymphomas,
poorly differentiated carcinomas and granulocytic sarcomas.
Supportive information, such as clinical history, bone marrow
or peripheral blood findings, immunohistochemistry or molecular/genetic testing are required to resolve the nature of the most
challenging cases.
Here below, we report a case of EMH in a mediastinal lymph
node in a patient with primary myelofibrosis (PMF) a clonal
stem cell disorder characterized by ineffective erytropoiesis and
dysplastic megacaryiocytic hyperplasia, usually accompanied by
reactive marrow fibrosis and sometimes EMH in the spleen or,
less frequently, in other sites.
Material and methods. A 72-years old man with PMF, followed
at the hematologic department, has been treated with cytoreductive therapy (hydroxyurea) since 2008. He also harbored the
JAK2 V617F mutation, that occurs in the majority of patients
with PMF. He underwent coronary artery bypass surgery due to
ischemic cardiomyopathy; in the pre-visit he showed no changes
in laboratory exams and clinical conditions. During the surgical
operation, an enlarged mediastinal periaortic lymph node was
found and sent for histopathological evaluation.
RESULTS: Grossly, the lymph node measured cm 1.5x0.9x0.5.
The histological analysis showed large cells, sometimes multinucleated, and eosinophilic cellular elements among the normal
nodal structure. At first glance these features were sospicious
for Hodgkin Lymphoma. A careful search revealed a multi-
lineage proliferation of all precursor cell lines of myelopoietic
elements.
Immunohistochemical analysis showed megakaryocytes positive for FVIII, while CD20, CD3, CD15, CD30, CD34 and
CD 117 were negative. MIB-1 proliferation index was < 1%.
Moreover the unexpression of CD34 and CD117 excluded the
possible progression towards a blast phase or acute myeloid
leukemia.
Conclusions. The immunohistochemical findings helped to
carry out the diagnosis of EMH. In fact, in case of infections,
along with immature myeloid series, necrosis should be seen.
Anaplastic large cell lymphomas may show large dissociated
abnormal lymphoid cells but a careful search may reveal erythroid and myeloid precurson cells. Dissociated megacaryiocytes
may be confused with poorly differentiated carcinomas and
eventually with Hodgkin Lymphoma cells. Above all EMH
should be distinguished from myeloid sarcoma, especially
with the differentiated-type, that consists of promyelocytes and
more mature granulocytic forms and would most frequently be
confused with EMH.
T-cell limphoma and nucleolar positivity for CD20:
a new prognostic marker?
E. Penitente, P. Viola, S. Malatesta, A. Di Lorito, C. Marinelli, E.
Dell’Osa, G. Lattanzio
UOC Anatomia Patologica, Ospedale SS Annunziata, Chieti, Italia
Background. The Non Hodgkin Lymphomas (NHLs) are
broadly divided into T-cell and B-cell types based on their
immunophenotyping by immunohisto-chemistry or flow cytometry. The markers, CD20 and CD3, are the most widely
used for B-and T-cell lineage, respectively, but coexpression
of various B- and T-cell markers is commonly encountered in
precursor T-cell lymphoblastic lymphoma and in low-grade
B-cell lymphomas such as mantle cell lymphoma and small
lymphocytic lymphoma/chronic lymphocytic leukemia. We report a case of large T cells histiocytic rich NHL with nucleolar
positivity for CD20.
Matherials and methods. A 79 year old man came to Ear Nose
and Throat (ENT) department for chest pain and sore throat. The
physical examination and the radiological findings showed a
large cervical mass that displaced the laringo-thracheal axis.
A fine needle aspiration has been done displaying atypical
elements suggestive of medullar carcinoma. The immunohistochemical coloration with calcitonin was not significant so a
biopsy was carried out.
Resultsts and conclusions. The histological examination of
the biopsy revealed a diffuse infiltrate composed by large lymphoid cells, several histiocytes and normal small lymphocytes.
To avoid misdiagnosis, a large panel of monoclonal antibodies
has been suggested for immunophenotyping. The calcitonin
came out to be negative as well as TTF-1, ALK and CKAE1/3.
P53 and MIB1 were positive in more than 90% of the neoplastic elements.
CD3 was positive in small lymphocytes but not in the neoplastic
population (as happens in more aggressive cases), CD68 highlighted the histiocytes elements and CD20 showed a diffuse (>
90%) nucleolar positivity. The morphological and immunohistochemical data oriented for a diagnosis of large T cells rich NHL
with an aggressive behavior.
In literature the expression of CD20 in T-cell lymphoma has been
described, although is quite rare and these types of lymphoma
always pursue an aggressive clinical course with poor response
to the therapy.
In our case the nucleolar positivity for CD20 helped us to make
the diagnosis of T-cell lymphoma despite the loss of CD3
marker.
Poster
Castleman’s disease in childhood
N. Scibetta*, P. Farruggia**, P. D’Angelo**, A. Trizzino**, E.
Unti*, L. Marasà*
*
Unit of Pathology and **Unit of Pediatric Hematology and Oncology,
“Civico, G. Di Cristina and Benfratelli” Hospital, A.R.N.A.S., Palermo,
Italy
Introduction. Castleman’s disease (CD) is a massive growth of
lymphoid tissue of unknown etiology, rarely reported in childhood,
classified into two clinical subtypes: a localized and a multifocal
subtype. Localized disease presents as a solitary mass and usually
shows a benign course. Multifocal disease is accompanied by systemic symptoms, progressive clinical course and worse prognosis.
Three major histological subtypes: the most frequent hyaline-vascular (HV) variant, the plasma cell variant, generally observed in
the multifocal subtype and the mixed form, plasmablastic variant.
Surgery is the optimal therapeutic approach in the localized form,
while for unresectable or disseminated disease steroid chemotherapy and radiotherapy have been employed with variable success.
We focused pathologic characteristics of three cases of localized
HV-CD in childhood observed in the last 4 years.
Case reports. GM, female, aged 3.3 years, showed an asymptomatic left axillary mass showing a slow progressive growth. She
was in good general conditions and underwent complete surgical
resection of this lymph node of 4 cm diameter. Histological assessments defined the diagnosis of HV-CD. No relapse occurred
in 48 months.
LG, male, 3.8 years, presented an isolated palpable subcutaneous
tumefaction, 1 cm diameter, on his right blade shoulder. He was in
good physical condition and routine blood tests, abdominal ultrasound and chest X-ray were all normal. After 3 months the mass
enlarged and a complete surgical resection of two lymph nodes,
sized 2.5 and 1.2 cm was performed. Histology revealed a classic
HV-CD. No recurrence was noted after a 36-month follow up.
DA, male, aged 13 years, developed a mass in the anterior part of
the neck. He was in good condition and the remaining examination was normal. A chest CT scan showed that the neck mass,
with high contrast-enhancement, in the anterior-superior mediastinum caused a serious right trachea dislocation. Abdominal ultrasound and 99mTc-MDP total body bone scan were both normal.
Since the mass was unresectable, an open biopsy was performed;
histology defined a HV-CD.
Pathologic findings. Histologically all our cases, diagnosed as
HV-CD, showed a mass of lymphoid tissue with scattered abnormal lymphoid follicles, which ranged in size from small to large.
The germinal centers were depleted and composed predominantly
of follicular dendritic cells and endothelial cells of hyalinized
capillaries. Their appearance corresponds to that of “hyaline
vascular nodules”. Another peculiar finding was the presence
of more than one small germinal center within a single follicle.
There was a concentric layering of lymphocytes at the periphery
of the follicles with an onion–skin appearance.
The interfollicular areas showed vascular proliferation with numerous hyperplastic vessels of the postcapillary venule type with
hyaline material between vessels and absence of sinuses. A small
number of lymphocytes, plasmacells, eosinophils and immunoblasts were present.
Immunohistochemically, positive CD21 and CD35 was seen in
the large cells with vesicular nuclei (follicular dendritic cells) in
the center of follicles, strong positive FVIII-related antigen was
seen in the endothelium of the interfollicular vessels, but only
weak and focal reaction was found in the hyaline vessels located
in the center of the follicles. The immunoglobulin production by
plasmacells was polytypic and in the interfollicular areas large
numbers of suppressor T cells were found.
In summary the diagnostic features were small hyaline germinal
centers within an expanded mantle zone, as well as a highly vascularized interfollicular network.
199
Conclusions. CD, although extremely rare in childhood, since
it can display a similar presentation as more frequent different
diseases (eg Hodgkin or non Hodgkin Lymphoma), must be take
in account in differential diagnosis, thanks to various peculiar
pathologic characteristics.
T-lymphoblastic lymphoma arising from the small
intestine in a child
E. Unti*, N. Scibetta*, F. Di Marco**, A.F. Carolina***, U. Burgio****, P. D’Angelo**
*
Unit of Pathology, **Unit of Pediatric Hematology and Oncology, ***Unit
of Pediatric Surgery and ****Unit of Radiology, “Civico, G. Di Cristina
and Benfratelli” Hospitals, A.R.N.A.S., Palermo, Italy
Introduction. T-cell Lymphoblastic lymphoma (T-LBL) is
a neoplasm of lymphoblasts committed to the T-cell lineage,
typically composed of small to medium-sized blast cells and a
primary site of presentation other than peripheral blood (PB) or
bone marrow (BM). With extensive BM and PB involvement,
lymphoblastic leukemia is the appropriate term. T-LBL comprises approximately 85-90% of all lymphoblastic lymphomas.
Although T-LBL may be present at any age, it is typically seen
in older children and young adolescent males. T-LBL frequently
shows mediastinal involvement, often exhibiting rapid growth
(sometimes presenting as a respiratory emergency), and a wide
variety of other sites may be involved, including peripheral
lymph nodes, skin, tonsil, liver, spleen, central nervous system,
and testis, although presentation at these sites without nodal or
mediastinal involvement is uncommon. T-LBL is highly aggressive, but frequently curable with current therapy. We report about
a rare case of T-LBL arising from the small intestine in a child.
Case report. Clinical and laboratory data. BP, male, aged 4
years and 2 months, was referred because of persistent abdominal
pain. Physical examination was unremarkable; in particular no
lymph nodes, liver or spleen enlargement was detected.
Diagnostic assessments. Abdominal ultrasound and CT total
body scan showed a heterogenous hypodense mass having
undefined margins, involving small intestine and determining
some signs of initial occlusion; a moderate ascites was also
detected; mediastinum was not enlarged. Laboratory assessments revealed only low blood levels of proteins and albumin,
that needed substitutive therapy; evaluation of PB smears was
normal. The child underwent open surgical procedure: the
whole ileum was diffusely infiltrated by multiple hemorrhagic
nodules; resection of two ileal segments of 13 cm and 15 cm in
length was performed; bilateral BM aspirate showed a normal
picture and flow cytometry.
Pathologic findings. The specimens of the ileum were fixed in
4% formaldehyde and embedded in paraplast. Sections 4 micron
thick were stained with H&E. Intestinal wall showed extensive
widespread infiltration by neoplastic cells, medium in size, with
high nuclear-cytoplasmatic ratio, nuclei round to ovoid in shape,
and the nuclear membranes thin, but distinct. The nucleoli were
either small and single or not discernable, and the chromatin was
fine. Mitotic figures were numerous. A starry sky appearance and
a multinodular pattern were prominent focally. The tumor cells
were positive for TdT, cytoplasmatic CD3 (cyCD3), CD2, CD5,
CD4, CD7, CD10, and negative for CD20,CD79a, CD13, CD33,
CD117, CD23, EMA, CD30, CD8, CD56, BCL6, CD34, CD1a;
Ki67 was 90%.
Histologically our case was diagnosed as T-LBL and classified
into stage medullary T of intrathymic differentiation (helper phenotype), according to the antigens expressed.
Treatment. The child was enrolled in the international Euro
LB02 Protocol, for the treatment of Non-B NHL in children and
obtained a complete remission after induction therapy. Now, the
child is receiving consolidation phase of therapy, and had no serious side effects.
200
Conclusions. We describe a very unusual case of primary T-LBL
arising from the small intestine in a child. The overall picture
strongly suggest a primary intestinal origin of this T-LBL, which
contradicts the conventional wisdom that T-LBL arises in the
thymus from primitive cortical lymphocyte before rapidly disseminating.
Extramedullary plasmacytoma of the tonsil:
an histological pathogenetic hypothesis
P. Viola*, A. Di Lorito*, S. Malatesta*, L. Citraro**, A. Croce**,
G. Lattanzio*
*
UOC Anatomia Patologica, Ospedale SS Annunziata, Chieti, Italia; **Clinica Otorinolaringoiatrica, Università “G. d’Annunzio” Chieti, Italia
Background. Extramedullary plasmacytomas (EMP) are an
immunoproliferative monoclonal disease of the B-cell line that
arises outside the bone marrow without evidence of existing
multiple myeloma. They are rare tumours which usually occur
in the upper aero-digestive tract. Although the most common
sites are sinonasal/nasopharyngeal areas, it is rare for them to
originate from the tonsil. In literature cases have been reported
in patients such farmers or people exposed to inhale irritants
for long time: the predisposition of this pathology for the sub
mucosa of the upper respiratory tract has led to the hypothesis that chronic stimulation may promote the development of
plasmacytoma. Also Interleukin-6 deregulated production has
been implicated in the pathogenesis of several diseases including autoimmune disorders and plasma cell dyscrasias. IL-6 is
an essential factor for the in vitro growth of plasmacytoma
cells and some authors have described its primary role in the
development of B-cell neoplasias also in mice. We report a
case of EMP arising in the tonsil with a coexisting actinomyces
infection.
Materials and methods. A 79 years old woman came to otorhinolaryngologist for ear pain and no other symptoms. During the
visit, a mild enlargement of her left palatine tonsil and small
white area has been noted by the clinician. No palpabile limp
nodes were present. A biopsy of the suspicious area has been
performed for further investigations.
Results. Histopathological analysis revealed a diffuse infiltration
of plasma cells beneath the surface epithelium. Plasma cells had
a prominent eccentric nucleus with a “spoke wheel” chromatin and abundant basophilic cytoplasm. Immunohistochemical
study showed positivity for both CD138, light chain λ and CD
56 (marker that identifies atypical plasma cells); negativity for
CD20, CD3, CD15, CD30, CD68 and light chain κ. Proliferation
index assessed by MIB-1 was < 1%.
The diagnosis of EMP was carried out and four months later
the patient underwent surgical removal of the palatine tonsil.
Grossly the specimen was brown-yellowish, measured cm 2,
6x1, 6x0, 7 and weighted gr.3. The cutting surface showed a
brownish area within the parenchyma with a maximum diameter
of cm 0, 8. Microscopically within an iperplastic tonsillary tissue, we noted groups of actinomyces inside the crypts (sign of
a chronically infection) with an inflammatory reactions. Along
with these finding, the plasmacitoma previously diagnosed was
still present.
Conclusions. To our knowledge this could be the first histological example of the effect of the chronically stimulation on immune system in the development of plasma cells disorder.
We postulate that the coexistence of chronically actynomices
infection may have led to the development of plasmacitoma in an
old patient throughout the secretion of the inflammatory mediators. Further analysis will be required to confirm the exact role of
IL-6 in this particular case.
ALK-positive anaplastic large cell lymphoma
with PAX-5 expression: report of a case
A. Zamò, E. Munari, A. Bertolaso, M. Bonifacio, M. Chilosi
Dipartimento di Patologia e Diagnostica, Sezione di Anatomia Patologica, Università di Verona
ALK-positive anaplastic large cell lymphoma (ALCL) is a welldefined entity included in the 2008 WHO classification, comprising about 5% of non-Hodgkin lymphomas and 10 to 30% of
childhood lymphomas. Although lacking several T-cell antigens,
it is generally considered a T-cell derived neoplasm. Since both
ALCL and Hodgkin lymphoma (HL) strongly express CD30, and
they can show overlapping morphological features, the B-cell
specific marker PAX5 is widely used for distinguishing the two
entities. PAX5 is considered the most specific B-cell marker, and
is defined as “the guardian of B-cell identity”.
We report an exceptional case of ALK-positive ALCL with PAX5
expression. A 38-years old patient presented with fever and generalized lymphadenopathy lasting for two months at the time of
biopsy. A basic cytofluorimetric analysis was performed, showing
the presence of a large cell population with dim CD5 and CD4 expression. Histopathological evaluation revealed an effaced lymph
node architecture, due to the presence of a neoplastic infiltration
by medium-sized cells in wide aggregates; occasionally larger cells
with “hallmark” morphology were present; intrasinusoidal involvement was evident. These atypical cells showed a strong staining
for CD30, as well as perforin, while granzyme B was only focally
expressed. Other T-cell markers (CD2, CD3, CD5, CD7, ZAP70)
and B-cell markers (CD20, CD79a) were negative, with the notable exception of PAX5, which showed a dim but specific staining,
similar to that observed in HL. ALK was diffusely expressed in
neoplastic cells, with a predominantly cytoplasmic granular staining. Double chromogenic stains and immunofluorescence were
also performed to confirm the findings. ALK-positive diffuse large
B-cell lymphoma was ruled out because this case didn’t show the
typical immunoblastic/plasmablastic morphology, didn’t express
plasma cell markers and was diffusely CD30-positive. The patient
was unfortunately lost at follow-up.
Four cases of PAX5-positive ALCL were recently described, of
which only one was ALK-positive, diagnosed on a vertebral lesion. A previous study had identified three PAX5-positive ALKnegative ALCL, but no ALK-positive cases. To our knowledge,
this case is the first description of lymph node involvement by
PAX5-positive and ALK-positive ALCL. Our data show that the
morphological presentation is totally overlapping with classical
ALCL; we also confirm that PAX5 can be rarely expressed in
ALCL and should not be taken as a final proof of the B-cell origin
of a neoplastic population.
Patologia pleuropolmonare
Mucinous Cystic Carcinoma of the lung with
divergent histology and imunophenotype shows
distinct molecular alterations: a case report
C. Bellevicine*, U. Malapelle*, P. Colonna**, M. Mangiapia**, G.
Troncone*, N. De Rosa**
Dipartimento di Scienze Biomorfologiche e Funzionali, Università Federico II, Naples, Italy; **AORN Vincenzo Monaldi, Naples, Italy
*
Background. Mucinous Cystic Carcinomas (MCC) of the lung
(so-called colloid carcinoma) is “a cystic adenocarcinoma with
copious mucin production” 1 and represents an histological
variant of lung adenocarcinoma with peculiar clinicopathological
features. Here we present a case of MCC with two components
whose histology, immunophenotype and molecular features diverged.
201
Poster
Case. Grossly, a well-circumscribed, multilocular mucoid cystic
mass was evident. Histological features were heterogeneous;
mucin lakes, lined by cuboidal to columnar cells, showing basal
nuclei and apical mucin, were associated to more solid glandular
areas lacking mucin; these latter immunostained in a “lung manner“ (CDX2-, TTF1+), while mucinous areas featured CDX2+
and TTF1-. Selective DNA extraction by laser capture microdissection showed mutually exclusive EGFR (solid areas) and
KRAS (mucinous areas) mutations.
Discussion. Mucin producing adenocarcinomas represent a distinct
CDX2 positive subtype with high rates of KRAS mutation 2. Here
we report a case of MCC whose invasive component lacked
mucus and showed divergent immunohistochemical and molecular phenotype. Further investigation are needed to uncover the
origins of this unusual lung adenocarcinoma variant, as either
divergent differentiation from a bipotential progenitor cell (e.g.
Clara cells) or an unusual collision tumor derived from two different progenitors could be taken into account.
References
1
Higashiyama M, Doi O, Kodama K, et al. Cystic mucinous adenocarcinoma of the lung. Two cases of cystic variant of mucus-producing
lung adenocarcinoma. Chest 1992;101:763-6.
2
Finberg KE, Sequist LV, Joshi VA, et al. Mucinous differentiation
correlates with absence of EGFR mutation and presence of KRAS
mutation in lung adenocarcinomas with bronchioloalveolar features.
J Mol Diagn. 2007;9:320-6.
The choice of using core biopsy under ct guidance
in peripheral lung masses for best application of a
diagnostic algorithm to determinate histotypes
D. Bellis*, G.C. Sarnelli**, M. Icardi***, G.C. Abbona*, L. Viberti *
ASLTO1, Ospedale Martini, Servizio di Anatomia Patologica, Torino;
ASLTO1, Ospedale Martini, Servizio di Radiologia, Torino; ***ASLTO1
Ospedale Martini, Servizio di Oncologia, Torino
*
**
The aim of this work is to stress the utility of a multidisciplinary
approach to afford the necessity of typing lung cancer before any
proposed therapy. The importance of distinguishing histological
types of lung cancer is predominantly dichotomous, based on the
distinction between small cell carcinoma and others and derived
from events subsequent to the election of surgery and/or medical
therapies. Recently, the distinction in the category of “non-small
cell carcinomas” in adenocarcinoma or squamous cell carcinoma
plays an important role in the choice of new target therapies.
Hence the need of a diagnostic immunohistochemical algorithm utilizing squamous differentiation markers (high molecular
weight cytokeratin CK5 / 6 and p63) and glandular differentiation markers (TTF1-1, cytokeratin 7, napsin-A) with a impact of
specificity in the diagnosis and positive effects on the response
to chemotherapy, resulting in better quality of life and improved
survival 1. Multidisciplinary team with oncologists, pathologists,
radiologists and thoracic surgeons allow to determine better the
correct diagnostic approach and the chose between fine needle
aspiration and core biopsy. In our experience, the pre-operatory
choice of using in some cases, core biopsy under CT guidance
with coaxial cutting system for peripheral lung mass allows easier
use of immune-histochemical markers. In comparison to the inclusion of a fine needle aspiration cytology a core biopsy improves the chances to obtain a specific morphology and a greater
number of sections in paraffin. A coaxial technique is used, with
needle-guide included and with razor sharpened cannula and trocar bevel point. The needle is clearly visible in its whole length.
This way it is possible to do 2-3 biopsy samples with only one
passage through the pleura. Adequacy of specimens is assessed
on-site by a cytotechnologist and the clinica-radiological date,
size of the nodules, number of specimens, complications encountered are recorded.
Fifteen patients with lung nodules underwent CT-guided tran-
sthoracic coaxial cutting-needle biopsy (18-20 G) (CNB) in
the first half of 2011. Findings were diagnostic in 100%. Cytopathologic evaluation of samples was immediate in all patients.
Follow up included chest CT (2/16 pneumothorax without the
tube insertion). Core biopsy reveals three small cell carcinomas, 1 metastatic tumor, a primary pulmonary lymphoma and
9 non-small cell carcinomas (2 squamous cell carcinomas and
7 adenocarcinomas). Two of these cases were not amenable to
surgical and the oncologist requested the determination of EGFR
Mutation Test (Epidermal Growth Factor Receptor), with negative results. This will, if positive, markedly reduce waiting time
for those patients, who most likely will have the greatest benefit
from EGFR TKI therapy.
This preliminary results shows high reliability and high rate of diagnostic performance, increasing the diagnostic success rate and
has been a reliable means of differentiating benign and malignant
pleura-pulmonary lesions.
Reference
1
Travis WD, Brambilla E, Noguchi M, et al. International Association
for the study of Lung Cancer/American Thoracic Society/European
Respiratory Society International Multidisciplinary Classification of
Lung Adenocarcinoma. J Thoracic Oncology 2011; 6:244-85.
