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Transcription

n o i t

the education program for the annual congress of the European Hematology Association
hematology education
education program for the
12th congress of the
european hematology
association
vienna, austria,
june 7-10, 2007
the education program for the annual congress of the European Hematology Association
hematology education
EHA Executive Board
E. Hellström-Lindberg, President, Sweden
W. Fibbe, President Elect, The Netherlands
E. Montserrat, Past President, Spain
A. Hagenbeek, Treasurer, The Netherlands
I. Roberts, Secretary, United Kingdom
EHA Councilors
E. Berntorp, Sweden
H. Döhner, Germany
U. Jäger, Austria
C. Lacombe, France
C. Mecucci, Italy
J. San Miguel, Spain
R. Skoda, Switzerland
I. Touw, The Netherlands
EHA Local Organizing Committee 12th Congress
H. Ludwig, Congress President, Austria
I. Pabinger, Austria
U. Jäger, Austria
EHA Scientific Program Committee 12th Congress
I. Roberts, Chair, United Kingdom
M. Alberich Jorda, USA
J. Bladé, Spain
N. Casadevall, France
H. Döhner, Germany
A. Green, United Kingdom
C. Mecucci, Italy
F. Peyvandi, Italy
G. Salles, France
N. Zoumbos, Greece
EHA Scientific Program Committee Advisory
Board 12th Congress
P. Beris, Switzerland
N. Borregaard, Denmark
M. Cavazzana-Calvo, France
J. Cools, Belgium
N. Cross, United Kingdom
T. Enver, United Kingdom
A. Falanga, Italy
J. Greil, Germany
C. Hershko, Israel
S. Izraeli, Israel
C. Lacombe, France
B. Mansouri, Switzerland
J. Melo, United Kingdom
W. Ouwehand, United Kingdom
R. Pieters, The Netherlands
A. Rosenwald, Germany
B. Schlegelberger, Germany
M. Theobald, The Netherlands
M. van Oers, The Netherlands
W. van Solinge, The Netherlands
C. Verfaillie, USA
EHA Education Committee
A. Green, Chair, United Kingdom
E. Berntorp, Sweden
C. Chomienne, France
C. Craddock, United Kingdom
L. Degos, France
H. Döhner, Germany
E. Hellström-Lindberg, Sweden
D. Jasmin, France
F. Lo Coco, Italy
A. Urbano Ispizua, Spain
EHA Publication Commitee
M. Cazzola, Editor, Italy
R. Foà, Editor, Italy
E. Hellström-Lindberg, Sweden
C. Lacombe, France
S. McCann, Ireland
European Hematology Association
EHA Executive Office
Westblaak 71, 3012 KE, Rotterdam,
The Netherlands
Tel.: +31 10 436 1760, Fax: +31 10 436 1817
E-mail: info@ehaweb.org,
Website: www.ehaweb.org
Word of welcome
On behalf of the EHA Education Committee and Scientific Program Committee, we are delighted to
welcome you to the beautiful city of Vienna. The EHA Congress is the largest and most comprehensive
hematology meeting in Europe with a world class line up of invited speakers. The Education Program covers the whole spectrum of clinical hematology and we have assembled a distinguished cast of internationally-recognised speakers. In addition to enjoying the talks, we hope you find the peer-reviewed papers
in the Education Book a useful source of information and references for the coming year.
Tony Green
Chair Education Committee
Irene Roberts
Chair Scientific Program Committee
Heinz Ludwig
Congress President
hematology education: the education program for the annual congress
of the European Hematology Association - 2007; volume 1, issue 1
Table of Contents
Iron Metabolism and Disease
1-8
9-17
18-23
Mechanisms of iron regulation and of iron deficiency
C. Hershko, A. Ronson, M. Souroujon, I.Z. Cabantchik,
J. Patz
Pathophysiology, diagnosis and treatment
of the anemia of chronic disease
G. Weiss
Screening hemochromatosis and iron overload
C. Camaschella, A. Pagani, E. Poggiali, L. Silvestri
83-88
Current treatment of T-cell lymphomas:
are we making any progress?