9p21 deletion in the diagnosis of malignant pleural
mesothelioma
T. Bensi, N.F. Trincheri, R. Libener, S. Orecchia, M. Salvio, P.
Barbieri, N. Mariani, P. Re, P.G. Betta
Pathology Unit A.O. Alessandria
Malignant Pleural Mesothelioma (MPM) is an aggressive neoplasm of the serosal cavities that usually portends a dismal shortterm prognosis. Definitive pathological diagnosis of MPM is
often difficult based on morphology alone.
Deletion of 9p21 locus, the locus harbouring the cell cycle
control-associated gene p16/CDKN2A, has long been known as
a common cytogenetic alteration in MPM and is also a negative
prognostic factor in surgically treated patients.
Fluorescent in-situ hybridization (FISH) to detect deletion has
recently been proposed as an ancillary test to differentiate benign
from malignant mesothelial proliferation both in histo- and cytopathologycal settings.
The aim of the present study was to further evaluate the purpoted
diagnostic contribution of 9p21 status assessed by FISH in 11
consecutive cases of primary pleural lesions, consisting of 7
MPM and 4 reactive mesothelial hyperplasias respectively.
FISH assay was carried on paraffin section from tissue biopsy
or cell-block effusion specimens. The kit (Vysis CKN2A/CEP9
FISH Probe Kit – Abbott Molecular) was used according to the
manufacturer’s instructions.
All MPMs exhibited at least 15% malignant cells harbouring
heterozygous or omozygous deletions of 9p21 locus, whereas no
deletion was found in 4 reactive cases. Half of the omozygously
deleted cases included concurrent eterozygous pattern.
FISH assay for p16/CDKN2A appears to be valuable test to confirm a probable diagnosis of MPM, especially when dealing only
with pleural effusion and/or small tissue specimens from needle
biopsies.
FISH assay is expected to allow a more timely diagnosis and to
spare suspected MPM patients more invasive diagnostic procedures.
(T.B. and N.F.T. are scholarship holders of the Italian League
against Cancer, Alessandria section).
202
Incidence of o6-methylguanine DNA
methyltransferase (MGMT) expression in lung
neuroendocrine cancer
M. Carosi*, R. Baldelli**, A. Francesconi*, P. Visca*, R. Covello*,
A. Faggiano****, A. Barnabei**, A. Colao****, M. Appetecchia**, F.
Facciolo***, E. Pescarmona*
*
Pathology, **Endocrinology, ***Thoracic Surgery, Regina Elena National
Cancer Institute of Rome; ****Department of Molecular and Clinical Endocrinology and Oncology, “Federico II” University of Naples, Italy
Neuroendocrine carcinomas (NECs) represent relatively rare and
heterogeneous malignancies. In most cases they are advanced at
diagnosis and slow-growing, therefore conditioning a better prognosis compared with non neuroendocrine carcinomas from the
same sites. Several systemic therapeutic options exist, including
chemotherapy, somatostatin analogs, interferon, peptide receptor
radionuclide therapy (PRRT) and molecular targeted drugs. New
somatostatin analogs, covering a higher number of SSTR subtypes, were developed, including pasireotide (SOM230), which
controls 25% of carcinoid syndromes resistant to full dose of somatostatin analogs. Chimeric analogs, which bind SSTR2/SSTR5
and dopamine-2 receptor subtype (D2), are in preclinical phase
of development. Among the numerous molecular targeted agents
investigated in NETs, mTOR inhibitors and VEGF/VEGFR/
PDGFR inhibitors are in most advanced clinical phase of investigation. In particular, everolimus, sunitinib, and bevacizumab
are all studied in phase III trials. Both everolimus and sunitinib
produced significant survival benefit versus placebo in advanced
progressing well-differentiated pancreatic NECs. In recent years,
the oral alkylating agent temozolomide (TMZ) has emerged as
an active agent in PNETs. Like dacarbazine, TMZ is converted
to the active alkylator MTIC that induces DNA methylation at
the O6 position of guanine. A phase II study investigating the
combination of TMZ and thalidomide demonstrated an objective
response rate of 45% in the PNET subset of patients. A recent
retrospective study of TMZ combined with capecitabine in 30
chemo-naive PNET patients reported an objective radiographic
response rate of 70% and median progression free survival of
18 months. Side effects were relatively tolerable, with a grade
3/4 adverse event rate of only 12%. The aim of this study was to
evaluate the expression of MGMT methylation in neuroendocrine
tumors of the lung (NET-lung); the method to determine the
hypermethylation status of MGMT, namely methylation-specific
PCR, allowing the selection of patients most likely to benefit from
TMZ treatment. Paraffin-embedded formalin-fixed tissue was obtained by surgical resection of NET-lung from 7 patients provided
by the Department of Thoracic Surgery of the “Regina Elena”
National Cancer Institute of Rome. DNA was isolated from the
paraffin-embedded tissue macrodissected from histologically
marked slides and fixed in 10% Formalin or non-crosslinking
fixatives. DNA was made accessible by protein digestion: DNA
was solubilized, while digested proteins were “salted out” and
spun to the bottom of the tube. DNA was precipitated, dried under
vacuum, and resuspended. DNA was subjected to bisulfite treatment. Unmethylated cytosine, but not its methylated counterpart,
was modified into uracil by the treatment. Purified DNA was
subjected to PCR amplification with specific primers designated
to distinguish methylated from unmethylated DNA. The PCR
products were separated on 3% agarose gels using DNA marker
of 100 bp and visualized by UV transilluminator. Previous results
seem to indicate that the MGMT methylation of the promoter is
present in 4 of 7 samples evaluated (57%). Considering the significant effect and the few adverse effects, there might be a wider
indication for TMZ treatment of aggressive NET-lung. However,
more data are necessary to decide whether MGMT methylation
should be used as a surrogate marker for predicting tumour TMZ
sensitivity.
Primary abdominal mesothelioma infiltranting
the spleen: case report
D. Di Clemente, G. arborea, T. Montrone, F. Gaudio, E. Maiorano, G. Ingravallo, A. Cimmino, R. Ricco
dipartimento di Anatomia e Istologia Patologica/Università degli Studi di
Bari “Aldo Moro”, Bari, Italia.
The spleen may be more frequently affected by hematopoietic
malignancies (malignant lymphoma and lymphoproliferative disorders), but also by vascular tumors (hemangiomas, lymphangiomas, littoral cell angiomas, angiosarcomas and other sarcomas)
and by follicular dendritic and interdigitated cells tumors. Splenic
localizations of other malignancies (e.g., malignant melanoma
and carcinoma of the lung, breast, stomach, liver, colon, pancreas
and ovary) are rare and unlikely to yield clinical manifestations
(splenomegaly or rupture).
We report on the case of a 69 y.o. man, a former railway worker,
who was admitted to surgery for severe anemia and abdominal
pain of unknown cause, in whom CT had highlighted severe architectural distorsion, with preservation of the the upper pole, due
to the presence of multiple hypodense areas of purported neoplastic origin. Following splenectomy, a 15x9x4 cm. mass, weighting
gr. 800, anf tightly connected with the spleen was sent for pathological examination. Grossly, the tumor showed a compact cut
surface, with alternating yellowish and grey areas. Histologically
the neoplasm was composed of sho frankly epithelioid cells, with
intensely eosinophilic cytoplasm, intermingled with malignantlooking spindle cells, inflammatory cells and extensive necrosis.
The neoplastic cells were immunoreactive for cytokeratins, vimentin, mesothelioma antigen HBME and calretinin but negative
for S-100 protein, HMB 45 and CD 117. MIB1-immunoreactivity
was detected in 10% of the neoplastic nuclei.
Based on the above features, the diagnosis of abdominal, bifasic
malignant mesothelioma invading the spleen was rendered.
The above clinico-pathological features of the case highlight the
unusual presentation of an uncommon neoplasm at a rare site:
the patient manifested non-specific symptoms and, based on the
tight connection of the neoplasm with the spleen, GIST, sarcomas and follicular dendritic and interdigitated cells tumors were
considered in the differential diagnosis. Immunohistochemistry is
mandatory in such cases and allowed the adequate characterization of a biphasic malignant mesothelioma involving the spleen,
a very uncommon presentation for such entity.
EGFR and KRAS mutation testings is feasible
on lung cancer liquid based cytology: a pilot study
U. Malapelle, C. Bellevicine, C. De Luca, M. Salatiello, S.
Russo, G. Troncone
Background. Epidermal growth factor receptor (EGFR) is a major therapeutic target in lung non small cell carcinomas (NSCLC).
EGFR and KRAS mutations respectively induce sensibility and
resistance to EGFR antagonists. In advanced
NSCLC gene testing is often performed on cytology. Cytopathologist’s on-site evaluation of the harvested material is not always
possible. Thus, liquid-based cytology (LBC), that eliminates the
need for slide preparation by clinicians,
may be very useful.
Methods. The study was addressed on the LBCs referred to our
laboratory for EGFR and KRAS mutation analysis. Forty-two
cases were selected. In each single case DNA was extracted
twice. One sample was obtained directly from CitoLyt solution,
whereas the other DNA sample was derived after smear preparation and laser capture microdissection (LCM) of Papanicolaou
stained cells. EGFR and KRAS mutational analyses were performed by direct sequencing.
Poster
Results. On CitoLyt derived DNA four EGFR (9%) and five
KRAS (12%) gene mutations were found. When direct sequencing was performed after LCM, the rate of cases that displayed
either EGFR or KRAS mutations increased from 21% to 40%.
Overall, EGFR and KRAS gene mutations respectively occurred
in 19% and in 21% of LBCs. EGFR and KRAS mutations were
reliably detected by microdissecting as few as 25 cells. This
thereshold was also confirmed in experiments on LBCs prepared
from NSCLC cell lines.
Conclusions. Although time-consuming, LCM makes direct sequencing highly sensitive even on a LBC preparations containing
only a few cells. This study data provide useful benchmarks for
routine EGFR and KRAS analyses on LBCs.
Histological pattern and grading of lung tumors
carrying braf mutations
S. Malatesta1, P. Viola2, L. Felicioni3, M.G. Sciarrotta1, A. Chella4, L. Guetti5, F. Mucilli5, F. Buttitta3, A. Marchetti1
Centro di Medicina Molecolare Predittiva, Fondazione-Università “G.
d’Annunzio”Università di Chieti-Pescara, Chieti, Italia; 2Unità Operativa
di Anatomia Patologica, Ospedale Clinicizzato di Chieti, Chieti; 3Medicina Molecolare Oncologica e Cardiovascolare, Fondazione-Università “G.
d’Annunzio”Università di Chieti-Pescara, Chieti, Italia; 4Dipartimento di
Chirurgia, Università di Pisa, Pisa, Italia; 5Dipartimento di Chirurgia,
Università “G. d’Annunzio”Università di Chieti-Pescara, Chieti, Italia
1
Background. BRAF is the kinase most frequently affected by
somatic mutations in human tumors. However, the reported
frequency of these mutations in lung cancer is very low (1-3%)
and little is known about the histopathological characteristics of
tumors harboring BRAF mutations. We recently assessed the mutational status of BRAF in a large series of 1046 non-small-cell
lung cancers (NSCLC) and found that BRAF mutations are most
likely to occur in adenocarcinomas (ADCs), with a frequency of
4.9%. Two main type of BRAF mutations have been reported in
lung carcinomas: BRAF V600E and non-V600E mutations. The
former is a hotspot mutation frequently found in other malignancies such as melanomas and thyroid tumors. Pushed by the recent
development of anti-BRAF therapeutic strategies, we decided to
evaluate the histopathological characteristics of ADC harboring
BRAF mutations for a more accurate selection of patients.
Matherial and methods. Seventeen ADCs carrying BRAF mutations, including 10 ADCs with V600E mutation and 7 ADCs
with non-V600E mutation, were revised histologically according
to the new International Multidisciplinary Classification of Lung
ADC (IMCLA). In addition, the recently developed 3-tier grading system of ADC based on the two major histologic subtypes
proposed by Sica et al. was applied to grade the tumors. Tumors
were classified as Grade I, corresponding to in situ ADC, Grade
II corresponding to acinar and papillary patterns and Grade III
corresponding to micropapillary, solid, and variants such as
cribriform, ragged-anastomosing glands, and dispersed intraalveolar tumor cells.
Results. According to the Sica scoring system, 8 out of 10 (80%)
of the tumors with V600E mutations were histopatologically
classified as infiltrating lung ADCs with a predominant (50% of
cases) or secondary (30% of cases) micropapillary component
and thereafter were included in the high-grade tier (grade III). On
the other hand, only 1 out of 7 (14,3%) ADCs with non-V600E
mutation showed micropapillary pattern; these tumors were
mainly classified as low or intermediate grade lesions, according
to the Sica scoring system.
Conclusions. Lung ADCs harboring V600E BRAF mutation are
high grade tumors, characterized by micropapillary features and
potential aggressive behavior. The identification of this pattern
of growth on histological sections could be useful to select those
patients more likely to carry this molecular change.
203
Churg-strauss syndrome: the value of skin biopsy.
A case report
S. Marasà*, A. Valentino*, E. Orlando*, T. Bellavia**, G. Costanza**, G. Andronico**, G. Cerasola**
*
Department of Human Pathology and **Internal Medicine, Policlinico Paolo Giaccone, University of Palermo, Italy
Introduction. Churg-Strauss syndrome (CSS) is a small-vessel
vasculitis characterized by asthma, hypereosinophilia, pulmonary
infiltrates. CSS can be diagnosed by the presence of four or more
of the six criteria(American College of Rheumatology,1990),
which include asthma, eosinophilia greater than 10%, paranasal
sinutis, pulmonary infiltration, histological proof of vasculitis
and mono- or poly-neuropathy. We report here on a case of 34year-old female who developed papular erythematous rash on
the extensor surface of the arms, particularly the left elbow and
hands. Concomitantly with the onset of cutaneous lesions she
was affected by asthmatic bronchitis, night fever, dry cough, leg
weakness and arthralgias.
Case report. A 34-years old female was admitted for malaise,
lassitude, night fever, dry cough, leg weakness and arthralgias, a
recent weight loss of 3 kg and skin lesions on the arms. She was
affected by bronchial asthma, for five years, poorly controlled
by usual therapy. A plain X-rays of the chest, performed in the
June 2009, showed a bilateral thickening of broncho-vascular
pattern in middle and lower lobes. She was underweight, with
high temperature, tachycardia, cervical lymphadenopathy, muscle hypotonia with hypotrophy and skin lesions that appeared as
papular erythematous rash on the exstensor surface of the arms,
particularly the left elbow and hands.
The laboratory tests showed eosinophilia, increased level of ESR,
CRP and D-Dimero, mild hypocromic microcytic anemia, hypergammaglobulinemia with an eleveted IgG level. The patient was
positive for p-ANCA, negative for c-ANCA and ANA. IgE, IgM,
IgA and C3 values were within the normal range, whilst C4 was
increased. Kidney function and urine microscopic examination
were within the normal range. The search in serum and blood cultures for bacterial and virus infections were negative. The chest
CT showed diffuse parenchimal consolidation and ground glass
opacities, mainly in the upper and lower right lobes, and diffuse
mediastinal adenopathy. A bronchoscopy with BAL showed aspects of non-specific marked inflammation. To avoid pulmonary
and renal biopsy, it was performed skin biopsy from one of the
hand lesions.
Discussion. We want to emphasize that skin biopsy is important
to establish an exact diagnosis and exclude other diseases. In fact,
the skin is the most easily accessible site for obtaining histological samples, thus making easier an early diagnosis. Cutaneous involvement is common, it is frequently observed in about 40-70%
of CSS patients.
The histological findings are very characteristic: there are one or
more extravascular palisading granulomas with an eosinophilic
core of necrosis with fibrinoid collagen degeneration surrounded
by histiocytic reaction, neutrophil infiltration and leukocytoclastic debris. In our case, the skin lesions, initially, were not
considered by the clinicians, but they were proved to be essential
for diagnosis of CSS.
References
1
Davis MD, Daoud MS, McEvoy MT, et al. Cutaneous manifestations
of Churg-Strauss syndrome: a clinicopathologic correlation. J Am
Acad Dermatol 1997;37:199-203.
2
Churg A. Recent advances in the diagnosis of Churg-Strauss syndrome. Mod Pathol 2001;14:1284-93.
204
A rare case of solitary peripheral mixed papilloma
of the lung
A. Nottegar*, E. Gilioli**, A. Eccher**, M. Brunelli*, E. Brunello*,
D. Segala*, G. Martignoni*, M. Chilosi*, A. Iannucci **
Italia; **Istituto di Anatomia Patologica, Ospedale di Verona, Verona, Italia.
*
Introduction. Solitary peripheral papillomas of the lung are
rare neoplasms, which arise from the bronchial epithelium. The
majority of the cases reported in Literature are squamous cell
papillomas, but glandular and mixed papillomas are also described. Exceptionally, pulmonary papillomas are situated in the
bronchioloalveolar district.
In this paper a rare case of solitary peripheral mixed (glandular
and squamous cell) papilloma is reported.
Case report. A 72-year-old woman underwent chest radiography
during a health check. A nodular lesion of 1.5 cm in size was
noticed in the left lower lobe. She was completely asymptomatic.
In her clinical history only a intestinal familiar poliposis was remarkable. In the suspect of a malignant disease, a video-assisted
thoracoscopic lung biopsy was performed. Three fragments of the
lesion were examined with intraoperative frozen section. Grossly,
the fragments appeared whitish and irregular. The tumour was
mainly located in the respiratory bronchiole with extension along
the alveolar walls. It consisted of squamous cells admixed with a
papillary proliferation made of ciliated cells, goblet cells and basal cells without infiltrative pattern of growth. Nuclear atypia and
mitoses were not evident. Based on this report, a mass excision
without lobectomy was performed. Histological exam confirmed
the frozen section findings.
Immunohistochemically, the lesion expressed cytokeratin 7
strongly and p63 and cytokeratin 5 were positive in the squamous
cells component. Also basal cells expressed p63.The glandular
part was immunoreactive for MUC-1 and MUC-5AC. The Ki-67
labeling index was low (2-3%). Cytokeratin 20, CDX2 and TTF-1
immunostainigs were negative.
A diagnosis of solitary peripheral mixed-type papilloma was
posed.
Discussion. Pulmonary papilloma is a rare condition that affects
mostly adults. It can be classified as squamous cells, glandular
or mixed squamous cells and glandular, when the latter exceeds
30% of the lesion. It usually presents as a solitary lesion. Multiple papillomas (a condition called lung papillomatosis) are more
common and related to HPV infection.
The majority of lung papillomas are central endobronchial masses
and only few of the reported cases are located in the bronchioloalveolar region. Central papillomas are often symptomatic
and cause hemopthysis, while peripheral papillomas are usually
discovered incidentally with chest-radiography.
Histologically, pulmonary papilloma could mimic a well differentiated adenocarcinoma. However, papilloma grows along
alveolar walls and lacks infiltrative pattern. The presence of
ciliated cells and basal cells is usefull to distinguish glandular
papillomas from well differentiated adenocarcinomas. In our
case, in addition to these benign characteristics, a combination
of squamous and glandular cells without atypia was helpful to
exclude an adenocarcinoma.
Expression of thrombomodulin, calretinin,
cytokeratin 5/6, D2-40 and WT-1 in primary lung
carcinomas of different types
M. Rotellini, L. Messerini, L. Novelli, C. Caporalini, C.E.
Comin
Patologica, Università degli Studi di Firenze, Firenze, Italia.
Introduction. Several immunohistochemical markers have proven to be valuable in the positive identification of mesothelioma.
Most investigations on this topic have concentrated mainly on the
differential diagnosis between epithelioid pleural mesothelioma
and lung adenocarcinoma. Within positive mesothelial markers, thrombomodulin (TM), calretinin, cytokeratin 5/6 (CK 5/6),
Wilm’s tumor 1 (WT-1) protein and D2-40 monoclonal antibody
seem to have high sensitivity and/or specificity in the positive
diagnosis of epithelioid mesothelioma. However, data on the
expression of these markers in tumours other than lung adenocarcinoma are very few. The purpose of the present study was
to evaluate the expression of the above five mesothelial positive
markers in a series of primary epithelial lung tumors.
Materials and methods. The study group consisted of 171 cases
of primary pulmonary carcinomas including, 55 squamous cell
carcinomas (SCC), 71 adenocarcinomas (ADC), 17 small cell
lung carcinomas (SCLC), 4 large cell carcinomas (LCC), 4 large
cell neuroendocrine carcinomas (LCNEC), 10 sarcomatoid carcinomas (SC) and 10 typical carcinoid tumors (CT). Seventy-five
unequivocal epithelioid pleural mesotheliomas were studied for
comparison. All cases were immunostained for TM, calretinin,
CK5/6, D2-40 and WT-1. The pattern of staining was recorded
and the immunostaining was graded on a scale of 1+ to 4+ according to the percentage of reactive cells (1+, 1%-25%; 2+,
26%-50%; 3+, 51%-75%; 4+, 76%-100%).
Results. Thrombomodulin (TM): SCC showed a highest percentage (71%) of TM-positive cases and a high score of immunoreactivity. Only 3 (4%) cases of ADC showed focal TM
immunostaining. TM was found to be focally positive in 2 (12%)
SCLC and in 1 (25%) LCNEC. Within LCC 1 case showed focal
immunostaining and 1 case was scored 4+. One SC was TMpositive with moderately diffuse (3+) immunostaining. CT were
all TM-negative. TM immunostained 58 (77%) mesotheliomas
showing heterogenous grading of reactivity. Calretinin: SCC
showed calretinin immunoreactivity in 11 (20%) cases. Focal
positivity was found in 2 ADC. The highest frequency (41%) of
positivity was observed in SCLC. Focal calretinin immunostaining was found in 1 LCC. Strong and diffuse immunostaining was
observed in 1 SC. CT were all negative. Strong and diffuse calretinin immunostainig was seen in all mesothelioma cases. Cytokeratin 5/6 (CK5/6): Most (85%) SCC were CK5/6-positive. Focal
(<5%) CK5/6 positivity was found in 3 (4%) ADC. LCC and SC
showed CK5/6-positive staining in 2 and 4 cases respectively.
No CK5/6 immunoreactivity was found in SCLC, LCNEC and
CT. Within mesotheliomas, 93% were CK5/6-immunoreactive.
D2-40: SCC showed D2-40 immunoreactivity in 23 (42%) of the
cases, whereas, only 2 (3%) ADC were D2-40-positive. No positivity was found in the remaining histologic subtypes. Concerning mesotheliomas, 93% were D2-40-positive. WT-1: WT-1 was
negative in all our study cases except for 1 SCC which showed
few tumour nests with WT-1 nuclear-positivity. Sixty-seven
(89%) mesotheliomas were WT-1-positive.
Conclusions. These results indicate that each of the most commonly used positive mesothelial markers reacts with different
subtypes of lung carcinomas with a variable frequence and
variable grading of immunoreactivity; this should be considered
when using these markers in the differential diagnosis of thoracic
tumours especially when dealing with small biopsy fragments.
Poster
Primary rhabdomyosarcoma of the lung following
radiation treatment for breast cancer: report of a
case
M. Rotellini*, L. Messerini*, D. Floridi**, L. Novelli*, F. Castiglione*, C. Caporalini*, G. Tancredi***, C.E. Comin*
Patologica, Università degli Studi di Firenze, Firenze, Italia; **Anatomia
Patologica Ospedale S. Donato, Arezzo, Italia; ***Unità di Chirurgia Toracica, Azienda Ospedaliera Universitaria Careggi, Firenze, Italia
*
Introduction. Rhabdomyosarcomas in adults are rare neoplasms
that most commonly present in the soft tissues. However, such
tumours may arise in any location, even where striated muscle is
not normally present. In fact, rhabdomyosarcomas have been reported in various sites such as the kidney, the urinary bladder, the
central nervous system, the ovary, and the anterior mediastinum.
Rhabdomyosarcomas arising in the lung are exceedingly rare
entities. We describe a case of pulmonary rhabdomyosarcoma in
a patient who received radiotherapy for breast carcinoma.
Case report. A 68-year-old woman was referred to the thoracic
division of our hospital with a history of progressive dyspnoea and
chest pain. The patient reported a past medical history of left breast
cancer treated with radiotherapy 4 years before (25 doses of 200
cGy each). A computed tomography scan showed an inhomogeneous 11-cm left-sided upper lobe lesion involving the mediastinum.
Multiple small nodules in the left lower lobe were described. Left
basal pleural effusion and pericardial effusion were also seen. Open
mediastinal biopsies were performed. Biopsy specimens were
processed for histological evaluation. Microscopic examination revealed a proliferation of bizarre atypical cells with large, pleomorphic, and irregular hyperchromatic nuclei surrounded by abundant,
deeply eosinophilic cytoplasm. Necrosis and abnormal mitotic
figures were observed. Immunohistochemical studies revealed cytoplasmic staining with vimentin, actin (HHF-35), smooth muscle
actin (1A4 clone) and nuclear staining with myogenin. Stains for
broad-spectrum keratin, CAM 5.2, EMA and S-100 protein were
negative. Since the disease was considered inoperable, the patient
underwent only one cycle of chemiotherapy with epirubicin, ifosfamide and MESNA; she died four months after diagnosis.
Discussion. Sarcomas are a rare, but recognized, complication
of radiotherapy for breast carcinoma. The diagnosis of radiationinduced sarcomas (RIS) is based on established criteria which include: 1) history of radiotherapy, 2) asymptomatic latency period
of several years, 3) occurrence of sarcoma within a previously
irradiated field, and 4) histologic confirmation of the sarcomatous
nature of the post-irradiation lesion. Our case fulfilled the above
criteria. For breast irradiation, the latency period has been reported to be from 2 to 50 years and it has been shown that the risk
increases with time. The most frequent locations of RIS for breast
irradiation are soft tissues and bones and the most frequently reported histotype is angiosarcoma. RIS tend to be diagnosed at an
advanced stage, which might explain the poor overall prognosis.
The treatment for most patients is late and ineffective. To the best
of our knowledge, only one previous case of radiation-induced
rhabdomyosarcoma of the lung after radiotherapy for breast carcinoma has been previously reported in the English literature.
Patologia fetoplacentare
Differential diagnosis of hydropic pregnancies in
first trimester miscarriages
V.R.L. Beltrami, G. Girardi, G.F. Zuccotti, G. Botta, M. Ribotta
Ospedaliera “O.I.R.M.-Sant’Anna”, Torino, Italia.
Histological diagnosis of hydropic pregnancies is very difficult
205
because of the overlap of histomorphologic features, especially
if they are detected in earlier gestation. Differential diagnosis
includes hydropic miscarriage, partial and complete moles.
Hydropic miscarriage is caused by early unknown elements; it is
diploid or biparental near diploid. Usually shows less voluminous
villi than mole, with rare and small cisterns; villi are edematous
and avascular with trophoblastic hypoplasia.
Hydatiform mole is the more frequent trophoblastic gestational
disease noticed in spontaneous abortions in first trimester of
pregnancy; it is clinically classified as gestational trophoblastic
disease according to clinical persistent risk and neoplastic degeneration; it is caused by conception defaults and it is divided in
partial and complete mole. Partial mole is triploid while complete
mole is uniparental diploid. Histological analysis is the main
diagnostic method for differential diagnosis of hydropic pregnancies. However histological diagnosis shows a considerable grade
of subjectivity and overlap of morphological features that reduces
exam reproducibility. Histological diagnostic parameters have
been studied in later molar pregnancies (after 12th gestational
week); currently revisions are made in earlier gestation while
classical histological features are not yet evolved: it is difficult
not only to differentiate partial from complete mole, but also to
discriminate early non molar hydropic pregnancies.
The aim of this work is to identify morphological features useful
to differentiate first trimester hydropic pregnancies. In 289 miscarriages (suspicious for mole or hydropic abortus) observed in
Pathologic Department of “O.I.R.M.-S. Anna” Hospital in Turin
between 01 january 2007 and 31 december 2010 we performed
cytogenetic, histologic and immunoistochemistry analysis for
Ki67 (proliferation index) and p57 (uniparental origin).
All the results are compared in order to reveal common morphological parameters useful to differential diagnosis defining
specific pathologic features in early miscarriage:
Hydropic abort: low intermediate trophoblast differentiation,
polar syncytiotrophoblastic distribution in villous surface and
in intervillous space, not homogeneous villous edema, central
hypovascularity, p57 always present and Ki67 low positive in
ciyotrophoblast (4-5%).
Partial mole: irregular and scalloped villous, invagination of villi
surface into the stroma, focal hyperplasia of syncytiotrophoblast,
p57 always present and Ki67 medium positive in cytotrophoblast
(10-15%).
Complete mole: cyto syncytiotrophoblast with hyperplasia and
atypia, cisterns and absence of embryonic elements, p57 always
absent and Ki67 high positive in cytotrophoblast (90%).
According to these histological and immunohistochemistry parameters all the cases observed would be correctly diagnosed if
compared to cytogenetic analysis.
Trophoblastic differentiation and vascularization
abnormalities in chromosomopathy
G. Girardi, V.R.L. Beltrami, G.F. Zuccotti, G.Botta, M. Ribotta
Ospedaliera “O.I.R.M.-Sant’Anna”, Torino, Italia
85% of spontaneous abortions occur at the first trimester of
pregnancy, and the most frequent cause is a chromosomal abnormality. It’s known that some chromosomal abnormalities lead
to trophoblastic growth and differentiation abnormalities. To
highlight common elements between first trimester abortions, we
performed histological analysis, immunohistochemistry (CK18,
Ki67, p63), and cytogenetic analysis on 442 cases of early abortion received by the Pathology Laboratory of the “Ospedale
Infantile Regina Margherita-Sant’Anna” of Turin, of which 62%
with chromosomopathy.
Morphological analysis: abortive material with chromosomal
abnormality is characterized by morphological abnormalities
that can be grouped into the following categories: cytotrophob-
206
last (CT) to intermediate trophoblast (IT) differentiation delay;
sincitiotrophoblast (ST) abnormal proliferation with “fingerlike” projections on villous circumference; large intervillous
spaces occupated by ST; uneven villous size caused by zonal
edema and along mayor’s axis, with “tadpole” structure. There
are also no recognizable arterialized central vessels, IT is almost
disappeared, and the villous profile is very irregular with invaginations of CT.
Normal villous have homogeneous diameter, IT and vases well
represented, intervillous spaces inhabited by small villous and
no by ST.
In abortive material with chromosomopaty 21, like 18, are visible
morphological characteristics similar to normal. IT is recognizable, although with the abnormalities of cellular distribution into
villous stroma which rarely has a central vessel.
Immunohistochemistry analysis: p63 stain highlight a CT uniform marking in every cases but not at the decidual level. CK18
stains every villous trophoblastic subpopulation and highlight
implant site differences in chromosomopaties: in aneuploidies
cases, trophoblastic cells are absent, (except in 21 trisomies),
while in normal karyotype TB is well recognizable in the decidual
localization.
Ki67 stain, shows high cellular proliferation in IT and low in CT
and ST.
Overall, in the majority of chromosomopaty abortions cases, a CT
and IT differentiation deficit and vascular maturation is observed.
This deficit can be responsible for both defective implant caused
by the absence of IT, and therefore implant site trophoblast (lack
of CK18+ IT cells in decidual site), and fibrosis with zonal edema
for insufficient vascularization.
These alterations can lead to ovular camera detachment, or
ipovascularization. In both cases the pregnancy can’t go on. In
particular, defective implant can lead to early interruption.
Placental site nodule of the cervix
T. Montrone, M. Palumbo, G. Fiore, D. Di Clemente, G. Arborea,
A. Scivetti, L. Resta
Dipartimento di Anatomia e Istologia Patologica, Università degli studi
di Bari, Bari, Italia.
Placental site nodule (PSN) is an uncommon, benign, generally
asymptomatic lesion that originates from chorionic intermediate
trophoblast and may often be detected several months to years
after the pregnancy from which it resulted. Although the majority
involve the endometrium, PSN occasionally be seen in the cervix
and rarely in the Fallopian tube and ovary. This entity may have
bizarre histological findings and necessitates differentiation from
other aggressive lesions of intermediate trophoblast (placental
site trophoblastic tumour or epithelioid trophoblastic tumour)
and from squamous cell carcinoma, especially when it is found
in unusual clinical situations or in localizations outside the endometrium. We report a case of a 37-year-old female who had an
abnormal PAP-test (L-SIL) and a cervical biopsy at subsequent
hysteroscopy, in an area of erosion of the epithelium. The histology showed in the sub-epithelial stroma a focus of anaplastic
cells, immunoreactive for CK pool, suggesting a carcinoma invasion. For this suspect, the patient had a cone biopsy that showed
a small, single nodule composed by fibrinoid material including
epithelioid cells, with eosinophilic cytoplasm and with mild nuclear pleomorphism. Isolated multinucleated cells were present.
No mitoses were recorded. These cells were immunoreactive for
CK pool, focally for hPL and negative for hCG. We concluded
for a placental site nodule of the cervix. In the anamnesis the
patient had an abortion 3 years before and a spontaneous vaginal
labour 2 years before, followed at one month by an uterine curettage for a chorion material retention. No further pregnancy or
abortion were noted. The serum levels of hCG and hPL were low
at 1 year after cone biopsy.
The clinical history pose several questions. The cervical lesion
is not similar to pseudotrophoblastic tumours (atypical chorioncarcinoma) or to an exaggerated placental site. The disease may
be correlated to the previous normal pregnancy (but it had not a
cervical implant), or an accidental implant during the endometrial
revision, or to a subsequent abortion not clinically evident. Presence of trophoblastic cells is a confounding finding either in PAP
smears and in histological examination of cervical biopsies.
Neuropatologia
TTF1 immunopositive supratentorial pnet.
A clinicopathologic study of a case
M. Bisceglia1, M. Bianco2, M. Maruzzi3, A. Spirito3, T. Parracino4, C.A. Galliani5
Unità Operativa di Anatomia Patologica, Ospedale “Casa Sollievo della
Sofferenza”, San Giovanni Rotondo, Italia; 2Unità Operativa di Neurochirurgia, IRCCS, Ospedale “Casa Sollievo della Sofferenza”, San Giovanni
Rotondo, Italia; 3Unità Operativa di Ematologia e Oncologia Pediatrica,
Ospedale “Casa Sollievo della Sofferenza”, San Giovanni Rotondo, Italia; 3Unità Operativa di Radiologia, Ospedale “Casa Sollievo della Sofferenza”, San Giovanni Rotondo, Italia; 4 Department of Pathology, Cook
Children’s Medical Center, Fort Worth, TX, USA
1
Background and aims of the study. Human thyroid-specific
transcription factor-1 (TTF-1), also known as thyroid-specific
enhancer-binding protein (T/EBP - or NKX2.1), is a 371 aminoacid long polypeptide (MW 38 to 40 kDa), homeodomaincontaining transcription factor of the NK-2 family. The known
molecular targets in the thyroid are thyroglobulin, thyroid
peroxidase, and thyrotropin receptor genes. TTF-1 expression
has also been demonstrated both in the bronchioloalveolar epithelium of lung, where it similarly functions as a transcriptional
activator of specific genes (alveolar type II cells surfactant
protein genes, and bronchiolar Clara cell secretory protein
gene), and in the ventral forebrain, where the responsive genes
are mostly unknown. TTF-1 expression in the central nervous
system (CNS) has been documented in third ventral ventricular neuroepithelium of the preoptic and hypothalamic areas,
including the infundibulum, site of derivation of the neurohypophysis (Kimura et al, 1996; Bingle et al, 1997). TTF-1 plays
a fundamental role in organogenesis by regulating expression
of additional genes that are directly involved in development
and differentiation. Experimental studies have demonstrated
that in the T/ebp knock-out homozygous mice, the embryonic
development of thyroid, lung, hypothalamus, and pituitary is
dramatically impaired (Kimura et al, 1996; Lee et al, 2001).
TTF-1 is extensively used in surgical pathology as a lineage
specific immunohistochemical marker, most often for primary
and secondary tumors of the lung and thyroid. Recently, nuclear TTF-1 immunostaining was documented in primary brain
tumors (Zamecnik et al, 2004; Prok et al, 2006; Galloway and
Sim, 2007; Lee et al, 2009). During the course of investigating
TTF-1 expression in embryonal tumors, both in children and
adults, we studied 24 embryonal CNS tumors: 14 infratentorial
PNET (medulloblastoma), 6 supratentorial PNET, 3 supratentorial neuroblastoma (including 1 olfactory neuroblastoma), 2 pineoblastoma, and 1 retinoblastoma. [personal unpublished data
of two of us (MB, CAG)]. All but 1 paediatric suprasellar PNET
failed to express TTF-1. We report on a TTF-1 immunoreactive
suprasellar PNET in a paediatric patient.
Case report. A 6½ year-old girl was brought in sub-comatous
state to the emergency department. CT scan and MRI of the head
disclosed an 8.0 cm, midline, contrast enhancing tumor of the suprasellar region, with endophytic protrusion into the 3rd ventricle,
and encasement of the intracranial carotid arteries. Due to technical infeasibility, the patient underwent partial resection of the
207
Poster
suprasellar tumor. Histopathologically, the neoplasma was made
up of small round-blue-cells with perivascular pseudorosettes,
Homer-Wright rosettes, brisk mitotic and apoptotic activity, and
foci of necrosis. Immunohistochemically, the tumor cells were
focally positive for synaptophysin, neurofilaments, and GFAP,
and negative for desmin and myogenin. INI-1 was normally
expressed. Proliferation index, as assessed by Ki-67/MIB-1, was
50%. Since our study of TTF-1 expression in small round cell
tumors was in progress, we included this case. It revealed immunostaining for TTF-1 in 40% of the tumor cells’ nuclei. The
patient received craniospinal radiation and chemotherapy with
temozolamide and VP16. Imaging performed 1-year after the
partial resection revealed seemingly quiescent residual tumor.
However, the patient died 20 months after the diagnosis.
Discussion. In the experimental rodent CNS model, TTF-1 expression is detected in the ependymal and subependymal cells
rd
of the ventral neuroepithelium of the 3 ventricle, including
neurons of selected hypothalamic nuclei, astrocytes of the median
eminence, pituicytes of the infundibular stalk and neurohypophysis, and in the adjacent extrahypothalamic (rat) suprachiasmatic
nucleus and subfornical organ (Kimura et al, 1996; Lee et al,
2001; Nakamura, 2001; Kim et al, 2006; Son et al, 2003). In
2004 Zamecnik et al using monoclonal antibody against TTF-1
(clone 8G7G3/1) documented TTF-1 expression in 2 cases of
ependymomas (1 gr. II and 1 grade III, both localized in the 3rd
cerebral ventricle, in a 5-year old boy and in a 12-year old girl,
respectively) out of 73 primary tumors of human brain, including
33 astrocytic tumors of various grades, 27 ependymomas (11
grade II and 16 grade III), 7 medulloblastomas, 3 gangliogliomas.
In 2006 Prok and Prayson using the same clone 8G7G3 studied
50 cases of glioblastomas, but did not prove immunopositivity in
any. In 2007 Galloway and Sim investigated TTF-1 expression
in 28 cases of glioblastomas using the 2 clones currently available, the 8G7G3/1 and SPT24: 14 glioblastomas proved positive
when clone SPT24 was used, while no case was immunopositive
with clone 8G7G3. Based on our own and others’ experience, it
must be said that clone SPT24 is more sensitive but less specific
than clone 8G7G3/1. Galloway and Sim also questioned the specificity of TTF-1 immunopositivity in their 14 glioblastomas. In
2009 Lee et al again using clone 8G7G3/1 studied a series of 5
pituicytomas and 4 granular cell tumors arising from the posterior
lobe of pituitary, all of which expressed TTF-1. We also used
clone 8C7C3/1 for assessing TTF-1 expression in a series of 24
embryonal tumors of CNS, leading to the discovery of the TTF-1
immunopositive suprasellar PNET presented herein (Bisceglia et
al, 2011).
Conclusions. The case presented herein is the only case from a
6½ year-old girl which demonstrated TTF-1 immunoreactivity,
among 24 embryonal CNS neoplasms analysed for the expression
of TTF-1. Primary brain tumors fail to express nuclear TTF-1 immunoreactivity as detected with clone 8G7G3/1, with the possible
exception of those originating in the periventricular regions of
the diencephalon. This is concordant with experimental studies.
TTF-1 immunopositivity may reflect a lineage marker.
Long-term survival in two patients
with glioblastoma
M. Bisceglia *, M. Bianco
D’Angelo **.
**
, I. Carosi *, M.A. Grasso
, V.
***
Unità Operativa di Anatomia Patologica, IRCCS, Ospedale “Casa Sollievo della Sofferenza”, San Giovanni Rotondo, Italia; **Unità Operativa
di Neurochirurgia, IRCCS, Ospedale “Casa Sollievo della Sofferenza”,
San Giovanni Rotondo, Italia; *** Farmacia Ospedaliera ASL-FG, Foggia
*
Background and aims of the study. Glioblastoma (GB) or
WHO grade IV astrocytoma is the most common and aggressive
of primary tumors of the brain in adults, with a peak incidence
> 60 years of age. The median overall survival in patients with
GB is ≤ 12 months, but in a single recent series of 39 patients
treated with multimodality-therapy it was 23.1 months. Those
patients that survive 3 years after diagnosis are defined long-term
survivors, and this most likely occurs in 3% - 5% of all the cases
(range between ≤ 2% and 18%). The prevalence of prolonged
survival rate at 5 years for GB in different series is consistently
dismal but variable from 0.5% in the series of Deb et al of 1296
cases (Neurol India 2005;53:329) to 4% in the series of McLendon & Halperin consisting of 766 patients with a minimum of 5
years of follow-up (Cancer 2003;98:1745), to 3.4% for classic
GB and 12.3 % for giant cell GB, respectively, in the ever largest series of Kozak & Moody (Neuro-Oncology 2009;11:833),
comprising 16,430 total cases. Giant cell GB represents 1%-5%
of all GB cases, is more common in the pediatric population and
young adults, and is known as a GB variant with possible clinical
relevance on prognosis. In the cited series by Kozak & Moody,
around 1% (171 cases in total) were of the giant cell variant with
an overall 5-year survival of 12.3%. So far, < 450 cases of longterm GB survivors have been recorded in the world literature as
living 3 to 9 years after the initial diagnosis (Krex et al, 2007 –
Brain 130:2596; and personal updated review), and 42 cases with
survival times of 10 years or longer (13 patients survived 15 years
or more, 4 of whom were alive at their 22 to 25 years follow-ups)
(Sabel et al, 2001 - J Neurosurg 94:605; and authors’ personal
updated review). Of the above 13 patients surviving 15 years or
longer, 3 were of the giant cell variant with 2 surviving 17 years
and 20, respectively. We present herein 2 personal cases of longterm survivor with GB, the former of the giant cell variant and
the latter of classic type.
Case reports. Case 1. A 28 year-old female was admitted because
of headache and vomiting for 1 month, eventuating in endocranial
hypertension. Neuroimaging showed a well circumscribed 6 cm
solid-cystic brain tumor in the left frontal lobe adjacent to the
lateral ventricle for which she underwent gross total resection
(GTR). Histopathologically, the tumor was a giant cell GB, a
diagnosis confirmed by several consultant neuropathologists.
Postoperatively, the patient received radiation therapy. After a
13-year disease free interval, a 2 cm intraventricular recurrence
was detected during her routine yearly image surveillance for
which a second GTR was achieved. Histopathologically, the
tumor retained the same giant cell characteristics. No adjunctive therapy was given. Currently, at the age of 47 and 19 years
after the initial diagnosis the patient is alive and well. Case 2. A
17-year old female was admitted because of worsening headache
and nausea. Neuroimaging disclosed a 6 cm right temporo-insular
cerebral tumor for which GTR was performed. Postoperative
radiotherapy was administered and chemotherapy with temozolomide was temporarily given but had to be discontinued due to severe toxicity. The patient is being followed regularly. Currently,
> 8 years after her initial diagnosis, the patient is alive and well.
Discussion and conclusions. Long-term survival in GB is very
rare and survival beyond 10 year is anecdotal. Our two patients
are the only examples among ~ 700 primary GBs treated at our
institution in the past 2 decades. Some clinical features (young,
female, high Karnofsky performance score), gross morphological
and anatomical appearances (relative circumscription & size <
4 cm), GTR with adjuvant radio- and chemotherapy (alkylating
agents) all are possible positive predictors of good prognosis.
Since 1980, the giant cell variant and giant cell component in a
given GB has been credited of being associated with a possibly
longer survival compared to classic GB (Burger & Vollmer. Cancer 1980; 46:1179). A new identified predictor of good prognosis
is hypermethylation of the MGMT gene promoter, in patients
treated with temozolomide.
208
Collateral trigone choroid plexus papilloma
with extreme stromal sclerosis
A. Cimmino*, G. Ingravallo*, R. Rossi*, P.I. D’Urso**, S.V. Scarcella*, L. Resta*
Dipartimento di Anatomia e Istologia Patologica/Università degli studi
di Bari, Bari, Italia; **Dipartimento di Neurochirurgia/Università degli
studi di Bari, Bari, Italia.
*
Choroid plexus papillomas (CPPs) are relatively rare and usually
benign neoplasms. CPPs account for 0,4 to 0,6% of all intracranial neoplasms. In rare instances they may be congenital and more
exceptionally bilateral. Stromal changes and unusual histological
features in choroid plexus papilloma, such as oncocytic changes,
mucinous degeneration, melanization and tubular glandular architecture may occur in choroids plexus papilloma, but massive
fibrovascular stroma collagenization of a choroid plexus papilloma has not previously reported.
A 60-year-olf female was referred with MRI evidence of a left
intraventricular collateral trigone lesion, manifested with symptoms of increased intracranial pressure. A smaller lesion was also
demonstrated in the contralateral ventricle. Patient underwent to
surgical removal.
Microscopic examination revealed a fibrosclerotic mass containing scattered islands of epithelial papillary fronds, covered
by a single layer of uniform columnar/cuboidal epithelial cells.
The tumor was almost entirely effaced by collagenized stroma.
Fibrous sclerosis extensively interested the lesion, reducing the
papillomatous component to nodular scars containing sparse
benign glandular elements. Neoplastic stroma was constituted
by fibroblasts, a conspicuous amount of collagen fibers, inflammatory cells and macrophages. There was no evidence of malignancy. Immunohistochemical analysis showed immunoreactivity
for S-100 protein and CK-pool, patchy positivity for CK7, and
absence of reactivity for GFAP and CK20 in the epithelial cells.
In the epithelial cells, there was not immunoreactivity for the
specific markers of more frequent metastatic carcinomas with
papillary architecture (TTF-1, estrogen receptor and CDX-2).