A. Delmer
89-96
Novel drugs for the treatment of T-cell lymphoma
O.A. O’Connor
Myeloma
97-101
Advances in myeloma biology: basis for new therapy
N. Munshi
102-107
Multiple myeloma: diagnosis, staging and
criteria of response
J. Bladé
108-114
Novel treatment approaches in multiple myeloma
A. Palumbo, I. Avonto, P. Falco, C. Federica, T. Caravita,
M.T. Petrucci, M. Cavo, M. Boccadoro,
Hemostasis
24-30
Epidemiology of coagulation disorders
F. Peyvandi, M. Spreafico
31-38
Recent advances in hemophilia management
C. Négrier, Y. Dargaud, J-L. Plantier
39-44
Gene therapy for hemophilia
M.K.L. Chuah, T. VandenDriessche
Chronic lymphocytic leukemia
115-121
Genetics in chronic lymphocytic leukemia:
impact for prognosis and treatment decisions
U. Jäger, M. Shehata, D. Heintel, R. Hubmann,
B. Kainz, E. Porpaczy, A. Hauswirth, A. Gaiger
122-128
State-of-the-art treatment of chronic lymphocytic
leukemia
M. Hallek
129-133
New Drugs for chronic lymphocytic leukemia
J. Gribben
Thrombosis
45-50
Vitamin K epoxide reductase (VKORC1):
pharmacogenetics and oral anticoagulation
J. Oldenburg, C.R. Müller, M. Watzka
51-55
Do arterial and venous thrombosis share common
risk factors?
G. Lowe
56-59
Venous thromboembolism in medical patients:
stratification and prevention
P. Prandoni
Hodgkin’s lymphoma
60-63
The role of PET in staging and response assessment
L. Specht
64-69
Tailoring the treatment for early-stage
M. André, O. Reman
70-75
Treatment of relapsing/refractory patients with
Hodgkin lymphoma
P. Brice
What’s new in T-cell non-Hodgkin’s
lymphomas?
76-82
Pathology and genetics of T-cell lymphomas
A. Chott, A-I. Schmatz, E. Kretschmer-Chott,
L. Müllauer, B. Streubel
Sickle cell disease
134-139
Hemolysis-associated pulmonary hypertension
in sickle cell disease and thalassemia
G.J. Kato, M.T. Gladwin
140-147
The contribution of asthma to sickle cell disease
related morbidity and mortality
M.R. DeBaun, J.E. Jennings, J.H. Boyd, J.J. Field,
C. Hiller, R.C. Strunk
148-153
Hydroxyurea: benefits and risks in patients affected
with sickle cell anemia
M. De Montalembert
Acute lymphoblastic leukemia
154-160
Genetics of T-cell acute lymphoblastic leukemia
C.J. Harrison
161-167
Treatment of Philadelphia chromosome positive
acute lymphoblastic leukemia
O.G. Ottmann, H. Pfeifer, B. Wassmann
education program for the 12th congress of the european hematology association, vienna, austria, june 7-10, 2007
hematology education: the education program for the annual congress
of the European Hematology Association - 2007; volume 1, issue 1
Table of Contents
168-174
Detection of minimal residual disease in adult
patients with acute lymphoblastic leukemia:
methodological advances and clinical significance
M. Brüggemann, T. Raff, S. Böttcher, S. Irmer, S. Lüschen
C. Pott, M. Ritgen, N. Gökbuget, D. Hoelzer, M. Kneba
Acute myeloid leukemia
175-182
Targeting critical pathways in leukemia stem cells
S. Anand, W-I. Chan, B.T. Kvinlaug, B.J.P. Huntly
183-192
Clinical use of molecular markers in adult
acute myeloid leukemia
K. Mrózek, P. Paschka, G. Marcucci, S.P. Whitman,
C.D. Bloomfield
193-199
Management of elderly patients with
H. Dombret, E. Raffoux, L. Degos
254-258
Conventional and new treatment modalities for
myelofibrosis
F. Cervantes
Cellular immunotherapy and vaccination
259-264
The molecular and cellular basis for
immunotherapeutic intervention in malignant
disease: dynamic imaging of the immune system
C. Lotz
265-269
Immunotherapy of leukaemia with TCR gene
modified T cells
E.C. Morris, D.P. Hart, J. King, S. Thomas,
M. Cesco-Gaspere, S. Xue, H.J. Stauss
270-277
Perspectives and limitations of vaccination strategies
against cancer
P. Romero, D. Speiser
Myelodysplastic syndromes