Electron microscopy showed a papillary structure lined by low
columnar cells with numerous short microvilli on the luminal
surface. The basal surface was followed by a well defined basal
lamina. The lateral surfaces had typical junctional complexes
near the luminal end and numerous interdigitations of the cell
membranes. The nuclei were rounded, contained finely granular
chromatin and small inconspicuous nucleoli. A moderate amount
of rough endoplasmic reticulum and mitochondria were uniformly distributed throughout the cytoplasm. Cytoplasmic filamentous
inclusions of variable morphology were present.
A diagnosis of CPP with extreme stromal sclerosis was made. Interestingly, massive fibrovascular stroma collagenization of human
CPP has not been previously reported. Similar event is common in
breast papilloma, a benign tumor of the epithelium of mammary
duct. In the current case, the massive collagenization may be due to
an ischemic injury, as consequence of an imbalance between tumor
growth and inadequate angiogenesis or blood flow.
Pituitary prolactinoma with extensive spherical
amyloid deposition
L. Ventura1, M.L. Jaffrain-Rea2 3, S. Marzi4, A. Catalucci5, M.
Anselmi5, R.J. Galzio4 6
U. O. C. di Anatomia Patologica, Ospedale San Salvatore, L’Aquila, Italia; 2Dipartimento di Medicina Sperimentale, Università, L’Aquila, Italia; 3Neuromed IRCCS, Pozzilli (IS), Italia; 4U. O. C. di Neurochirurgia,
Ospedale San Salvatore, L’Aquila, Italia; 5U. O. C. di Neuroradiologia,
Ospedale San Salvatore-Università, L’Aquila, Italia; 6 Dipartimento Chirurgico, Università, L’Aquila, Italia
1
Pituitary adenomas show accumulation of amyloid in the 70% of
cases, more commonly in the form of stellate/perivascular type
and rarely as the spherical type, occurring as spheroid accumulations of variable diameter (40-1500 mm). The first type may be
encountered in all endocrine pituitary adenomas, the second form
is almost exclusively found in prolactin-producing adenomas. We
present a case of a giant prolactinoma with extensive spherical
amyloid deposition in a man.
A 57-year-old male patient was operated on for a giant tumor
of the sellar region. He came to our observation because of progressive visual defects, with a long standing history of reduced
libido and erectile dysfunction. Pre-operative hormone evaluation revealed very high plasma prolactin (PRL) levels (2627 ng/
ml). Magnetic resonance imaging (MRI) showed the presence of
a sellar and suprasellar lesion, invading the sphenoid sinus, both
cavernous sinuses and extending up near to the floor of the left
lateral ventricle and, posteriorly, into the mesencephalic cistern.
The lesion, measuring about 5,5x3,5x4 cm, was spontaneously
hyperintense in T1-weighted sequences and hypointense in T2weighted sequences, with low intensity spots suggesting the
presence of extensive calcifications, as well documented also
on plan CT examination. Because of the atypical aspect of the
lesion on MRI and severe visual field defects, the patient underwent debulking surgery by a trans-sphenoidal route, without
dopamine-agonist therapy. He recovered from surgery without
complications, showing improvement of visual field 3 months
after the operation.
Multiple fragments of soft, yellowish tissue containing tiny spheroidal bodies were formalin-fixed, routinely processed and embedded in paraffin, to obtain sections stained with hematoxylineosin, reticulin, Congo red and immunohistochemically stained
with antibodies against AE1/AE3 cytokeratins, chromogranin A,
synaptophysin, PRL, GH, FSH, LH, TSH, ACTH, and MIB-1.
Microscopy revealed a large number of round, eosinophilic,
concentrically laminated bodies, with adjacent clusters of
epithelial cells in the peripheral portions of the specimen. Such
bodies stained positively with Congo red and revealed greenyellow birefringence under polarized light. They also showed
positivity for PRL and cytokeratins. Tumor cells showed diffuse cytoplasmic expression of cytokeratins, PRL and synaptophysin, and focal positivity for chromogranin A and GH in the
cytoplasm. Other hormones resulted negative. The proliferation
index (MIB-1) was around 3%. Multifocal areas of xanthogranulomatous inflammatory reaction were also present. The final
diagnosis was pituitary prolactinoma with extensive spherical
amyloid deposition.
The presence of spheroidal amyloid deposition has already
been described in prolactinomas and might be explained by an
abnormal processing of PRL, which presents structural motifs
similar to other amyloid-related proteins. Amyloid deposits do
not cause any characteristic clinical or biochemical features,
and intrasellar amyloid deposition is not usually recognized
before surgery.
In our case, the long standing history of reduced libido and
erectile dysfunction, coupled with high plasma PRL levels
were suggestive for hypogonadism due to prolactinoma, but
MRI findings were not typical of pituitary adenoma. Interestingly, such MRI features were similar to some previously
reported case, suggesting that amyloid deposition can be suspected preoperatively.
Patients with suspected amyloid deposition in prolactinoma may
be treated surgically in order to confirm the diagnosis histologically and remove the intrasellar amyloid deposits. Pathologists
should be aware of such issues, in order to correctly diagnose this
particular entity and allow planning further therapy.
209
Poster
Paleopatologia
Prostatic hyperplasia in italian mummies
(XV-XIX century)
L. Ventura*, V. Giuffra**, G.L. Gravina***, F. Marampon***, C.
Mercurio*, G. Fornaciari**
limited to the prostate but easily extensible to other organs on a
larger number of mummies, could solve some important medical problems as, for example, the origin and diffusion of some
sexually transmitted diseases and genital tumors, whose natural
history is still unclear.
The mummified bodies from the church
of San Michele Arcangelo in Sermoneta (LT).
A preliminary survey
U. O. C. di Anatomia Patologica, Ospedale San Salvatore, L’Aquila,
Italia; **Divisione di Paleopatologia, Storia della Medicina e Bioetica,
Dipartimento di Oncologia, Trapianti e delle Nuove Tecnologie in Medicina, Università, Pisa, Italia; ***Divisione di Radioterapia e Radiobiologia,
Dipartimento di Medicina Sperimentale, Università, L’Aquila, Italia
*
Prostatic hyperplasia represents a very common condition today,
but it was well known in the past as a cause for bladder distension. At autopsy of natural or artificial mummies, the difficulty
in identifying even a normal-volume prostate is likely to be due
to putrefaction processes as well as dramatic reduction in size.
We report three ancient cases of prostatic hyperplasia recently
observed in natural and artificial mummies from central Italy.
The first case regards Pandolfo III Malatesta (1370-1427), Lord
of Fano and a leading figure of the Italian Renaissance. He was
a valiant soldier and horseman with a very active life style. The
monumental tomb containing his naturally mummified body, was
explored in Fano. After careful X-ray and videographic examination, autopsy showed good preservation of the skeletal muscles,
cartilage, internal and external organs, including prostate gland
and penis. Macroscopic examination revealed a renal stone and
severe enlargement of the prostate, with calcifications detected
by X-ray and large nodules protruding in the lumen of an ectatic
urethra. Histology showed fibrous and muscular tissue surrounding circular lacunae, without preservation of epithelial structures.
The macroscopic and histological picture allowed to diagnose
prostatic hyperplasia.
The second case, regarding the artificial mummy of Salimbene
Capacci (1433-1497), Rector of the Medieval hospital of S.
Maria della Scala in Siena, revealed well preserved pelvic organs
at X-ray and CT scans. At autopsy, the thoracic and abdominal
cavities appeared filled with vegetable material, but some organs
were still in situ, namely remains of bladder, prostate and the
terminal segment of the intestinal tract. The prostate consisted
of a central fibrous structure, surrounded by perineal tissues.
Histology revealed dense fibrous tissue with muscular fibers and
roundish cavities of variable size. Such histologic findings, the
distended bladder, and te age of the subject support the diagnosis
of prostatic hyperplasia.
The third case (XIX century) concerns the natural mummy of
an anonymous 50-60 year-old-man, recovered in a friary near
L’Aquila and undergone complete CT and autopsy study. Pelvic
CT scans showed distended urinary bladder and a ring of dense
tissue at the site of the prostate. At autopsy the bladder measured
7x6x5 cm, the prostate was 4x5x3 cm and the prostatic urethra
had a 2 cm diameter. Histology revealed fibrous tissue containing muscular fibers and roundish cavities of variable size, filled
with eosinophilic, PAS-positive material, also immunoreactive
for PSA. The presence of a prostate with such histologic appearance, a preserved and distended urinary bladder and the age of the
subject support the diagnosis of prostatic hyperplasia.
To the best of our knowledge, so far neither benign nor malignant
forms of prostatic enlargements have appeared in paleopathology literature. Therefore, the Italian cases of the Renaissance
and Modern ages represent the only known reports of prostatic
hyperplasia in mummies and clearly demonstrate that paleopathological studies on prostate gland using diverse and modern technologies are possible. The good preservation of the external and
internal genitalia of these three individuals may be related to the
supine position of the bodies after death, which allowed rapid
dehydration of these structures. This type of approach, currently
The village of Sermoneta is of medieval origin and situated in the
province of Latina (southern Lazio region). The church of San
Michele Arcangelo in Sermoneta dates back to XII century and is
located in the oldest part of the village. Recent restorations of the
entire complex allowed to find different hypogeal burial environments within the so-called Chapel of the “Battenti”, belonging to
the most ancient part of the church. Five distinct burial ambients
were present near the altar of the chapel, and two of them appeared in close contact with the external walls of the village.
Most of the crypts had been used as ossuaries, containing skeletal segments without anatomic connection mixed together, but
inside one of these burials 7 partially mummified individuals
were found. Moreover, at the top of one ossuary an incomplete,
well-preserved mummy, featuring head, neck, arms and trunk,
could be recovered.
The subjects underwent external inspection with anthropological measurements and pathological examination. Radiographic
investigation were planned for selected bones and computed
tomography (CT) scanning was performed in the mummified
subject. Object of the present study are the results from the preliminary anthropological and paleopathological survey on these
individuals.
From an anthropological point of view, the series of individuals
recovered from the same burial included 6 adults (1 male and 5
females), between 18 and 60 years of age at death, and 1 infant
with an age at death of 3-4 years. The stature of the adult subjects
ranged from 148 to 171 cm. The examination of small fabric
fragments from clothes allowed to date the individuals back to
the end of XIX century. The incomplete mummy belonged to an
undeterminate subject of adult age, wearing clothes dating back
to XVIII-XIX century.
Occasional macroscopic evidence of organs was observed, represented by pelvic viscera in one subject and the left breast in another, whereas skin, skeletal muscles and other superficial structures (eyes, ears, hair, nails) appeared well preserved throughout
the whole series. This suggests the preminent role of desiccation
in the mummification process and confirms the environment capability in tissue preservation.
The initial paleopathologic analysis allowed to identify various
pathologic conditions, including poor dental status (caries in 5
individuals, periodontal disease in 2 and dental wear in 4), 1 case
of dental anomaly (unerupted upper canines) associated with sacral spina bifida occulta. The marked cutaneous folds in partially
mummified individuals indicated well nourished subjects and the
possible presence of obesity.
The partially mummified individual underwent CT scanning by
using a Philips Brilliance 16 scanner with 2 mm thick sections,
obtained at reconstruction intervals of 1 mm. CT scans displayed
remnants of encefalic tissue and good preservation of thoracic
internal organs. Of pathological significance were also an upper
right molar radicular cyst, bilateral shoulder osteoarthritis, more
*
L. Ventura*, G. Miranda**, C. Mercurio*, I. Trombetta***, G. Fontecchio***, V. Urbani****
U. O. C. di Anatomia Patologica, Ospedale San Salvatore, L’Aquila, Italia; **Dipartimento di Scienze Ambientali, Università, L’Aquila, Italia;
***
Centro Regionale di Immunoematologia e Tipizzazione Tissutale, Ospedale San Salvatore, L’Aquila, Italia; ****Radiologia, Casa di Cura Privata
Villa Serena, Città S. Angelo (PE), Italia
210
prominent in the right side, and diffuse right pleural adhesions
with partial right displacement of the mediastinum, related to
previous episodes of pneumonia.
In conclusion, this small series of individuals dating back to
XVIII-XIX century identifies a population in a good nutritional
status, as demonstrated by the presence of high rates of caries
and obesity. Example of osteoarthritis and pneumonia were also
detected, and the coexistence of a dental anomaly with sacral
spina bifida in the same subject may be related to developmental
abnormalities.
Further investigations, featuring radiological, histological and
molecular analyses are still in progress, in order to obtain additional information.
Patologia renale
Renal sinus pseudolymphoma in a patient
with multiple carcinomas: a case report
and brief review of the literature
nancies. The pathogenesis of PL has not yet been elucidated, and
it may involve infectious agents, allergic responses, autoimmune
reactions and tumors. The relatively high prevalence of Sjogren
syndrome in PL, as described in the literature, suggests a possible pathogenetic involvement of autoimmunity. In our case no
systemic immunologic abnormalities were detected. PL has been
reported also in association with malignancies, and putatively
interpreted as a immunoreactions to neoplastic cells. In our case
PL was associated with bladder urothelial carcinoma and prostatic adenocarcinoma. Differential diagnosis of PL include space
occupying lesions, lymphoma, and inflammatory pseudotumor
(IPT). The absence of mitotic figures, pleomorphism and nuclear
atypia as well as the polyclonality of lymphoid cells rule out malignancy. On the other hand, no clinical or pathological features
of IPT were observed. However, in spite of the fact that there
are specific criteria for the diagnosis of PL, a morphological examination associated with immunohistochemistry and molecular
biology is mandatory in order to avoid pitfalls.
Atipical Hemolytic Uremic Syndrome (aHUS):
a Case Report.
M.R. Ambrosio, M. Onorati, B.J. Rocca, M.T. del Vecchio, C.
Bellan, S. Mannucci, N. Palummo, S. Tripodi
R. Arena*, E. Unti*, V. Azzolina**, M.C. Sapia**, N. Scibetta*, S.
Maringhini**
*
Unit of Pathology and **Unit of Neprhology, “Civico, G. Di Cristina and
Benfratelli” Hospital, A.R.N.A.S., Palermo, Italy
Background. pseudolymphoma (PL) is a rare disease, characterized by the proliferation of non-neoplastic, polyclonal lymphocytes forming follicles with active germinal centers, often
mimicking lymphoproliferative disorders. Unlike lymphomas,
most PL undergo spontaneous remission. We report a case of PL
of the renal sinus in a 70-year-old man, associated with a bladder
high grade urothelial carcinoma and with a prostatic adenocarcinoma. A brief review of the literature is also added. To the best
of our knowledge, this is the second case of PL of the renal sinus,
but the first one with these characteristic associations.
Methods. the surgical specimens consisted of kidney, ureter,
bladder and prostate. Representative samples were stained with
haematoxylin and eosin. Immunohistochemistry for CD20, CD3,
CD4, CD8, CD5, CD30, CD79a, CD138, Bcl-2, kappa and
lambda light chains was performed. The rearrangement of the
immunoglobulin heavy chain genes was also carried out.
Results. the renal sinus showed a whitish mass (5 cm) stenosing
the ureter. In the right wall of the bladder, an ulcerate, grayish
lesion was also observed. Microscopically, the lesion of the
renal sinus consisted of a proliferation of small to medium size
lymphocytes (CD20 and CD79a positive and Bcl-2 negative)
sometimes arranged in follicular structures with germinal centers. Interfollicular areas were expanded by a diffuse infiltrate of
lymphocytes (CD3 and Bcl-2 positive) and plasma cells (CD138,
kappa and lambda light chains positive). Inflammatory cells were
scattered in a hyalinized fibrous tissue involving the ureter. Mitotic figures and nuclear atypia were absent. Neither myofibroblasts nor cells positive for follicular dendritic cell markers were
observed. A diagnosis of PL was made and it was confirmed by
molecular studies that showed oligopolyclonal IgH rearrangement. The lesion of the bladder was a high grade flat urothelial
carcinoma infiltrating all the layers of the wall and the fat tissue (pT3bNxMx). An acinar adenocarcinoma, Gleason score 6
(pT2aNoMx) was also observed in the prostate.
Conclusions. in the kidney and upper urothelial tract PL is uncommon. Seven cases have been described, including that here
illustrated. Three of them were in the renal parenchima, two in
the perirenal space, one in the upper ureter and one in the renal
pelvis. All patients had single lesions. The median age was 62
years, with a female predominance. The average diameter of
the lesion was 2.6 cm. Three patients were affected by Sjogren
syndrome and only the patient herein described presented malig-
Introduction. Atypical Hemolytic Uremic Syndrome is rare in
the child (about two cases per million population / year in the
United Kingdom) and, unlike the typical form, is burdened by
high mortality, risk of recurrence and evolution to renal failure
may occur. The most common causes are represented by disorders of the complement (genetic or acquired), changes in factor H
and I and more rarely by the deficiency of ADAMTS 13.
The aim of this study is to evaluate a case of Atypical HUS with
diriment histological examination.
Methods. 4 year-old patient, male gender, sent by the dispensary
for hematuria and proteinuria. Laboratory tests have shown at admission renal insufficiency (serum creatinine 2.1mg/dl, BUN 68
mg/dl creatinine clearance -according to Schwartz- 23.5 ml/m),
anemia (HB 7.6g/dl), slight increase in indirect bilirubin (1.1g/dl)
and in LDH. Coombs’test (direct and indirect) negative, platelets
143,000 increasing in the following days with subsequent reduction in the 17 th day to 76,000 (minimum reached value) and
low haptoglobin (<6 mg/dl). Normal C3, C4, IgA. At 14 th day
of hospitaolization percutaneous renal biopsy was performed,
and at 19 th day because oligo-anury occurs, peritoneal dialysis
was initiated. Received biopsy results after infusion of frozen
fresh-plasma plasmepheresis daily for 7 days was initiated, and
then every other day. The clinical conditions have improved
considerably, but persisting oligoanuria and after the detection
of abnormalities of factor H, therapy with Eculizumab (anti-C5
monoclonal antibody) was initiated with reduction of serum creatinine and renewal of diuresis.
Results. Two chips of the renal parenchyma containing 44
glomeruli, tubules, interstitium and vessels of small and medium
caliber reach. Two glomeruli are in complete sclerohyalinosis, have some of the remaining aspects of mesangiolisis with
dilatation of glomerular capillaries to take up aneurysmal aspects,
others are characterized by variable degrees of mesangial proliferation with aspects of focal and segmental sclerosis and “binary
inspectress” of capillary’s walls.
There are endothelial swelling and intimal hyperplasia and fibrosis, paid by some medium and small caliber vessels and arterioles
with occasional evidence of thrombotic occlusion of the lumen.
There is moderate tubular atrophy, associated with interstitial
fibrosis. The lumen of distal tubules is sometimes dilated and occupied by voluminous cylinders (granular-hyaline and hematic).
Direct immunofluorescence showed a mesangial positivity of
211
Poster
granular type, for C3c (+) and IgG (+), negative for IgM, IgA, K
and Lambda light chains, C1q, C4c and fibrinogen.
Conclusions. Early histological diagnosis prompted us to undertake in the short term plasmapheresis with rapid improvement in
patient’s clinical condition despite the renal failure persisted; that
has shown sensitivity to the drug therapy.
Extracapillary proliferation as an independent
predictive factor in IgAN
K. Giannakakis1, R. Polci2, I. Serriello3, A. Gigante3, M. Rosa5, S.
Feriozzi2, M. Galliani4, M. Morosetti6, F. Pugliese3, T. Petitti8, T.
Faraggiana1, A. Onetti-Muda7
1
Anatomia Patologica, “Sapienza” Università di Roma; 2Nefrologia,
Ospedale Belcolle, Viterbo; 3Medicina Clinica, “Sapienza”Università
di Roma; 4 Nefrologia, Ospedale Pertini, Roma; 5Nefrologia, Ospedale
San Camillo-Forlanini, Roma; 6 Nefrologia, Ospedale GB Grassi, Roma;
7
Anatomia Patologica, Università Campus Bio-Medico, Roma; 8 CED,
Università Campus Bio-Medico, Roma
Background. The predictive value of the Oxford classification of
histological lesions in IgA nephropaty has been validated; attention has been placed on its predictive value of the decline of renal
function. The aim of our work was to correlate active glomerular
lesions at biopsy and progression of renal damage.
Methods. We have studied 473 renal biopsies with a diagnosis of IgAN; of these, 184 had availability of clinical data at
follow-up (Scr and eGFR by CKD-EPI formula) up to maximum of 25 years. The median age at diagnosis was 36.7 years;
70% of patients were males. Histological parameters were
from the Oxford classsification (mesangial and endocapillary
proliferation, segmental glomerulosclerosis, tubular atrophy,
extracapillary proliferation, interstitial fibrosis); in addition,
glomerular fibrinoid necrosis was also considered. Data were
analyzed by univariate and multivariate analysis, according to
linear regression of longitudinal data, taking into account the
distance between time of biopsy and time points of acquisition
of clinical data.
Results. Statistical analysis showed a correlation between progression of renal damage (eGFR) and segmental glomerulosclerosis (p=0.001), cellular crescents (p=0.01), fibrous crescents
(p=0.02), fibrinoid necrosis (p=0.04) and interstitial fibrosis
(p=0.03); no correlation was evident with fibrocellular crescents
and endocapillary proliferation.
Conclusions. Our preliminary results suggest that active glomerular lesions as cellular crescents and fibrinoid necrosis, correlate
with decline of renal function, differently from the Oxford classification. These histological paremeters should therefore be taken
into account to classify histologically cases of IgAN, and for the
appropriate treatment.
Renal infarction mimicking a neoplastic lesion
in a patient with angioimmunoblastic T- cell
lymphoma
M. Onorati, M.R. Ambrosio, B.J. Rocca, M.G. Mastrogiulio, A.
Barone, A. Ginori, L. Vassallo, M. Cintorino, S. Tripodi
Background. Angioimmunoblastic T-cell lymphoma (AITL)
represents one of the most common subtypes of peripheral T-cell
lymphoma. It mainly affects elderly people and, in advanced
stage, it is characterized by generalized lymphadenopathies, hypergammaglobulinemia, skin rash and immunological disorders.
The prognosis is poor, although some patients seem to benefit
from high dose chemotherapy and autologous stem cell transplantation (ASCT). We describe a case of a 45-year-old patient treated
with a cytarabine-based regimen and ASCT, which developed a
renal infarction due to the onset of panarteritis nodosa (PN). To
the best of our knowledge, this is the second case described in the
literature of a patient with AITL and secondary visceral PN.
Methods. The patient presented with myalgia, arthralgia, fever
and cough. A chest X-ray examination showed bilateral lung
nodules and enlarged mediastinal lymph nodes, suspicious for
sarcoidosis. Physical examination revealed cervical, axillary and
inguinal lymphadenopathies and skin rash. Blood cell count, renal and liver function and angiotensin converting enzyme (ACE)
were normal. Lactate dehydrogenase, immunoglobulin and β-2
microglobulin were increased. A cervical lymph node biopsy was
performed and representative samples were stained with haematoxilin and eosin; a panel of antibodies for immunohistochemistry
were checked (CD20, CD3, CD4, CD10). EBV-encoded RNA
(EBER) and rearrangement of the T cell receptor (TCR) genes
were also performed. Bone marrow biopsy was negative. After
two cycles of chemotherapy, the patient had neither systemic
symptoms nor superficial lymphadenitis but a whole body CTscan showed a lesion in the upper pole of the left kidney. A renal
neoplasm was suspected, hence nephrectomy was performed.
Results. The kidney showed a large pale yellowish area at the
upper pole (4 cm) with a triangular morphology, centered on the
renal cortex and consistent with an infarction. Microscopically,
coagulative necrosis of renal parenchyma and multiple segmentary inflammatory lesions of small and middle renal arteries were
found. The earlier arteriolar lesions showed a rupture of internal
elastic lamina and aneurismatic dilatation of the wall; the older
ones showed obliteration of the lumen by trombotic material and
fibrosis, which, in some vessels, was recanalized by thin vascular
channels. Accordingly to the American College of Rheumatology (ACR) criteria, PN was diagnosed. The architecture of the
cervical lymph node was completely effaced by a polymorphous
lymphoid infiltrate, predominantly composed of atypical, small to
medium size lymphocytes with a clear cytoplasm (CD3+, CD4+,
CD10+, EBER+), admixed with plasma cells, immunoblasts
(CD20+), eosinophils and numerous high-endothelial venules.
The diagnosis was angioimmunoblastic T-cell lymphoma associated with panarteritis nodosa.
Conclusion. Only two cases of renal failure due to PN have been
reported associated with AITL. This possibility is to kept in mind
since PN can simulate a tumor, so delaying the correct diagnosis
and the treatment.
Renal failure due to a malignant lymphoma
infiltration uncovered by renal biopsy
R. Passantino*, G. Li Cavoli**, A. Ferrantelli**, U. Rotolo**, L.
Marasà*
Unità Operativa di Anatomia Patologica, Ospedale ARNAS Civico Di
Cristina Benfratelli di Palermo, Palermo, Italia; **Unità Operativa di
Nefrologia e Dialisi, Ospedale ARNAS Civico Di Cristina Benfratelli di
Palermo, Palermo, Italia
*
Background. The incidence of lymphomas, especially nonHodgkin’s lymphoma (NHL), has shown a steady increase over
the last decades. At the same time, the prognosis has improved.
Given the longer survival of lymphoma patients, pathological
manifestations related to malignancy might become more frequent. In this setting, the kidney is one of the most important solid
organ affected by direct or indirect lymphomatous involvement.
Kidney involvement can be related to obstruction or treatmentinduced toxicity, but more intriguing are 1) direct infiltration,
2) association with kidney malignancies, 3) association with
glomerular diseases. Primary infiltration is rarely seen, while
secondary infiltration is described most frequently in autopsy
series, even in the absence of renal failure. These alterations may
mimic glomerular and/or interstitial diseases. Renal biopsy is
often needed in this setting. In this report, we describe a patiente
whose presentation of lymphoma was renal failure and bilateral
enlarged kidneys.
212
Case. In August 2009 a 72-year-old Caucasian man, suffering
from hyperfunctioning nodular goiter and arterial hypertension,
was admitted to a local hospital for fatigue and weigth loss. It was
found hypoalbuminemia and proteinuria in nephrotic range, mild
renal failure, normochromic-normocytic anemia and thrombocytopenia. Physical examination, TC scan and ultrasonography showed
splenomegaly, pleural effusion, edema in the lower extremities,
latero-cervical, axillary, ilo-mediastinic, abdominal lymphadenopathy and increased dimensions of kidneys with changed parenchymal echogenicity. He was carried out axillary lymphonode
biopsy with diagnosis of non-necrotizing granulomatous process
with epithelioid cells and rare giant cells and bone marrow aspiration with diagnosis of B lymphoproliferative process. The
search of amyloid in abdominal fat was negative. Immunological
and virological screening resulted negative. The search for tumor
markers and monoclonal paraprotein was negative. In October
2008 because of partial response to steroidal, albumin and diuretic
therapy and worsening of renal function, the patient was transferred to Nephrology Department where he began haemodialysis
treatment with regression of dyspnea and significant reduction of
pleural effusion and legs edema. In November 2008 it was possible
perform renal biopsy with evidence of lymphoproliferative process. Histologically, the normal kidney architecture was extensively
replaced by lymphoma with a diffuse pattern. The neoplasm was
composed predominantly of small lymphoid cells with mildly irregular nuclear contours and moderate cytoplasm. The neoplastic
cells were CD20+, CD5-, CD10- e CD23-. The renal function
gradually improved and in December 2008 it was possible the
withdrawal of dialytic treatment. The patient was admitted to
Hematology Department with diagnosis of marginal lymphoma
stage IV°. He began the first cycle of CVP (CyclophosphamideVincristina-Prednisone). At this moment fair general conditions
and creatinine value 2.1 mg/dl. The patient did not show up the
next cycle of chemotherapy and was lost to follow-up.
Conclusions. This report shows the importance of renal biopsy
in the work-up of the renal failure even in patients with known
malignant diseases.
References
Besso L. et al. Il coinvolgimento renale nei linfomi. G Ital Nefrol 2010;
27(S50): S34-S39. Garcia M. et al. Malt lymphoma involving the
kidney. A report of 10 cases and review of the literature. Am J Clin
Pathol 2007;128:464-73.
logical and microbiological laboratory tests were unremarkable.
Renal biopsy disclosed signs of TMA: among 43 glomeruli light
microscopy revealed focal ischemic signs and mild mesangial
cell proliferation, vessel narrowing with thrombi and thickening
of arteriolar walls and intimal onion skin-like swelling, mild interstitial lymphomonocitic infiltration and focal tubular atrophy.
Immunofluorescence showed mesangial IgM (+), k (+), C1q (+)
and fibrinogen (+) staining. A diagnosis of TMA was made. She
was treated with transfusions, haemodialysis, plasma exchange
and methylprednisolone i.v. followed by oral prednisone. Cardiac
function improved and haematological signs progressively disappeared but renal function didn’t recover. IFN-β treatment was
discontinued. Now she is on peritoneal dialysis treatment.
Conclusions. TMA is a rare side effect of Alpha-Interferon treatment. The mechanism for the development of TMA associated
with IFN is not clear. IFN, such as TNF, IL-1 and free radicals,
can participate in tissue injury and endothelial cell damage with the
resulting deleterious effects. It can exert complex immunomodulatory effects on endothelial cells with differential effects on various
endothelial cell surface markers, including the mayor histocompatibility complex antigens and intracellular adhesion molecules. It
can induce modulation of fibrinolitic response of endothelial cells
through a prothrombotic way. The release of platelet-aggregating
agents from the damaged endothelial cells is probably the final
event, resulting in intraluminal thrombus formation and organ damage. In conclusion, in our patient the temporal association suggests
a direct causal effect between IFN therapy and TMA. To our knowledge, this observation is the first report of TMA induced by IFN-β.
References
1
Aravindan A, et al. Minimal change disease with interferon-beta
therapy for relapsing remitting multiple sclerosis. NDT Plus 2010;3:
132-4.
2
Magee CC. Renal thrombotic microangiopathy induced by interferon
alpha. Nephrol Dial Transplant 2001;16:2111-2.
3
Badid C, et al. Renal thrombotic microangiopathy induced by interferon-alpha. Neprhol Dial Transplant 2001;16:846-8.
4
Vacher-Coponat H, et al. Thrombotic microangiopathy in a patient
with chronic myelocitic leukemia treated with alpha-interferon. Nephrol Dial Transplant 1999;14:2469-71.
5
Galesic K, et al. Thrombotic microangiopathy associated with alphainterferon therapy for chronic myeloid leukemia. Nephrology (Carlton) 2006;11:49-52.
Thrombotic microangiopathy beta-interferon
induced
Benign glomus tumour of urinary bladder: a new
mimicker of urothelial neoplasms
R. Passantino*, G. Li Cavoli**, A. Ferrantelli**, C. Tortorici**, L.
Bono**, C. Giammaresi**, U. Rotolo**
B.J. Rocca, M.R. Ambrosio, M. Onorati, F. De Luca, M. Cintorino, S. Tripodi
Unità Operativa di Anatomia Patologica, Ospedale ARNAS Civico Di
Cristina Benfratelli di Palermo, Palermo, Italia; **Unità Operativa di
Nefrologia e Dialisi Ospedale ARNAS Civico Di Cristina Benfratelli di
Palermo, Palermo, Italia
*
Background. Many drugs have been associated with Thrombotic
MicroAngiopathy (TMA). More than 50 drugs (particularly cyclosporine, tacrolimus, anti-VEGF therapy, mitomycin-C, cisplatin, quinine, vaccines) and other substances have been associated
with the development of TMA, but many case reports are difficult
to interpret because there is uncertainty regarding the diagnosis of
TMA and the relation of drug exposure to the onset of TMA. We
report here a case of TMA Beta-Interferon (IFN-β) induced.
Case. A 36-year-old Caucasian woman, with a 3-year history of
multiple sclerosis and normal blood pressure and renal function,
was admitted for acute renal failure and pulmonary edema. Three
months previously she started subcutaneous IFN-β-1a treatment
of 22 μg thrice weekly. On admission physical examination
showed high blood pressure and severe pleuro-pericarditis without neurological or dermatological findings. We found laboratory
features of microangiopathic haemolytic anaemia. Other immuno-
Department of Human Pathology and Oncology, Anatomic Pathology Section, University of Siena, Italy
Background. Glomus tumors, also defined as glomangiomas,
are rare soft tissue tumors, which occur in both the sexes with
equal frequency mostly from 20 to 40 years of age. The great
majority are benign but some cases with atypical/malignant behaviour have been reported. They are believed to originate from
glomocytes, which are modified smooth muscle cells present
in the walls of specialized structures engaged in thermoregulation. Most often glomangiomas occur in the subungueal region,
as small blue-red painful nodules, or in the lateral areas of the
digits and the palm, which are sites where glomus bodies are
abundant. They are rare in the internal organs, not having glomus
bodies, such as is the stomach, the mesentery, the pancreas and
the lung. Reports of glomus tumours in the genitourinary regions
are extremely rare and involve mostly clitoris, vagina, cervix,
periurethral soft tissues, kidney and renal pelvis. To the best of
our knowledge, only one case of glomus tumour of the urinary
bladder has been described so far, it was malignant; we herein
report the first case of a benign one.
213
Poster
Methods. A 63-year-old patient presented with hematuria. The
endoscopic appearance was that of a polypoid lesion (12 mm
in maximum diameter) with a smooth surface, located in the
posterior wall of the bladder. During cystoscopy, a transurethral
resection was performed and the lesion was easily and completely
removed. All the fragments were routinely treated and stained
with haematoxilin and eosin; special stains (Alcian blue, collagen
IV) and immunohistochemistry (Cytokeratin 20, CKAE1/AE3,
p53, CD31, CD34, S100, chromogranin, synaptophisin, desmin,
Ki-67) were also performed.
Results. Microscopic examination, at low power, showed a
well circumscribed but not encapsulated neoplasm covered by a
thinned layer of normal urothelium with some von Brunn nests.
The tumour was composed of trabecula with an endophytic
growth that were organized around branching and dilated thin
vessels. The stroma was thin and presented mixoid areas. The
cells were monomorphic, with not well-defined cell borders,
abundant light eosinophylic cytoplasm (sometimes vacuolized)
and round to oval bland nuclei without nucleoli. Some vacuoles
had a light mucin content confirmed by Alcian-blue staining.
Mitoses were absent. Immunohistochemically, the glomus cells
did not expressed cytokeratin 20, CKAE1/AE3 and p53 whereas
these markers extensively stained superficial urothelium and von
Brunn nests. Glomus cells strongly expressed smooth muscle
actin. Collagen type IV decorated some tract of cell membrane
of individual glomus cells. Endothelial markers CD31 and CD34
were positive. Synaptophysin, chromogranin and S-100 were
negative. Proliferation index (Ki-67) was about 10%. Considering the little or absent atypia and that none of the criteria of the
Folpe’s classification of malignant glomus tumours was present,
the tumor was diagnosed as benign. The main differential diagnosis included: inverted papilloma and papillary urothelial neoplasm
of low malignant potential and urothelial carcinoma of low grade
(both excluded by CK20, CKAE1/AE3, p53 negativity), carcinoid (excluded by chromogranin and synaptophysin negativity),
nephrogenic adenoma (characterized by cuboidal and columnar
cells), paraganglioma (S100 was negative), hemangiopericytoma
(excluded due to the positivity of CD34), and smooth muscle
neoplasms (desmin was negative).
Conclusions. To the best of our knowledge, this is the first case
of benign glomus tumor of the bladder. This report widens the
spectrum of the differential diagnosis with bladder urothelial
neoplasms.
Carcinosarcoma of the kidney with concurrent
adenoma of the adrenal gland in a MEN1 patient
R. Santi*, T. Cavalli**, F. Giudici**, M. Pepi*, M.L. Brandi***, F.
Tonelli**, G. Nesi*
*
Department of Pathology, University of Florence, Italy; **Department of
Clinical Physiopathology, University of Florence, Florence, Italy; ***Department of Internal Medicine, University of Florence, Florence, Italy
Multiple neuroendocrine neoplasia 1 (MEN1) is a rare autosomal
dominant disorder characterized by primary endocrine abnormalities involving the pituitary, parathyroid, endocrine pancreas
and duodenum. Adrenal cortex is affected in up to 40% of MEN1
patients, generally with non-functional hyperplastic lesions. Nonendocrine neoplasms have been described in MEN1 patients as
uncommon and possibly coincidental findings. Among them, renal tumours are exceedingly rare. A case of carcinosarcoma of the
kidney with concurrent adenoma of the ipsilateral adrenal cortex
in a patient affected by MEN1 syndrome is herein described.
A 36-year-old man, subjected to radiological follow-up for MEN1
syndrome, was diagnosed with a 3-cm mass in the left kidney. On
Magnetic Resonance Imaging (MRI) with contrast medium, the
renal mass showed irregular enhancement suggestive of malignancy. Also evident was a 1.5-cm nodule in the left adrenal gland,
indicative of cortical adenoma. The patient’s past medical history
gave duodeno-pancreatectomy for multiple endocrine tumours
of the pancreas and total parathyroidectomy for primary chief
cell hyperplasia. Biochemical and hormonal profile was normal.
Tumour markers, i.e. alpha-fetoprotein, CA 125, CEA and CA
19-9, were within the normal range. The patient underwent left
nephrectomy with ipsilateral adrenalectomy.
Grossly, the renal neoplasm was grey-white in colour, with
haemorrhage, necrosis and bony hard areas. The adrenal lesion
appeared as a small, encapsulated, golden-yellow nodule. On
histological examination, the kidney tumour consisted of an
epithelial component with tubulo-papillary morphology and a
mesenchymal component with manifested areas of osteosarcoma.
No neoplastic invasion of the perirenal fat and vascular structures was detected. The adrenal cortical tumour was composed
of compact cells with eosinophilic cytoplasm, arranged in cords
and nests. There was no evidence of capsular and vascular invasion. Necrosis and atypical mitotic figures were not identified.
The proliferative fraction was immunohistochemically assessed
with Ki-67 at less than 1%. A diagnosis of renal carcinosarcoma
and adrenal cortical adenoma was established. Somatic loss of
the wild-type allele (loss of heterozygosity - LOH) at 11q13 was
demonstrated in the cortical adenoma, but not in the kidney tumour. The patient is free of disease at nine months follow-up.
Sarcomatoid differentiation in renal cell carcinoma, first described by Farrow et al. in 1968, may be found in all of the
major renal cell carcinoma subtypes. It is characterized by highly
pleomorphic spindle cells and/or giant cells resembling sarcoma
and is indicative of an aggressive tumour. In a few reports, the
sarcomatoid component consists of areas of chondrosarcoma and
osteosarcoma. Such entities could be designated as carcinosarcoma. In the present case, pathological and genetic findings
suggest that the occurrence of renal tumour was unrelated to the
inherited condition. Contrariwise, benign adrenal tumours, although uncommon, are considered among the features of MEN1
syndrome.
Worrisome histologic features in benign
renal oncocytoma: immunoistochemical and
cytogenetic analysis
D. Segala, S. Gobbo, E. Munari, C. Cannizzaro, M. Ficial, M.
Chilosi, M. Brunelli, G. Martignoni
Italia
Renal oncocytoma in a benign epithelial neoplasm that accounts
for about 7% of kidney tumors and affects patients aged between
20 and 80 years, with a peak incidence in the seventh decade.
Sometimes renal oncocytoma shows atypical histological features
such as macroscopic central scar composed of worrisome tumor
cells with either clear cell changes or basophilic type 1 papillary renal cell carcinoma-like appearance, cytological atypia,
oncoblasts, necrosis, perirenal fat infiltration, lympho-vascular
invasion. These worrisome histological features can represent
a potential source of misdiagnosis of malignancies, especially
when the pathologist has limited material avaiable for the diagnosis, such as in the context of kidney tumor biopsies where the
choice of different treatment options are important.
The aim of the study is to characterize the immunophenotypical
and cytogenetic profile renal oncocytomas with atypical features,
to verify if molecular characteristics can help the pathologist in
the their differential diagnosis with malignancies.
Seventy-eight renal oncocytomas were retrieved from the archive
of the department of Pathology and Diagnostic, University of
Verona. Revision of the whole histological slides were performed, with morphological identification of atypical features
(central scar, perirenal fat infiltration, lymphovascular invasion,
cytological atypia, oncoblasts, mitosis, necrosis, calcification,
entrapped tubules). Ten cases representative of the group of wor-
214
risome histologic features were selected for immunophenotypical
and cytogenetic analysis. Immunohistochemistry was performed
using antibodies against Parvalbumin, CD10, CD13, Vimentin,
Cytocheratin 7 (CK7), Racemase and S100A1. Fluorescence In
Situ Hybridization (FISH) was used to detect chromosome 1, 2,
6, 7, 10, 17, Y and 11q13 abnormalities in “classical” and worrisome patterns.
The results showed that 51% of the tumors had at least one
atypical morphological pattern. Among renal oncocytomas with
atypical features, the tumor component with “classical” morphology had overlapping immunophenotypical and cytogenetic characteristics compared with oncocytomas without atypical aspects.
Cell proliferations in the context of central fibrous scar had an
immunophenotype similar to that observed in papillary renal cell
carcinoma (constant expression of CK7 and variable expression
of CD10, CD13 and Racemase), but the entire chromosomal
profile tested showed disomies. All other atypical morphological
features had a disomic chromosomal profile, with the exception
of the areas of cytological atypia, that demonstrated frequent
trisomies (67% of cases).
In conclusion, immunohistochemistry and cytogenetic investigations could be a useful tool in differential diagnosis between
benign renal oncocytoma with atypical feature and malignant
epithelial tumors of the kidney, especially when the diagnosis
should be done on limited material.
Role and distribution of pentraxin 3 (PTX3)
in glomerular lesions of HIV positive patients
mal renal parenchyma was used to test PTX3 distribution in non
inflamed renal tissue.
IHC was performed on formalin-fixed paraffin embedded biopsies, by using affinity-purified rabbit IgG against human PTX3
(raised in our laboratory).
Results. Normal renal tissue was negative for PTX3 expression. In HIV positive subjects, the higher PTX3 positivity was
observed in cases of HIVAN, IgAN, MGN. NC-FSGS cases had
a very low PTX3 expression. The positive staining was mainly
in the interstitium; glomerular positivity was found in rare cases,
with a mesangial pattern. The correlation between PTX3 positive
areas and interstitial inflammatory infiltrate, sclerosis and C3c/
C1q immunofluorescence deposition was lacking. Comparing
PTX3 expression in HIV positive and negative subjects (even
excluding HIVAN) was higher in HIV patients.
Conclusions. These preliminary findings seem to support the role
of HIV infection, and the following systemic immunomodulation,
in the expression and distribution of the proinflammatory protein
PTX3 in renal parenchyma of patients with glomerular disease.
Patologia mammaria
Colon Metastasis of Ductal Breast Cancer:
Description of a case
V. Arena*, I. Pennacchia*, R. Ricci*, G. Palazzoni**, F. Marazzi,
F.M. Vecchio*
Istituto di Anatomia Patologica; **UOC Radioterapia, Università Cattolica del Sacro Cuore – Roma
A. Vitale*, A. Tosoni**, L. Zawada**, F. Genderini*, S. Caruso*, G.
Vago**, G. Barbiano di Belgiojoso*, M. Nebuloni**
*
*
U.O. Nefrologia, Osp. L. Sacco; **Anatomia Patologica, Dip. Scienze Cliniche L. Sacco, Università degli Studi di Milano, Milano, Italia
Colonic metastases of breast cancer, although rare, may mimic
other disease states, which may impair the clinical diagnosis and
delay treatment, resulting in earlier mortality. We herein report a
case of a 55-year-old woman who presented to our hospital complaining of weakness, abdominal pain and diarrhea. 7 years before
she had undergone a partial mastectomy followed by adjuvant
chemotherapy for a left infiltrating ductal breast carcinoma with
cervical lymph nodes metastasis (pT2N3M0, stage IIIc). Laboratory tests showed high levels of Ca 15.3 and CEA, whereas routine
radiological examinations of the thorax, abdomen and pelvis were
all normal. A PET/CT showed hypercaptation in the ascending
colon and a colonoscopy was performed. In the same area of the
abnormal hypercaptation, a friable, ulcerated lesion was observed
and a biopsy was performed. Histological examination of the colon biopsy specimen displayed surface epithelium with the lamina
propria infiltrated by tumor cells with abundant eosinophilic cytoplasm, many with eccentric nuclei. Immunohistochemically,
the tumor cells were positive with cytokeratin 7, GCFDP-15 and
estrogen receptor and negative with cytokeratin 20. Based on the
histological and immunohistochemical findings a diagnosis of colon metastasis from ductal breast cancer was made.
The patient is currently being treated with preoperative chemotherapy and hormonal therapy. A surgical treatment with a right
hemicolectomy is planned after the chemotherapy has finished.
Breast cancer is the most common cancer and leading cause
of cancer deaths among women worldwide. Common sites of
metastasis for breast cancer include bone, liver, lung and brain.
Gastrointestinal metastasis from breast cancer are rare and usually arise from lobular variety, with the upper gastrointestinal
tract more frequently involved than the colon. An early diagnosis
of gastrointestinal metastasis of breast cancer is difficult to make
due to the nonspecific nature of the symptoms so the prognosis
is usually poor. Surgical resection of gastrointestinal metastasis
could increase survival rates only in women in whom this is the
unique site of metastatic involvement.
PTX3 is a prototypic member of the long pentraxin family. PTX3
is involved in innate resistance to pathogens, controlling inflammation and extracellular matrix remodelling. PTX3 binds to C1q
and activates the complement cascade, stimulates fibroblasts,
thus favouring collagen matrix deposition. PTX3 is produced in
inflammatory conditions by different cells, such as fibroblasts,
monocytes/macrophages, dendritic and endothelial cells.
Glomerular involvement in HIV positive patients is characterized
by a wide spectrum of lesions. The most peculiar glomerular
lesion is HIV-associated nephropathy (HIVAN), which is characterized by focal and segmental glomerular sclerosis with prominent tuft collapse, hypertrophy /hyperplasia of podocytes, marked
tubular/interstitial changes.
Immune-complex glomerulonephritis (GN), such as membranoproliferative GN (MPGN), membranous GN (MGN), IgA nephropathy (IgAN), and other types of glomerular lesions not related
to immune-complex deposition (i.e. non-collapsing focal segmental glomerusclerosis –NC-FSGS, minimal changes disease
and other minor glomerulopathies) are also described in HIV
population. Non-HIVAN glomerulopathies in seropositive patients are histologically similar to those observed in non HIV subjects, although some peculiar aspects are found. Immunological
mechanisms, not completely clear so far, have been speculated to
explain these particular differences. For this reason, glomerular
disease in HIV patients could be an interesting field of research
about the role of PTX3.
The aim of the study was to assess PTX3 distribution in different types of HIV–related glomerular lesions, describing cell type
production and correlation with histological lesions.
Materials and methods. Thirty-one biopsies of cases with HIV
glomerulopathies were tested for PTX3 expression by immunohistochemistry (IHC): 7 HIVAN, 2 MPGN, 4 MGN, 4 IgAN, 9
NC-FSGS, 4 end stage renal disease (ESRD). Twenty-two cases
of glomerular diseases in HIV negative subjects, with comparable
histological diagnosis, were selected as controls. Moreover, nor-
215
Poster
Simultaneous fluorescence immunophenotyping
and her-2/neu genotyping (fiction) in breast
carcinomas candidates to target therapy
HER2 status in operable her2 positive breast
cancer patients treated with neoadjuvant
chemotherapy with or without anti-HER2 therapy
P. Balzarini, L. Benerini Gatta, M. Cadei, S. Simoncelli, P.
Grigolato
S. Bettelli*, G. Ficarra*, P.F. Conte**, V. Guarneri**, F. Piacentini**, L. Reggiani Bonetti*, A. Maiorana*
2nd Department of Pathology /University of Brescia, Brescia, Italy.
Laboratori, Sez. di Anatomia e Istologia Patologica, Azienda Ospedaliero
Universitaria, Policlinico di Modena, Italia; **Dipartimento di Oncologia,
Ematologia e Malattie Respiratorie, Azienda Ospedaliero Universitaria,
Policlinico di Modena, Italia
The use of FISH to study the status of proto-oncogene Her-2/neu
in routinely fixed paraffin- embedded tissue has become commonplace over the past decade. The Her-2/neu evaluation using
FISH technique is necessary for the characterization of breast
lesions expressing c-erbB2 protein with the score 2+, candidates
for treatment with the biological drug Trastuzumab / HerceptinTM.
The choice to the biological therapy is given using the Her-2/neu
proto-oncogene amplification (ratio Her-2/neu/CEP-17 > 2.2).
While the exclusion is declared in the absence of Her-2/neu gene
amplification (ratio Her-2/neu/CEP-17 < 1.8) in according to ASCO/CAP recommendations. However there are borderline cases
(1.8 < ratio Her-2/neu/CEP-17> 2.2) that need to be investigated;
ductal carcinoma in situ with microinvasion cases, metastasis and
such cases with Her-2/neu genetic heterogeneity, in which the
count of nuclear signals in the areas of invasive tumor, is difficult
to perform with fluorescence. The availability of a FICTION
technique, including the simultaneous evaluation of cytokeratins
(CK AE1/AE3; CK19), and FISH for Her-2/neu gene status, it
is therefore useful and of current applicability. For the first time
immunophenotyping and Her-2/neu FISH technique combined
have been successfully applied in breast cancer (formalin fixed
and paraffin-embedded samples) thus we were able to visualize
the antigen expression of neoplastic cells with Her-2/neu status
gene directly.
PI3KCA mutations in HER2-positive breast
carcinomas treated with trastuzumab
M. Barbareschi1 2 3, L. Cuorvo3, S. Girlando1 3, E. Leonardi1, C.
Eccher4, A. Ferro5, A. Caldara5, R. Triolo5, C. Cantaloni2, E. Galligioni5, P. Dalla Palma1.
Unit of Surgical Pathology; 2Trentino Biobank; 3Laboratory of Molecular Pathology, Unit of Surgical Pathology; 5 Unit Of Medical Oncolgy, S.
Chiara Hospital, Trento, Italy; 4 Kessler Foundation, Trento, Italy 1
Introduction. Aberrations of the components of the PI3K-AKT
pathway are frequent in infiltrating breast carcinomas (IBC) and
activating mutations of the PI3KCA gene mainly occur at hotspot
in exons 9 and 20.
Material and methods. We evaluated 127 Her2-positive IBC
treated with trastuzumab at the S. Chiara Hospital of Trento,
including 21 primary IBC treated with neoadjuvant trastuzumab,
47 advanced metastatic IBC, and 59 early stage IBC treated with
adjuvant trastuzumab therapy. Genomic DNA was extracted from
each paraffin-embedded tumor block using QIAamp DNA MiniKit (Qiagen Inc., Hilden, Germany). The sample were analyzed
with the Real-Time PCR and the pyrosequencing reaction was
performed according to the manufacturer’s instructions PyroMark™ IDQ96 V2.0 kit (Qiagen). Pyrosequencing™ was performed using the PyroMark™ Gold Q96 reagent kit (Qiagen).
Results and discussion. In our series PI3KCA gene mutations
were observed in 19% of locally advanced IBC, 27 % in metastatic IBC and 11% in early stage IBC. PI3KCA gene mutations
were not associated with tumor size, grade, ER and PgR status
and proliferative activity and were not predictive of response to
trastuzumab treatement.
Aknowledgemets. This study has been supported by grants of
the Provincia Autonoma di Trento and of the Fondazione Cassa
di Risparmio di Trento e Rovereto.
*
Introduction. A different HER2 expression from primary breast
carcinomas to metastatic deposits has been reported in the recent
literature. Tumor heterogeneity, genetic drift and the effect of the
adjuvant therapy might explain this phenomenon.
Methods. We evaluated the change in HER2 expression in two
consecutive cohorts of HER2+ breast cancer patients treated with
neoadjuvant therapy. The first cohort (Group A) included 38
patients enrolled before 2005, treated with chemotherapy alone.
The second cohort (Group B) included 48 patients treated with
neoadjuvant chemotherapy in combination with antiHER2 agents
(trastuzumab or lapatinib). HER2 expression was evaluated by
IHC on pre-treatment core biopsy (tru-cut with 14 gauge needle)
and on surgical specimen after neoadjuvant therapy. FISH analysis was performed on IHC 2+ samples.
Results. The two Groups were balanced in respect of tumor stage,
patient age, and HR expression. In particular, a co-expression of
HER2 and HR was observed in 60% of the patients in Group A
and in 70% of the patients in Group B (p=0.2). Patients of the
Group B had a significantly higher rate of pathologic complete
response (pCR) in comparison to those of Group A (45% vs
11%, p=0.001). A change in HER2 expression from biopsy to
post-therapy samples was observed in 39% of the patients of the
Group A vs 12% of the patients of the Group B (p=0.02). No
patient with pCR had recurrences so far, vs 25% of the patients
with no pCR (p=0.005). The rate of recurrence was significantly
higher in patients experiencing a change in HER2 expression
(50% vs 19%, p=0.018).
Conclusion. Contrary to our expectations, patients not receiving
anti-HER2 therapy as part of neoadjuvant therapy were more
likely to have a change in HER2 status vs patients receiving antiHER2 neoadjuvant therapy. The change in HER2 status has a
negative prognostic impact.
Changes in receptor status between primary and
recurrent breast cancer: prognostic significance
S. Bettelli*, G. Ficarra*, F. Piacentini**, M. Dominici**, P.F.
Conte**, V. Guarneri**, L. Reggiani Bonetti*, A. Maiorana*
Laboratori, Sez. di Anatomia e Istologia Patologica, Azienda Ospedaliero
Universitaria, Policlinico di Modena, Italia; ** Dipartimento di Oncologia, Ematologia e Malattie Respiratorie, Azienda Ospedaliero Universitaria, Policlinico di Modena, Italia
*
Introduction. The main determinants of treatment selection in
breast cancer are the expression of hormone receptor and HER2
status. The reassessment of tumor phenotype in recurrent disease
might have an impact on patient’s management and prognosis.
Aim of this analysis is to evaluate the impact of discordance in
triple-receptor status between primary and recurrent tumors in
patients with relapses.
Methods. 103 primary tumors and paired local recurrences or
metastases were studied. HER2 status was evaluated by immunohistochemistry (IHC) and/or fluorescent in situ hybridization
(FISH). Estrogen receptor (ER) and Progesteron receptor (PgR)
status were assessed by IHC; samples were considered as HRpositive in case of ER and/or PgR > 10%. Tumor specimens
were classified as triple negative (HR- and HER2-negative) or
216
non-triple negative (HR-positive and/or HER2-positive). The
impact of triple-receptor status changes between primary and
recurrent tumors on post-distant progression survival (PDPS)
were evaluated.
Results. Recurrent disease included distant metastases in 79
cases (81%) and local relapses in 24 patients (19%). Seventyeight percent of primary tumors were HR-positive; 19% were
HER2-positive. Seventy-four percent of recurrent tumors were
HR-positive and 25% were HER2-positive. A change in HER
status between primary and recurrent matched samples was observed in 15.5% of cases. Of 103 patients, 78 (75.7%) maintained
a non-triple negative phenotype in both primary and recurrent
disease (concordant non-triple negative), whereas 10 (9.7%)
showed a triple-negative phenotype in both primary and recurrent
sites (concordant triple negative). Eight patients (7.8%) had their
tumor status changed from non-triple negative (primary tumor)
to triple negative (recurrent disease). Seven patients (6.8%) had a
non-triple negative primary tumor that became triple negative at
recurrence. Of 103 patients with recurrent breast cancer, a distant
progression was observed in 93 cases. Patients with concordant
non-triple negative disease had significantly longer PDPS than
the other subgroups (p=0.0002). In particular, the patients with
tumor phenotype that had changed from non-triple negative to
triple negative had the worst prognosis compared to the concordant non-triple negative subgroup (HR 5.26, 95%CI 2.13-12.96;
p=0.0013).
Conclusions. In this analysis, tumors maintaining positivity for
HR and/or HER2 experienced a better outcome, reflecting the
correct use of available targeted agents as well a less aggressive
tumor phenotype. Patients with a changed phenotype, from nontriple negative (primary site) to triple negative (recurrent site)
have a particularly poor outcome. This might be due to the lack
of targeted therapies for metastatic/recurrent disease and to a biological change of the disease to more aggressive phenotypes.
Literature review and personal experience
on ttf-1 expression in carcinomas of the breast
M. Bisceglia *, F. Fiordelisi *, G. Falconieri **, G. DeMaglio **, L.
Andreini ***,. R. Nannini ***, M. Ricci ****, M. Brisigotti ****.
Unità Operativa di Anatomia Patologica, IRCCS – Ospedale “Casa
Sollievo della Sofferenza”, San Giovanni Rotondo (FG), Italia; ** Unità
Operativa di Anatomia Patologica, Ospedale Generale “S. Maria della
Misericordia”, Udine, Italia; ***. Unità Operativa di Anatomia Patologica, Azienda USL Imola, Imola, Italia; **** Unità Operativa di Anatomia
Patologica, Azienda USL Rimini, Rimini, Italia.
*
Background. TTF-1 is a nuclear transcription factor which plays
a fundamental role in morphogenesis and functionality of the
thyroid, lung, and diencephalon. Currently, TTF-1 is commonly
used as a lineage specific immunomarker in surgical pathology to
ascertain lung or thyroid origin in metastatic tumors of unknown
primary. However, diminishing the claims of TTF-1 as a specific
marker of lung and thyroid tissue, there is an increasing number
of reports documenting its sporadic and, in some circumstances,
frequent expression in tumors of disparate origins, mostly of neuroendocrine, but also of non-neuroendocrine nature (Table 3 in
Bisceglia et al - AJSP 2009;33:454). TTF-1 negativity in breast
cancer has traditionally been considered to be a mainstay of practical immunohistochemistry interpretation and its expression in a
given metastatic tumor was thought to exclude the possibility of
breast origin. Most recently Robens et al (AJSP 2010;34:1881),
using the antibody clone SPT24, reported TTF-1 expression in 13
of 546 usual breast carcinomas (2.4%). Accordingly the presence
of TTF-1 immunoreactivity in a tumor of unknown origin cannot
rule out breast origin.
Materials and methods. We reviewed the world literature
pertaining to TTF-1 immunoexpression in breast carcinomas
and conducted an immunohistochemical investigation of TTF-1
nuclear staining using antibody clone 8G7G3/1 in 30 cases of
ordinary invasive ductal carcinoma (IDC) (20 primaries, 10
metastases), 14 nonsmall cell neuroendocrine breast carcinomas (NON-SC-NEC, including 5 so-called carcinoid tumors, 2
neuroendocrine carcinomas not-otherwise-specified, 2 invasive
ductal carcinomas with neuroendocrine features, 1 invasive lobular carcinoma with neuroendocrine features, and 4 type B colloid
carcinomas). Of the 2 small cell (neuroendocrine) carcinoma
(SCC) of the breast, one was tested with antibody clone 8G7G3/1
and the other one with clone SPT24.
Results. From the literature. Ordinary carcinoma - Of the over
200 cases of primary and metastatic breast carcinomas of the
usual type that had been tested with either clone before the report
of Robens et al, none showed TTF-1 expression (Bisceglia et al,
AJSP, in press [Letter]), although there is a questionable report
in the literature claiming positive TTF-1 immunoreactivity in 4
of 5 IDC studied (Yamamoto, Oncol Rep 2004;11:825). NONSC-NEC – We have found only one (questionable) study assessing TTF-1 expression in carcinomas of breast with endocrine
features, in which 5 of 5 proved positive for TTF1 (Yamamoto).
Further 2 additional such tumors are mentioned in the article by
Robens et al, aside from the main study, as expressing TTF-1.
SCC: of a total of 47 known cases of SCC of the breast reported
in the literature, 9 were studied with TTF-1, and 6 of them were
positive (personal updated review). Personal cases. With regard
to our personally evaluated 30 ordinary invasive breast carcinomas and 14 NON-SC-NEC, we tested with 8G7G3/1 all were
negative, and of the 2 SCC the one which was tested with clone
8G7G3/1 was negative, while the other one, tested with clone
SPT24, was positive.
Discussion and conclusions. Of the 2 main antibody clones commercially available, SPT24 is more sensitive but less specific than
clone 8G7G3/1. This has been recorded in several publications
documenting TTF-1 immunoreactivity with SPT24 in colorectal
carcinomas, glial neoplasms, and gynecologic tumors, findings
which were not confirmed or were otherwise not in complete
agreement with those obtained with 8G7G3/1 (Bisceglia et al,
AJSP, in press [Letter]). In regard to the issue of TTF-1 in ordinary breast carcinoma, there is strong evidence that the main
factor influencing the prevalence of TTF-1 expression in tumors
(other than those of pulmonary or thyroid origin) is the type of
clone that is used, and as we believe that some positive results
obtained with SPT24 may not be confirmed with 8G7G3/1, we
will accordingly continue to use with caution clone 8G7G3/1 as
the choice marker in problematic cases. As to NON-SC-NEC of
the breast they are likely analogous to well differentiated NEC
of the GI tract and pancreas, most of which are negative, while
SCC of the breast are equivalent to SCC of other sites. From the
literature TTF-1 is often positive with either clone used, although
in our 2 cases only 1 was positive (with clone SPT24). TTF1
cannot be used to differentiate primary SCC of the breast from
metastatic pulmonary SCC.
Clinical impact of her-2 assessment in minor
morphological subtypes of breast carcinoma
by asco/cap guidelines
E. Brunello, M. Brunelli, A. Nottegar, E. Bragantini, M. Barbareschi, P. Dalla Palma, E. Manfrin, M. Chilosi, S. Gobbo, G.
Martignoni, F. Bonetti.
Department of Pathology and Diagnostic, University of Verona, Italy.
Background. Minor morphological subtypes (non-ductal, nonlobular) breast carcinomas represent around 10-15% of cases. To
address the paucity of information concerning HER-2 amplification for minor subtypes according to new ASCO/CAP cut-offs,
we studied a serie of non-ductal non-lobular breast cancer.
Methods. 41 cases of minor subtype of breast carcinoma: 6 med-
217
Poster
ullary, 2 adenoid cystic, 5 apocrine, 5 signet ring, 5 mucinous,
5 tubular, 3 small cell, 10 papillary were studied. We evaluated HER-2/neu status by comparing the original FDA and new
ASCO/CAP scoring systems.
HER-2 immunoexpression was analyzed by using Hercept Test
and in cases (2+) we assessed the Her-2/neu status by FISH.
Results. All tubular, mucinous, squamous, medullary, papillary, adenoid-cystic did show a negative Her-2 value at both
IHC (0-1+) and FISH(-) levels using both FDA and ASCO/CAP
cut-offs. Signet ring, apocrine and small cell carcinomas showed
discordance between cut-offs with an heterogeneous selection to
therapy.
Conclusions. The Her-2 assessment in minor (non-ducta, nonlobular) subtypes of breast carcinoma by ASCO/CAP guidelines
does not overall change the rate of patients candidated to Trastuzumab therapy. A better selection of these cohort of patients
to targeted therapy resides in new cut-offs or probably in other
correlated biological characters.
FGFR1 in metastatic and primary lobular breast
carcinoma. A biological rationale for new
therapeutic option.
E. Brunello, M. Brunelli, A. Nottegar, E. Manfrin, G. Bogina,
G. Zamboni, S. Pedron, E. Vittoria, M. Chilosi, G. Martignoni,
F. Bonetti
Department of Pathology and Diagnostic, University of Verona, Italy
Background. Lobular breast carcinoma usually shows poor
responsiveness to chemotherapies and often lacks targeted therapies. Since FGFR1 expression has been shown to play pivotal
roles in breast cancer tumorigenesis and FGFR1 inhibitor has
been recently developed, we sought to analyze the status of
FGFR1 gene in lobular breast carcinoma.
Methods. Twenty infiltrative lobular carcinomas where recruited
and tissue microarrays were constructed (3 cores for each case),
11 of which had available matched loco-regional lymph-nodal
metastasis. FGFR1 gene (8p12) amplification was evaluated by
cromogenic in situ hybridization (CISH) (Zytovision) analyses.
Results. Three (15%) primary lobular breast carcinomas showed
FGFR1 amplification (cluster of signals), whereas in 11 (55%)
was not observed any abnormality. Six cases (30%) had three
cromogenic signals. Two of 11 metastasis (18%) were amplified,
2/11 (18%) did not. The seven remaining cases (64%) showed
three cromogenic signals.
Conclusions. 1) A subset of lobular breast carcinoma, either
primary either metastatic, show FGFR1 amplification or gains of
cromogenic signals; 2) there is heterogeneity in matched primary
and metastatic carcinomas; 3) in the era of tailored therapies,
patients affected by the lobular subtype of breast carcinoma with
FGFR1 amplification could be approached to the new target biological therapy such as FGFR-1 inhibitor.
Quantitative measurement of cytokeratin 19
MRNA by one step nucleic acid amplification
(OSNA) is predictive of non-sentinel lymph node
status in breast cancer with a micrometastatic
sentinel node
S. Buglioni*, M. Mottolese*, B. Casini*, E. Gallo*, L. De Salvo*,
B. Claudio**, F. Di Filippo**, I. Terrenato***, A. Russo*, F. Marandino*, E. Pescarmona*
Servizio di Anatomia, Istologia Patologica e Citodiagnostica, Istituto Nazionale Tumori Regina Elena, Roma, Italia; **Chirurgia Generale, Istituto
Nazionale Tumori Regina Elena, Roma, Italia; ***Epidemiologia, Istituto
Nazionale Tumori Regina Elena, Roma, Italia
*
Background. The current standard of care for breast cancer
patients with a positive sentinel lymph node (SLN) is the com-
pletion of level 1 and 2 axillary lymph node dissection (ALND).
However, 40-70% of patients with positive SLN are undergoing
unnecessary ALND. Accurate estimates of the likelihood of
additional nodal metastases may be helpful in decision making
about further treatment, especially in the setting of patients with
minimal disease in the SLN (i.e., ≤2mm). To predict non sentinel
lymph nodes (NSLN) metastases in patients with a positive SLN,
different nomograms have been created, but they are not accurate
for SLN micrometastasis. In this context, the new molecular
OSNA method, based on the quantitative measurement of Cytokeratin 19 (CK19) mRNA in SLN, could represent a helpful diagnostic tool. In our Institute we validated the OSNA method on a
large series of 900 breast cancer patients in parallel with standard
histology (concordance rate 96%), then we started to analyze the
entire SLN by OSNA. The aim of this study was to correlate the
copy number of CK19 mRNA with the risk of additional positive
NSLN focusing on micrometastatic SLN
Material and methods. The intraoperative clinical study was
conducted on 250 fresh SLN from 185 consecutive patients with
clinically node negative breast cancer. A CK19 mRNA copy
number/mL lysate less than 250 copies/mL was regarded as
negative; copy number between 250 and 5000/mL was regarded
as micrometastasis, and copy number greater than 5000/mL as
macrometastasis. In patients with positive OSNA result, the probability of having a positive lymph node axillary dissection was
calculated by the unconditional logistic regression model.
Results. OSNA positivity for micro or macrometastasis was
found in 47/250 cases (18,8%). All these patients underwent axillary dissection in the same surgery and the axillary lymph nodes
were analyzed post-operatively by standard histological procedures. Twenty out of the 47 positive cases had a CK19 mRNA
copy number between 250 and 5000/mL and were regarded as
having a micrometastatic SLN. In this subset of patients the
metastatic involvement of NSLN is significantly associated with
the highest copy number (3000 ≤ copies < 5000 mRNA/µL) in
SLN (3 out of 5 cases had a positive ALND). In contrast, none
of the 15 patients with a micrometastatic SLN presenting a copy
number between 250 and 3000, had a positive axillary dissection
(p<0.0001).
Conclusions. Our data confirmed that the semiquantitative OSNA
method enables accurate automated intraoperative diagnosis with
the advantage of being reproducible, standardized and objective.
Of particular clinical interest, we showed that molecular driven
analyses may be useful to build new models highly predictive of
breast cancer axillary status in patients with a SLN positive for
micrometastasis.
Aberrant expression of cancer stem cell markers
in a low grade tubulobular breast carcinoma:
a correlative study between quantitative
mrna expression, flow cytometric and
immunohistochemistry analysis
F. Collina*, M. Di Bonito*, M. Cantile*, R. Camerlingo**, M. Cerrone*, L. Marra*, G. Liguori*, G. Pirozzi**, G. Botti*
SC Anatomia Patologica e Citopatologia, INT Fondazione G. Pascale,
Napoli; **Dip. Oncologia Sperimentale, INT Fondazione G. Pascale, Napoli
*
Recent concepts for cancer development suggest that a minority population of cancer stem-like cells (CSCs) may determine
the biologic behavior of tumors, including response to therapy.
Recently it was demonstrated a consistent presence of CSCs in
residual breast cancers after both neoadjuvant chemotherapy, and
endocrine therapy.
The initial reports about breast cancer stem cells describe the use
of CD44+CD24−/low cell-surface antigen signature to select CSCs.
However it was recently shown that CD44+CD24−/low phenotype
detection is not sufficient, alone, to characterize breast CSCs.
218
In cancer stem cell research has recently included Prominin-1,
CD133, a pentaspan transmembrane glycoprotein with a molecular weight of 120 kDa, that was initially considered to be a
marker of hematopoietic stem cells. Recently has been reported
the detection of CD133 expression in invasive ductal breast carcinomas and it was showed, mainly in triple-negative invasive
ductal breast carcinoma patients, that the expression of CD133
protein could be correlated with tumor size, metastasis of the
axillary lymph nodes and the clinical stage.
In all cases documented the percentage of stem cells selected by
CSCs markers immunostaining varies generally from 2 to 40%
and is strongly connected to grade and aggressiveness.
In this study we have identified a low grade tubulobular variant
of breast cancer showing an aberrant expression of prominin-1
marker (>70%). The hyperexpression of CD133 was evaluated by
Flow cytometry analysis, confirmed by immunohistochemistry
and for gene expression by Quantitative Real Time PCR.
Overexpression of cell cycle progression inhibitor
geminin is associated with tumor stem-like
phenotype of triple negative breast cancer
M. Di Bonito, M. Cantile, F. Collina, G. Scognamiglio, M. Cerrone, G. Liguori, G. Botti
Pathology Unit, National Cancer Institute, Pascale Hospital, via Mariano
Semmola 80131, Naples, Italy
Introduction. Triple-negative breast cancer, characterized by
tumors that do not express estrogen receptor (ER), progesterone
receptor (PR), and HER-2 genes, has a significant clinical relevance being associated with a shorter median time to relapse and
death and do not respond to endocrine therapy or other available
targeted agents.
It was been suggested that the increased aggressiveness of certain
types of cancer as well as resistance to standard drug therapies
may be associated with the presence of stem cell populations
within the tumor. Some molecular pathways associated with cell
cycle regulation may be directly linked to the preservation and
propagation of cancer stem cells.
Geminin is a nuclear protein that, during specific phases of the
cell cycle, is able to negatively regulate the function of Cdt1,
inhibiting the cell replication. In several studies geminin appears
frequently overexpressed, in vivo, in a variety of human tumors
(Kidney, colon, breast, lung cancer, salivary gland and lymphoma) and, in vitro, siRNA suppression of geminin is able to arrest
proliferation only of tumoral cells.
Aim. In this study we investigated the role of geminin in Triple
Negative breast cancers and its potential correlation with stemlike phenotype of this neoplasia.
Methods. We used tissue microarray technology building a specific Triple Negative Breast Cancer TMA. Geminin and cancer
stem cell marker CD133 expression was further investigated at
mRNA level for selected breast tumor samples through real-time
quantification.
Results and conclusions. Our results, made at gene and protein
level for both CD133 and geminin expression, showed a strong
correlation between these markers suggesting their potential
role in the tumor evolution and progression of this breast cancer
subtype.
Cancer stem cell marker CD133 and related geminin expression
could represent new molecular markers ables to better stratify
subsets of patients with triple-negative disease for different treatment approaches of subtypes with differential responsiveness to
specific agents.
Collagenous spherulosis and adenoid-cystic
carcinoma of the breast: immunohistochemistry
and usefulness of CD10 and HHF35 actin
A.G. Giannone, D. Cabibi
Studi di Palermo, Palermo, Italia.
Collagenous sferulosis of the breast (CS) is an uncommon benign lesion 1 usually representing an incidental finding in breast
samples removed for other benign and malignant lesions. Due to
its rarity and to its morphological aspects, it is sometimes overlooked or misdiagnosed as cribriform carcinoma or as adenoid
cystic carcinoma (AdCC) 2 but the differential diagnosis with
AdCC represents the most difficult challenge 3. Both CS and
AdCC, in fact, consist of cribriform proliferation of epithelial and
myoepithelial cells forming a sieve-like pattern with pseudocystic
spaces containing acellular basal membrane material 1 2 3 and the
immunophenotypic overlap of CK5/6 and of some myopithelial
markers, such as p63 and smooth muscle actin, could induce to
potential diagnostic pitfalls 3 4 5.
In this study, we assessed the expression of CD10, HHF35 actin,
smooth muscle actin (SMA), CEA, p63, c-kit (CD117), CK5/6,
Estrogen and Progesteron receptors (ERs, PRs) in 6 cases of CS
and 9 cases of AdCC with the aim of evaluating their usefulness
in the differential diagnosis.
Our results confirmed the overlapping expression of SMA, p63
and CK5/6, but we found different expression of CD10, HHF35
actin, c-kit, CEA, ERs and PRs in AdCC and CS.
CD10 and HHF35 actin were expressed only in myoepithelial
cells of CS and were absent in AdCC. C-kit highlighted ductular
structures of AdCC, but was rarely expressed in CS; on the contrary, CEA was extensively positive in CS, but rarely expressed
in AdCC. Finally, ERs and PRs were extensively positive in CS,
but negative in AdCC. So our study confirms the usefulness of
ERs, PRs, C-kit and CEA, already reported in the literature, and
highlights the importance of CD10 and HHF35 actin that, to our
knowledge, have not been studied in this setting. Our data confirm the different histogenesis of the two lesions suggesting that
CS could be a reactive lesion and not a true neoplasia, because
it consists of a mixture of two different, mature, cell types (epithelial, probably luminal, CEA+/C-Kit- cells, and myoepithelial,
SMA, P63, CD10 and HHF35 actin positive cells, as the normal
myoepithelium). On the other hand, the two different histotypes
of AdCC, probably arise from a basal stem line tending to divergent and incomplete differentiation toward mioepitelial-like cell
type (with an incomplete SMA/p63+, CD10/HHF35 actin- immunophenotype) and toward epithelial basal-like cell type (ERs/
PRs-, CK5/6+, C-kit+, CEA+/- immunophenotype). So, AdCC
could be a true basal-like neoplasia, even if with a more favorable
prognosis. In conclusion, this study evidences the importance of a
broad immunohistochemical panel, including CD10 and HHF35
actin, for the pathogenetic understanding and the differential
diagnosis of CS and AdCC that, due to their different prognosis,
need a different treatment.
References
1
Clement PB, Young RH, Azzopardi JG. Collagenous spherulosis of
the breast. Am J Surg Pathol 1987;11:411-417.
Mooney EE, Kayani N, Tavassoli FA. Spherulosis of the breast. A
spectrum of mucinous and collagenous lesions. Arch Pathol Lab Med
1999;123:626-30.
2
Rabban JT, Swain RS, Zaloudek CJ, et al. Immunophenotypic overlap
between adenoid cystic carcinoma and collagenous spherulosis of
the breast: potential diagnostic pitfalls using myoepithelial markers.
Modern Pathology 2006;19:1351-7.
3
Due W, Herbst WD, Loy V, et al. Characterization of adenoid
cystic carcinoma of the breast by immunohistology. J Clin Pathol
1989;42:470-6.
219
Poster
4
Divaris DXG, Smith S, Leask D, et al. Complex collagenous spherulosis of the breast presenting as a palpable mass. Breast J 2000;6:199203.
Angiosarcoma secondary to breast cancer
with contralateral dorsal recurrence
A. Labate*, E. Mazzon**, M. Mesiti***, D.M. Taglieri****, G.
Certo*
*
Section of Pathology, ***Breast Unit, Clinica Cappellani-Giomi, Messina,
Italy; **Department of Clinical and Experimental Medicine and Pharmacology, Pharmacology Section, University Hospital, University of Messina, Italy; ****Department of Physiology and Biophysics and Center for
Cardiovascular Research, College of Medicine, University of Illinois at
Chicago, 835 S. Wolcott Ave, M/C 901, Chicago, Illinois 60612-7342,
USA
Introduction. The secondary angiosarcoma of the breast is a rare
occurrence, with an incidence estimated between 0,002% and
0,005% per year. It can develop several years after conservative
treatment for breast carcinoma. Since the first case described in
1987 until today, only 66 published reports are retrievable when
searching “secondary breast angiosarcoma” online. Secondary
angiosarcoma usually develops on skin irradiated after surgery or
it appears in the proximity of lymphedema. Usually, radiotherapy
appears to be the main cause. Diagnosis is often late due to the
“benign” appearance of the lesion. Treatment consists of salvage
mastectomy. Long-term prognosis is often poor given the very
aggressive biological behavior. Hereafter we describe a case of
angiosarcoma with secondary contralateral recurrence. Case report. An 82-year-old patient received left quadrantectomy to treat invasive ductal carcinoma, followed by chemo- and
radiotherapy. After ten years, a purple swollen lesion in close
proximity of the surgical wound was identified. Salvage mastectomy was performed. Histological report I. Subcutaneous vascular proliferation with
lacunar and capillary patterns, lined with coarse, hyperchromatic
and polymorphic endothelial cells, occasionally interspersed with
muscle bundles, infiltrating lymphocytes and blood. Mitosis 5 x
10 HPF. • Low grade angiomatous proliferation.
A year later, we noted a two-centimeter wide lesion in the right suprascapular region, which appeared hyperechoic, heterogeneous,
characterized by uneven margins and deepening in the fascia,
as assessed by sonography. The lesion was surgically removed. Histological report II. Subdermal proliferation of multinucleate perivascular cells, endothelial cells with hyperchromatic
nuclei, gaps and vascular lacunae, and pseudopapillary structures. Mitosis 10-15 x10 HPF.
• Intermediate grade angiosarcoma.
Medical sonography evidenced multiple liver metastases. Discussion. Surgery, chemotherapy and radiotherapy are conventionally used to treat breast cancer. Angiosarcoma, a rare cancer,
can develop after several years after such treatment. Here we report a case where the angiosarcoma develops in the contralateral
dorsal skin as a recurrence of the primitive disease. Reference
1
Scow JS, Reynolds CA, Degnim AC, et al. Primary and secondary
angiosarcoma of the breast: the Mayo Clinic experience. Source Department of Surgery, Mayo Clinic. J Surg Oncol. 2010;101:401-7.
2
Biswas T, Tang P, Muhs A, Ling M. Angiosarcoma of the breast:
a rare clinicopathological entity. Source Department of Radiation
Oncology, University of Rochester Medical Center. Am J Clin Oncol
2009;32:582-6.
DNA repair mechanisms in triple negative breast
cancer: a target for combined therapies
with selective inhibitors and alkylating agents
D. Lepanto*, P. Possanzini*, O. Biasi*, M. Barberis*, B. Bonanni**, C. Fumagalli*.
Div. of Pathology and ** Div. of Cancer Prevention and Genetics, European Institute of Oncology, Milan, Italy
*
Introduction. Triple negative breast cancers (TNBC) are immunohistochemically characterized by the absence of estrogen and
progesterone receptors and Her2-neu overexpression. Clinically,
they have limited treatment options, aggressive course and poor
prognosis. In 10% of the cases, TNBC develop in BRCA1 mutation carriers, and 90% of cancers harbouring a BRCA1 mutation
are TNBC. BRCA1 plays a pivotal role repairing DNA doublestrand breaks via homologous recombination (HR) and BRCA1
mutations affect this DNA repair pathway. Therefore, other DNA
repair mechanisms protect cells against the accumulation of
mutations that could lead to the development of tumors, like the
Base Excision Repair (BER) and Direct DNA Repair pathways.
The most important components of these pathways are PARP1
(Poly-ADP-ribose polymerase) and MGMT (O-6-methyl 06methylguanine-DNA methyltransferase), respectively.
PARP1 acts in single-strand breaks repair and can be down-regulated by PARP inhibitors. These drugs are actually considered
potentially useful in TNBC carriers of BRCA-1 mutations.
MGMT cleaves mutagenic alkyl adducts within DNA and
MGMT loss of expression, almost connected to promoter methylation, is associated with tumor progression. However, MGMT
epigenetic silencing confers susceptibility to DNA-damaging
alkylating agents.
The goal of this study was: a) to evaluate the MGMT methylation
status and the quantitative expression of PARP1 in TNBC patients,
BRCA1-wild type (wt) and BRCA1-mutated, with deficient doublestranded DNA break repair; b) to offer potentially useful assays in
predicting response to alkylating agents and PARP inhibitors.
Patients and methods. We studied the MGMT methylation status and PARP expression of 58 TNBCs (26 BRCA1-wt and 32
BRCA1-mutated), by nucleic acid extraction from formalin-fixed
and paraffin embedded tumor specimens. The DNA was treated
with sodium-bisulfite and amplified by methylation specific PCR
(MSP), with primer pairs specific for methylated and unmethylated
MGMT promoter region, and evaluated by gel electrophoresis.
The RNA was reverse transcribed in cDNA and PARP1 relative
quantity (RQ) was evaluated by real time assay.
Results. The methylation status of MGMT was significantly
different among the two TNBC groups with prevalence of unmethylation in BRCA1-mutated tumors (76.9%) and methylation
in BRCA1 wt tumors (59.4%). p-value of the Χ2 test between the
two populations was 0.0055.
The PARP1 RQ values ranged from 3,29 to 34,39 in BRCA1-wt
samples and from 0 to 140 in BRCA1-mutated samples. Although
different RQ range, the two groups show similar distribution and
RQ values overlap on log10 scale graph.
Conclusions. BRCA1-wt TNBC frequently have the direct DNA
repair system silenced by MGMT methylation, leading response
to alkylating drugs. Moreover, the expression of PARP1 was similar and heterogeneous in the two groups, regardless of BRCA1
status. The absence of significant difference in PARP1 expression
between BRCA1 wt and BRCA1 mutated TNBC cohorts, favours
the hypothesis of offering PARP inhibitors to all TNBC as it is
proposed in BRCA1 mutated patients.
These new observations need further confirmations on a larger
number of cases but suggest the combined use of alkylating
agents and PARP1 inhibitors in treatment of TNBC.
In addition MGMT-MSP and RT-PCR PARP assays could be
used prospectively as a predictive parameter for response to these
treatments.
220
Inter-observer diagnostic reproducibility in needle
core biopsies of the breast: enhancement
of concordance using immunohistochemistry
for protein P63
ambiguous morphology at H&E. Both overall concordance among
10 observers and individual kappa coefficients of 9 of them in comparison to the reference diagnosis increased when their diagnoses
were provided after evaluating p63-stained slides.
F. Maletta*, L. Macrì*, D. Fenocchio**, L. Viberti***, A. Sapino*,
S. Guzzetti***
Peritumoral vascular invasion and nherf1 for
histological refinement of grade 2 breast cancer
Dipartimento di Scienze Biomediche ed Oncologia Umana, Ospedale S.
Giovanni Battista di Torino, Università di Torino, Torino, Italia; **Struttura Semplice Dipartimentale di Istologia e Citologia Diagnostica, Ospedale Santa Maria della Misericordia, Perugia, Italia; ***S.C. di Anatomia
Patologica, Ospedale Evangelico Valdese, ASL TO1, Torino, Italia.
*
Introduction. Needle core biopsies (NCB) have been introduced
with the purpose of obtaining accurate and definitive preoperative diagnoses in breast lesions. However, as reported elsewhere
(Bianchi et al, Pathol Oncol Res 2009; Kluttig et al, BMC Cancer
2007), few data are available about reliability and validity of
diagnoses made on NCB, with studies reporting higher levels
of inter-observers discordance in intermediate categories (B3).
To improve diagnostic accuracy, new instruments would be
welcomed and recent studies (Dewar et al, Arch Pathol Lab Med
2011) described the role of myoepithelial markers to provide
assistance in accurately classifying breast proliferations in core
biopsies, by playing an important role in distinguishing invasive
carcinoma from its histologic mimics. One of these myoepithelial
markers is the nuclear protein, p63, a member of the p53 gene
family.
Aim. Aim of this study was to evaluate the diagnostic concordance on breast NCB diagnoses, according to the B-diagnostic
categories of European Guidelines for Breast Cancer among 10
pathologists with different levels of experience in the diagnosis of
breast lesions; we selected cases with ambiguous morphology at
H&E; we then calculated and compared concordance in diagnoses
based on morphology (H&E) alone and diagnoses based on both
H&E and immunohistochemistry (IHC) for p63, in order to assess
the potential role of p63 in improving diagnostic accuracy.
Material and methods. Fifty NCBs of lesions with morphologically-ambiguous features were selected. Original diagnoses were:
B2 in 11 cases, B3 in 22 cases, B4 in 3 cases, B5a in 2 cases and
B5b in 12 cases. Ten observers (both general and expert pathologists) were asked to evaluate the H&E-stained slides of these 50
cases and to provide a diagnosis using such categories; they were
then asked to re-evaluate the 50 cases, with the addition of IHC
for p63-stained slides. The 10 pathologists made their diagnoses
blindly of the original diagnoses, of the diagnoses of the other
observers and of their own first diagnosis based on morphology
alone. Overall concordance was calculated with K of Cohen
Fleiss statistic, among 10 observers and for 6 categories (B2, B3,
B4, B5a, B5b, B5c). In addition, concordance (K of Cohen) was
calculated between the diagnosis of each observer and the reference diagnosis provided by an experienced breast pathologist
(SA), both before and after IHC for p63.
Results. Considering the total number of observations (10
observers, each of them evaluating 50 cases), in 23.8% of observations (119/500) diagnosis changed after evaluations of
IHC-stained slides. Overall concordance (K of Cohen Fleiss)
was of 27% (p<0.05) when calculated on evaluations based on
morphology alone (H&E slides) while increased to 41% (p<0.05)
when evaluations were based on IHC for p63 as well. Concordance of each observer with the reference diagnosis in the 50 cases
was calculated: for H&E slides, it ranged from 0.09 to 0.83 with
a mean K of Cohen of 0.44 (p<0.05); for evaluations based on
morphology and IHC, concordance ranged from 0.03 to 0.86 with
a mean K of Cohen of 0.56 (p<0.05). After evaluation of IHCstained slides, 8/9 observers increased their concordance with the
reference diagnosis.
Conclusion. IHC for p63 proved useful in improving inter-observer reproducibility and diagnostic accuracy in NCBs of lesions with
A. Malfettone, C. Saponaro, C. Salvatore, R. Daprile, A. Paradiso, G. Simone, A. Mangia
National Cancer Centre, Bari, Italy.
Background. Traditional determinants proven to be of prognostic importance in breast cancer include the TNM staging, histological grade, proliferative activity and hormone receptor status.
One of the limitations of the histological grading scheme is that
a high percentage of breast cancers are still classified as grade 2,
a category with ambiguous clinical significance. The aim of this
study was to better characterize tumors scored as grade 2.
Material and methods. We investigated traditional prognostic
factors and tumor marker expression signature, such as NHERF1,
VEGFR1, HIF-1α and TWIST1 proteins, in 187 primary invasive
breast cancers by immunohistochemistry, stratifying patients into
good and poor prognostic groups by the Nottingham Prognostic
Index.
Results. We found cytoplasmic NHERF1 expression positively
correlated to VEGFR1 (r = 0.382, P = 0.000). Multivariate logistic regression analysis in the whole series revealed the worse
prognosis correlated with PVI and MIB1 (P = 0.001 and P =
0.014, respectively). Grade 2 subgroup analysis showed that PVI
(P = 0.026) and loss of membranous NHERF1 (P = 0.033) were
adverse prognostic factors. The 73% of grade 2 tumors were
significantly associated to PVI+/membranous NHERF1- phenotype, characterizing an adverse prognosis (P = 0.001). The PVI+/
membranous NHERF1- phenotype identifies a category of grade
2 tumors with the worst prognosis, including patients with a family history of breast cancer.
Conclusions. These observations could support idea of the PVI+/
membranous NHERF1- phenotype as a discriminating expression
profile in grade 2 tumors, which could improving the accuracy of
predicting clinical outcome.
Detection of human epidermal growth factor
receptor 2 protein. Can cytology specimens take
the place of tissue sections?
E. Manfrin, A. Remo, A. Parisi, L. Marcolini, C. Lucchini, M.
Macario, F. Bonetti
Dipartimento di Patologia e Diagnostica, Sezione di Anatomia Patologica, Università di Verona
Background. Overexpression of HER2 protein and HER2 gene
amplification in breast cancer are prognostic factors for the
response to specific medical treatments. HER2 expression is
generally examined in tissue sections. We investigated whether
specimens from fine needle aspiration cytology (FNAC) are adequate for this analysis.
Material and methods. HER2 protein overexpression was assessed in both FNAC specimens and tissue sections from 18
women who underwent surgery for primary invasive breast cancer at Verona G.B Rossi Hospital. The tumors included 10 ductal
carcinomas, 6 mixed invasive and in situ ductal carcinomas and
2 invasive lobular carcinomas. Immunohistochemistry (IHC)
was done with the HercepTest (Dako). HER2 protein levels were
determined as Score 0, no staining or staining of <10%of cancer
cells; 1+, slight staining; 2+, intermediate staining; and 3+, strong
staining; of >30% of cancer cells. Correlation between data from
cytology specimens and tissue sections was evaluated with Chisquare contingency test.
221
Poster
Results. Sensibility and specificity value for FNAC-Her2 overexpression IHC-test was 100% and 80% respectively. Positive Predictive Value (VPP) was 43% for Score 3+ and 66.5% for score
2+; Negative Predictive Value (NPV) for score 0/1+ was 100%.
Conclusions. These findings suggest that for cases with HER2
protein scores of 0/1+ FNAC specimens may be used instead of
tissue sections without a loss of accuracy. However, the low accordance between FNAC specimens and tissue sections for cases
with a score of 2+ and 3+ indicates that FNAC cannot be substituted for tissue sections under these conditions.
Differential diagnosis of synchronous bilateral
breast cancers by analysis of x-chromosome
inactivation pattern – a pilot study
R. Mangerini*, S. Salvi*, P. Ferro** ***, M.C. Franceschini**, A.M.
Ferraris*, S. Boccardo*, F. Carli*, M. Truini*, S. Colli**, D. Gianquinto**, F. Fedeli**, M.P. Pistillo*, S. Roncella**.
National Institute for Cancer Research (IST) Genova; **ASL5 “Spezzino”, La Spezia; ***AIL F. Lanzone, La Spezia, Italy.
*
Introduction. Synchronous bilateral breast cancers (SBBC) are
independent malignant lesions detected in both breasts at the
same time or within six months of each other. The incidence of
the SBBC is reported in the range of 0.3% to 12% of all breast
cancers (BC). The controlateral BC may be considered either a
metastatic lesion or a second primary neoplasm and this issue is
of great relevance for the process of clinical decision making 1.
In BC with similar histopathologic characteristics of the synchronous tumours, differentiation between the two modalities is not
easy and other approaches are to be evaluated 2.
Following inactivation of one X chromosome in somatic cells of
females during embryogenesis, women heterozygous for polymorphic X-linked genes carry a mosaic of cells expressing one or the
other allele in their normal tissues. A technique has been developed
that takes advantage of a highly polymorphic short tandem repeat
situated in the proximity of four methylation sites within the coding region of the X-linked human androgen receptor gene, with a
heterozygosity rate approaching 90%. Analysis of X chromosome
inactivation pattern (XCIP) may therefore be useful for differential
diagnosis, since cells derived from clonal expansion of a single progenitor cell will show the same allelic inactivation. In fact, clonality
analysis with XCIP has already been used to study the origin and development of various neoplastic disorders in affected heterozygous
women, and its applicability to the characterization of SBBC lesions
constitutes the aim of the present study 3 4.
Materials and methods. We evaluated 4 cases in which the
histology and immunophenotype of both SBBC carcinomas were
similar (1 neuroendocrin/neuroendocrin, 2 invasive ductal (IDC)/
IDC, 1 invasive lobular (ILC)/ILC and 4 cases of SBBC in which
the histologic subtype was different (2 ILC/IDC, one mixed/IDC
and one ILC/cribriform).
DNA was extracted using QIAamp DNA Kits (Qiagen) from five
10µ thick paraffin-embedded tissue sections (Invitrogen). Before
extraction, the sections were deparaffinized with xilene, washed
in ethanol (twice 70 % and once in 50%) and treated with proteinase K (Invitrogen).
To assess clonality, PCR amplification of genomic DNA was performed with primers specific for HUMARA STR. PCR products
were separated by capillary electrophoresis on the ABI PRISM
3130XL Genetic Analyzer and analyzed with a GeneMapper
software (Applied Biosystems).
Results. Three out of 4 cases of SBBC with the same istotype
also expressed the same XCIP in primary and controlateral breast
cancer, while one case (IDC/IDC) showed different allelic inactivation. In contrast, 2 out of 4 cases with different istotype also
showed a different XCIP, confirming the histology, while 2 cases
showed similar X-inactivation ratio in both lesions (duttolobular/
IDC and IDC/IDC).
Conclusions. In spite of the small number of cases investigated
in our study, XCIP analysis may provide additional information
in order to understand the process of early metastatization and/or
simultaneous transformation of synchronous breast tumours.
References
1
M Intra, Rotmensz N, Viale G, et al. Clinicopathologic characteristics
of 143 patients with synchronous bilateral invasive breast carcinomas
treated in a single institution. Cancer 2004;101:905-12.
2
RS Saad, Denning KL, Finkelstein SD, et al. Diagnostic and prognostic utility of molecular markers in synchronous bilateral breast
carcinoma. Diagnostic and prognostic utility of molecular markers
in synchronous bilateral breast carcinoma. Mod Pathol. 2008;21:
1200-7.
3
AM Ferraris, Mangerini R, Pujic N, et al: High telomerase activity in
granulocytes from clonal polycythemia vera and essential thrombocythemia. Blood. 2005;105: 2138-40.
4
AM Ferraris, Mangerini R, Racchi O, et al: Heterogeneity of clonal
development in chronic myeloproliferative disorders. Am J Hematol.
1999;60: 158-60.
“How to prepare” a micropapillary breast
carcinoma cell line
C. Marchiò*, L. Annaratone*, D. Balmativola*, L. Macrì**, A.
Sapino*
Dipartimento di Scienze Biomediche e Oncologia Umana/Università di
Torino, Torino, Italia; **Servizione B di Anatomia Patologica/Ospedale
San Giovanni Battista-Molinette di Torino, Torino, Italia
*
Introduction. Micropapillary carcinomas (MPCs) represent an
uncommon and aggressive variant of oestrogen receptor positive
breast carcinomas, as demonstrated by clinicopathological data
and by recent genomic analysis. A unique feature of MPCs is represented by the ‘inverted polarity’ of neoplastic cells that leads to
baso-stromal MUC1 expression, which has been hypothesized to
be responsible at least in part for the detachment of the cells from
the stroma and for the dissemination of cancer cell clusters. So
far no functional investigation has been performed due to the lack
of a commercially available MPC cell line. Recently, neutrophilderived proteases, such as elastase and cathepsin-G, have been
shown to induce formation of highly aggregated multicellular 3D
spheroids of MCF-7 cells. In this study we sought to investigate
whether 3D spheroid formation in breast cancer cells showing
inverted polarity is induced by serine proteases such as elastase
and cathepsin-G and whether this may represent an artificial cell
line model of MPC.
Methods. Six human mammary adenocarcinoma cell lines, three
ER positive/HER2 negative (MCF-7, T47D, ZR-751), one ER
positive/HER2 positive (BT-474), one HER2 positive/ER negative (SK-BR-3) and one ER/HER2 negative with a basal phenotype (MDA-MB-231) were cultured with the addition of elastase
or cathepsin-G to the medium and in ultra-low attachment flasks.
Cell blocks of every cell line in different experimental conditions
were obtained. Immunohistochemistry (IHC) for ER, Ki-67,
EMA, MUC-1, HER2 and E-Cadherin (E-CAD) was performed.
To assess whether embolization in conventional breast cancers
may follow cell cluster detachment with inversion of polarity, we
tested by IHC for MUC-1 a series of invasive ductal carcinomas
not otherwise specified (IDC-NOS) showing angioinvasion.
Results.
1. Addition of elastase and cathepsin-G to ER positive/E-CAD
positive breast cancer cell lines lead to 3D spheroid formation
whilst ER negative/E-CAD negative cells grew mainly as single
cell suspension. Overlapping results were obtained by growing
these cells in ultra-low attachment plates. Out of the breast cancer cell lines analysed MCF-7 cells were the sole, in any of the
experimental conditions, to acquire a growth pattern that highly
resembled pure MPC by showing the so called “inverted polarity”, as demonstrated by EMA and MUC1 expression lining the
222
external border of the spheroids. In addition, in a way akin to
micropapillary carcinomas, E-CAD and MUC-1/EMA were expressed in a mutually exclusive way with E-CAD being expressed
along the basolateral membrane of cells and EMA/MUC1 lining
the external border of cell clusters.
2. Tumour emboli derived from ER-positive IDC-NOS showed
inversion of polarity with MUC-1 expression on the external
border.
Conclusion. Cell growth as suspension of either single cells or
cell clusters/spheroids is likely to be regulated by adhesion protein via ER activation. Both addition of serine proteases to the
medium of MCF7 breast cancer cells and MCF-7 cell culturing
in ultra-low attachment plates induce formation of 3D spheroids
showing the typical morphology and immunophenotype of
MPCs. The 3D spheroid model we have here produced is a good
model to study both MPCs and tumour embolization processes.
Studies are warranted to identify those molecules responsible for
regulating cell–cell or cell–extracellular matrix interactions leading to rotation of polarization of cancer cells in vivo.
How pathologist can collaborate
in the development and validation
of instruments for image analysis?
L. Molinaro
Dipartimento di Scienze Biomediche e Oncologia Umana, III Servizio di
Anatomia Patologica, AO-U San Giovanni Battista Università di Torino
Background. During the last 20 years, the automatic recognition
of structures (image analysis) based on shapes and colours (segmentation, colour deconvolution...) has been used in industrial
and security fields and today represents a milestone in astronomy
and also in scientific police and forensic medicine. The technical progresses obtained in such areas can be applied as well in
pathology. Automatic image analysis in surgical pathology can
be performed both in bright and dark-field, in single image acquisition or after a previous scan on digital slides. Our aim was
to collaborate in the development and validation of a system of
image analysis that uses the results of bright-field immunohistochemical analysis to optimize the dark-field FISH analysis of the
same specimen.
Methods. A scientific collaboration project was set up with Menarini Diagnostics and Hesp Technology for the improvement
and validation of HER2 analysis in breast cancer using a device
composed of a digital slide scanner and the analysis software DSight. The role of the pathologist was to highlight possible biases
and to propose improvement in the system. From the pathology
files of the San Giovanni Battista Molinette Hospital we selected
30 breast carcinomas scored 2+ by traditional microscopy, half of
which were HER2 amplified by FISH (Abbott Vysis HER/CEP17
probes) analysis. All cases were subjected to automatic analysis
by the Menarini D-Sight device, with the aim to set up a reproducible analysis algorithm of cases with heterogeneous HER2
expression. Briefly, the slides stained by immunohistochemistry
were first previewed at 4X and then scanned at 40X magnification. The areas with higher intensity of staining were then manually selected with a digital pencil tool and the automatic scoring
was finally performed. Afterward the corresponding FISH slide
was scanned with mercury lamp light at 4X magnification. Three
fiducial markers were selected on the bright-field preview to automatically merge the dark-field preview. This allows identifying
the previously analyzed IHC region of interest on FISH slides
releasing pathologists from a troublesome search in DAPI mode.
Dark-field analysis was performed by using a z-stack mode of 20
fields at 100X magnification with oil immersion. The algorithm
of bright and dark-field was reviewed several times to obtain a
final optimal product.
Results. We developed together the following options: a)
creation of a JPEG2000 reconstructed image of each acquired
dark-field. These FISH images corresponded to specific areas
previously selected within the immunocytochemical stainings.
These large images are browsable on the screen and allow a rapid
selection of the tumor cells avoiding the non-cancer cells; b) the
cell overlapping has been considered as a problem of the analysis
and explained to the engineers that modified the parameters; however this problem remained unsolved on 2D analysis; c) technical problems related to the time of scanning were discussed and
improved; d) a previous region mapping with cartesian coordinate
on FISH slides for multiple specimen analysis was suggested and
planned.
Conclusion. The collaboration of the technical staff developing
devices for image analysis with the “user” (pathologist) of the
system is an interesting and stimulating experience that allows the
pathologist to know how much is achievable from an instrument
and its limits. On the other hand engineers collaborating with
pathologists may better understand the target and the variability
of the biological problem. The D-Sight is a valuable system that
allows obtaining an easer and faster assessment of HER2 gene
status in cases with heterogeneous protein expression.
Amyloidosis of the breast
T. Montrone*, M. Liuzzi*, G. Ingravallo*, A. Napoli*, A.M. Guerrieri**, G. D’Eredità***, C. Giardina*
Dipartimento di Anatomia e Istologia Patologica/Università di Bari,
Bari, Italia; **SARIS/Policlinico di Bari, Bari, Italia; ***Dipartimento di
Chirurgia Generale e Speciale/Università di Bari, Bari, Italia
*
Amyloidosis of the breast is unusual, presenting clinically as localized mass (amyloid tumor) either in isolation or in association
with malignancy. Mammography may show microcalcifications,
a mass lesion or a combination of both features. We reported two
cases of amyloidosis of the breast both diagnosed on core biopsy.
The first patient, a 69 years-old woman presented a mass, in right
breast, with periferic, coarse calcifications, in an area 1 cm large,
radiologically considered as a dysplastic lesion; the second one,
a 52 years-old woman, showed mixed microcalcifications in an
area of 1 cm in diameter, radiologically suspicious for DCIS.
In both patients, mammotome core-biopsy (11G) showed rare
glandular structures, homogeneus eosinophilic deposits in the
stroma, with large calcifications. In the first case the amorphous
deposits were surrounded by a lot multinucleated giant cells and
macrophages. In none of them there was evidence of cancer.
These deposits had a green birefringence due to Congo-Red staining and were immunoreactive for λ chain in the first case and for
AA amyloid in the second one.
The first patient had Hashimoto thyroiditis while the second had
a primary Sjögren syndrome; serum protein electrophoresis was
normal in both patients.
The results confirmed the diagnosis of secondary amyloidosis of
the breast connected with chronic auto-immunitary inflammatory
disease. In both these patients the use of core-biopsy allowed to
avoid a unuseful surgery.
Squamoid eccrine ductal carcinoma (eccrine
ductal carcinoma with intraductal squamous
metaplasia)
D. Morichetti, T. Pusiol, M.G. Zorzi
Introduction. We present a case of eccrine ductal carcinoma with
squamoid features (SEDC). with critical review of the literature.
Case report. A 54-year-old woman presented with a slowly
growing nodular lesion on right tibial region. The lesion measured 12 x 11 mm. The tumor was well circumscribed and was
composed of numerous tubular structures, lined by one or several
layers of atypical basaloid cells with focal finding of a cuticular
223
Poster
luminal border. Diffuse intraductal squamous metaplasia was
present. The tumor was associate with a fairly dense sclerotic
stroma and focally showed an infiltrative appearance at the periphery, consistent with a low-grade malignancy. The patient is
free of recurrence or metastasis.
Discussion. By definition SEDC is composed by typical eccrine carcinoma with intraductal squamous metaplasia. The
case report by Terushkin et al 1 is incorrectly defined SEDC. In
the histological description the neoplasm consist of aggregates
with squamoid features, connect to the overlying epidermis, but
lumina are not identify within the squamoid islands. The ductal
component is separate by squamoid aggregates. In our opinion
the diagnosis should be eccrine ductal carcinoma associate with
squamoid nodules. In the three cases of SEDCs reported by Wong
et al 2 the squamoid differentiation is squamous cell carcinoma
(SCC). The Authors do not specify if the two components are
intermingled or separated. The diagnosis should be eccrine carcinoma mixed or associated with SCC. In the case of Herrero et
al 3 three neoplastic components are been described. The first is
composed by invasive sheets and islands of atypical keratinocytes
with squamous eddies, horn cyst, and the occasional presence of
intercellular bridges. The description and the microphotograph B
show the malignant nature of squamous proliferation. The second
component show ductal differentiation. The third component is
the presence of in situ SCC within the eccrine sweet ducts or
glands. The final diagnosis should be mixed SCC and eccrine
carcinoma with in situ intraductal SCC. The neoplasm described
by Kim et al 4 is mostly composed of atypical keratinocytes with
prominent squamous differentiation and ductal component at the
periphery of the tumor. The microphotograph A of the figure
2 not illustrate with clearly the composition of the tumor. It is
probable that the neoplasm is composed by eccrine carcinoma
with squamous component whose benignant or malignant nature
can not be establish by histological description and microphotographs. The Authors not specify if two components are separated
or intermingled and the microphotographs are indicative for two
separated components. The diagnosis of SEDC is not convincing.
The case reported Chhibber et al 5 is well documented as ductal
eccrine carcinoma with squamous differentiation. Urso et al 6
have described seven case of eccrine ductal carcinoma. The first
case is described as ductal proliferation with keratinizing squamous cysts, cellular cords, and squamous solid nests. The microphotographs not permit to establish if the two componets are
separated or intermingled. These Authors propose that SEDC is
a tumor that shows folliculosebaceous-apocrine unit differentiation rather than eccrine differentiation. In the diagnosis of SEDC
the histological criteria should be very stringent. Our case show
clearly the squamous metaplasia in the ductal structures of eccrine carcinoma and may be considered the first case convincing
of SEDC. We prefer the terminology of eccrine ductal carcinoma
with intraductal squamous metaplasia in order to emphasize that
two component are intermingled.
References
1
Terushkin E, Leffell DJ, Futoryan T, et al. Squamoid eccrine ductal
carcinoma: a case report and review of the literature. Am J Dermatopathol 2010;32:287-92.
2
Wong, TY, Suster S, Mihm MC, et al. Squamoid eccrine ductal carcinoma. Histopathology 1997;30:288-93.
3
Herrero, J, Monteagudo C, Jordá E, et al. Squamoid eccrine ductal
carcinoma. Histopathology 1998;32:478-80.
4
Kim, YJ, Kim AR, Yu DS, et al. Mohs micrographic surgery for
squamoid eccrine ductal carcinoma. Dermal Surg 2005;31:1462-4.
5
Chhibber V, Lyle S, Mahalingam M, et al. Ductal eccrine carcinoma with squamous differentiation: apropos a case. J Cutan Pathol
2007;34:503-7.
6
Urso C, Paglierani M, Bondi R. Histologic spectrum of carcinomas
with eccrine ductal differentiation (sweat-gland ductal carcinomas).
Am J Dermatopathol 1993;15:435-40.
Breast Core Needle Biopsy and B classification
F. Pagni* MD, F.M. Bosisio** MD, D. Salvioni*** MD, P. Colombo**** MD, B.E. Leone* MD, C. Di Bella* MD.
*
Department of Pathology, Desio-Seregno Hospital, Italy; ** Department
of Surgical Sciences, Pathology section, University Milano-Bicocca, Milan, Italy; ***Department of Radiology, Desio-Seregno Hospital, Italy;
****
Department of Surgery, Carate Brianza Hospital, Italy
Aim. to provide a detailed overview of the professional management of breast cancer diagnostic preoperative phase and validate
the British National Health Service Breast Cancer Screening Programme (NHSBSP) classification of Core Needle Biopsies (CNB)
Materials and methods. 226 CNB were performed over a period
of fifteen months between April 2009 and June 2010. A restrospective study was planned correlating the diagnosis made on CNB
with the diagnosis made on the final surgical specimen. Statistical
analysis evaluated sensitivity, specificity, positive and negative
predictive values of the NHSBSP diagnostic categories. Cohen’s
kappa (K) evaluated the agreement between the diagnosis on
CNB versus the final pathological diagnosis in “clinically positive
cases”. Finally a comparative analysis between CNB method and
Fine Needle Aspiration Biopsy (FNAB) is discussed.
Results. The distribution of our cases for each diagnostic category
reflects the literature guidelines with minor differences in the B2
and B4 groups (Fig.1). Statistical data about the patients’ follow up
revealed a little number of false negatives cases in the B1 and B2
categories and no false positive case in the B4 and B5 groups (Table
1). Uncertain malignant lesions (B3 category) was divided into 3
major areas (papillary lesions, fibroepithelial proliferations with
cellular stroma and intraepithelial atypical lesions such as DIN1/
LIN1). 26/29 patients in the B3 category underwent surgery (Table
2). Cohen’s K analysis showed strong statistical correlation (K=0.77;
Z=4.3; significance>1.96; alpha 0.05) between CNB diagnosis and
surgical pathology final results in the subgroup of high-risk patients
Figure 1 Distribution of the NHSBSP category among 226 CNB.
The majority of our cases was classified as malignant (B5), followed by certainly benignant lesion (B2). Suspicious cases (B3, B4)
together account for the 15.4% of the diagnosis, a percentage
quite higher than the optimum found in literature.
Percentage distribution of the categories and expected values from
literature
DIAGNOSTIC
CATEGORIES
OUR DATA
LITERATURE
GUIDELINES
B1
4%
3.9%
B2
36.8%
50.9%
B3
12.8%
7.6%
B4
2.6%
0.5%
B5
43.8%
37.1%
224
Tab. I. Statistical indicators in the case study (226 patients).
Minimum
Preferable
Our data
ABSOLUTE SENSITIVITY
(B5/malignants)
Quality index
>70%
>80%
88.3% (99B5/87B5+6B4+13B3+
3B2+3B1)=99/112
COMPLETE SENSITIVITY
(malignants- false negatives)/
malignants
>80%
>90%
94.6% (112-6)/112
SPECIFICITY
(B2/benigns)
>75%
>85%
72.8% 83/(226-112)=83/114
PPV B5
(malignants/B5)
>99%
100%
100% (12 patients lost not
included)
PPV B4
(malignants/B4)
>99%
100%
100%
NPV B2
(benigns/B2)
>90%
95%
96.3% (80/83)
False Positive Rate
<0.5%
<0.1%
0
False Negative Rate B2
< 5%
1%
3.9% (3/83)
Suspicious rate (B4+B3)
<10%
<5%
15.4% (29+6)/226
Tab. II. Uncertain malignant lesions and surgical follow up (category B3).
B3
DIN1/LIN1
Number of cases
Surgery
Fibroepithelial
Papillary
Total
12 (41%)
4 (13.8%)
12 (41%)
28 (96.5%)*
11/12 (91.6%)
4/4 (100%)
10/12 (83.3%)
25/28 (89.3%)
Up-graded (IDC)
1/11 (9%)
1/4 (25%)**
1/10 (10%)
3/25 (12%)
Confirmed non invasive
8/11 (72.8%)
3/4(75%)
9/10 (90%)
21/25 (84%)
Down-graded
2/11(18.2%)
0
0
1/25 (4%)
*in the B3 group (29 total cases) we collected also 1 atypical salivary-gland type lesion which was not include in the major areas.
** phyllodes malignant tumour
Tab. III. Cohen’s kappa analysis in cases with surgical indication (diagnosis on CNB >=DIN1).
CNB
DIN1/LIN1
DIN2/3-LIN2
IDC/ILC
Benign*
DIN1/LIN1
8
-
1
2
11
DIN2/3-LIN2
-
12
5
-
17
IDC/ILC
-
-
48
-
48
Tot
8
12
54
2
(11 cases from
B3 category)
(13 cases from
B5a, 4 from B5c)
(48 cases from
B5b)
Total
76
Tab. IV. CNB Vs FNAB.
B5a
B5b
B5c
B4
B3
B2
B1
Total
C1
2
2
-
-
-
2
1
7
C2
1
-
-
-
1
2
-
4
C3
-
1
-
-
5
5
1
12
C4
-
1
1
-
1
-
-
3
C5
-
12
-
1
1
-
2
16
TOT
3 (23%)
16 (20%)
1 (17%)
1(17%)
8 (27%)
9 (11%)
4 (44%)
42
Poster
(diagnosis >=DIN1 on CNB, Table 3). Global diagnostic power of
CNB in all of the 226 cases revealed high sensitivity (88.3%), lower
specificity (72.8%). In 42 “doubtful and insidious” cases synchronous FNAB and CNB were performed showing complementary role
in the diagnostic phase of breast lesions (Table 4).
Conclusions. CNB represents the gold standard method in the
diagnostic phase of many breast tumours; the NHSBSP classification is a useful reporting system in order to standardize the
pathological diagnosis and give a clear indication for the correct
management of the patients.
Warthin-like breast tumor
E. Orvieto, M. Lo Mele, L. Alessandrini, E. Vassarotto, V. Guzzardo, V. Belardinelli, M. Rugge
UOC Anatomia Patologica & Clinica Chirurgica II, Breast Unit, Azienda
Ospedaliera di Padova - Università degli Studi di Padova
Background. Salivary-type breast tumors include three variants,
i.e. acinic, oncocytic and mucoepidermoid cancers. We report a
case of a Warthin-like breast tumor coexisting with homolateral
(non-adjacent) ductal adenocarcinoma (pT2, N0).
Case report. A 52-year-old woman underwent radical (right)
mastectomy for infiltrating ductal adenocarcinoma of the external
quadrants. Assessed on the surgical specimen, the breast cancer
was 2.3 cm in size. The upper/inner quadrant (5 cm away from
the cancer) also revealed a rounded, well-circumscribed nodular
lesion (1.5 cm wide) that was mainly cystic on its cut surface,
interspersed with solid areas.
Postoperatively, histology confirmed the (preoperative) diagnosis of
NOS ductal adenocarcinoma (G2), with no lymph node metastases
(pT2, N0). Histological examination of the nodule from the upper/
inner quadrant demonstrated a rounded lesion with pushing margins, which consisted of papillary fronds covered with cubic-columnar epithelia with a granular eosinophilic cytoplasm (oncocytes);
neither mitotic activity nor cytological atypia were documented.
No myoepithelia were detected and their absence was confirmed by
appropriate immunostain. The tumor’s stroma showed a prominent
lymphoid infiltrate. Both the mitochondria-rich oncocytes and the
polyclonal lymphoid populations were confirmed by immunohistochemistry. The epithelial component exhibited positive immunostain for CK 7, 18, 19. Taken together, the gross and histological
findings were consistent with a Warthin-like tumor of the breast.
Discussion. A Warthin-like tumor of the breast could theoretically originate from salivary glands included within the breast
(salivary tissue heterotopy). It should be noted, however, that
the breast and the salivary gland are both tubulo-acinar exocrine
glands, and this similarity can result in phenotypically similar
tumors. Salivary-type breast tumors (which are not listed as a
separate category in the 2003 WHO classification) are grouped
according to whether they have a ME cell component or not.
According to the current literature, the latter group includes only
three cancer variants, i.e. acinic, oncocytic, and mucoepidermoid.
Consistently with this nosography, we postulate that the Warthintype breast tumor described herein is an additional variant of
salivary-type breast neoplasia, with no ME component.
Assessing proliferative activity (Ki67) in breast
cancer: inter-method variability and intra-tumor
consistency
E. Orvieto, G. Boccuzzo, M. Lo Mele, L. Giacomelli, R. Sangapur, G. Marchelle, A. Di Francesco, L. Alessandrini, C. Spoladore, M. Rugge
UOC Anatomia Patologica Azienda Ospedaliera, Dipartimento di Scienze
Statistiche, Università degli Studi di Padova
Background. Cancer’s proliferative activity (PA) is a clinically
relevant biological information. Ki67 nuclea

BIO-OPTICA PAG. Path Settembre 2011_BIO

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