Transfusion - Diagnosis and management of
alloimmune thrombocytopenia
200-204
Molecular pathogenesis of myelodysplastic
syndromes
C.M. Niemeyer, C.P. Kratz
278-284
Human platelet alloantigens: heterogeneity of
platelet alloantibodies
I. Soche, T. Bakchoul, S. Santoso
205-214
Epigenetic therapy in myelodysplastic syndromes
P. Fenaux, C. Gardin
285-293
215-218
New developments in curative approaches
in myelodysplasia
T. de Witte, R. Brand, A. van Biezen, S. Suciu, L. Baila,
P. Muus, M. Schaap, A. Schattenberg, R. Martino,
N. Kröger, S. Amadori
Laboratory diagnosis of neonatal
alloimmune thrombocytopenia
L. Porcelijn
294-298
Treatment and prevention of alloimmune
thrombocytopenias
A. Husebekk, M. Kjær Killie, J. Kjeldsen-Kragh,
B. Skogen
Chronic myeloid leukemia
New and evolving techniques in diagnostic
hematology
219-225
Clinical implications of ABL mutational screening
N. von Bubnoff, J. Duyster
299-305
226-230
Decision making at diagnosis in chronic myeloid
leukemia
M. Baccarani, F. Palandri, F. Castagnetti, A. Pusiol
Genome-wide approaches to identify new subtypes
of acute myeloid leukemia
P. Valk
306-310
231-239
Management of patients with imatinib resistance
E. Olavarria, J.F. Apperley
Which new tests should be offered by clinical
haemostasis laboratories?
M. Greaves, H.G. Watson
311-315
The role of molecular analysis in investigation
of inherited anemias
A. Iolascon, L. Boschetto, L. De Falco, S. Scianguetta,
R. Russo, C. Piscopo, I. Andolfo
a
Index of authors
Myeloproliferative disorders
240-247
Molecular pathogenesis of the myeloproliferative
diseases
W. Vainchenker, F. Delhommeau, J-L. Villeval
248-253
Diagnosis and classification of myeloproliferative
disorders in the JAK2 era
C. Harrison
education program for the 12th congress of the european hematology association, vienna, austria, june 7-10, 2007
Mechanisms of iron regulation and of iron deficiency
C. Hershko1,2
A. Ronson1,3
M. Souroujon2
I.Z. Cabantchik4
J. Patz3
1
Department of Hematology, Shaare
Zedek Medical Center,
2
Hematology Clinic and Central
Clinical Laboratories , Clalit Health
Services
3
Hematology and Gastroenterology
Clinics, Meuhedet Health Services
4
Department of Biological Chemistry,
Institute of Life Sciences, Hebrew
University of Jerusalem, Israel
Hematology Education:
the education program
for the annual congress of the
2007;1:1-8
A
B
S
T
R
A
C
T
Basal cellular iron handling in enterocytes is performed by the interaction of ferric
reductase, two distinct transport proteins for ferrous iron located on the duodenal brush
border (DMT1) and the basolateral membrane (ferroportin 1) respectively, and ferroxidase facilitating iron egress. The fine tuning of iron regulation and its adaptation to body
iron requirements is performed by hepcidin. Increased levels of serum diferric transferrin activate an iron sensor and signal transduction effector complex, consisting of TFR2, HFE and HJV for downstream regulation of hepcidin production. Despite these elegant
regulatory mechanisms, iron deficiency remains one of the the most common nutritional deficiencies of mankind. Physiological or nutritional iron deficiency is the result of an
interplay of increased host requirements, limited external supply, and increased blood
loss. By contrast, pathological iron deficiency is most often the result of gastrointestinal disease associated with abnormal blood loss or malabsorption. If gastroenterologic
evaluation fails to disclose a likely cause of IDA, or in patients refractory to oral iron
treatment, screening for celiac disease, autoimmune gastritis, and H pylori is recommended. Recent studies indicate that 20 to 27% of patients with unexplained IDA have
autoimmune gastritis, about 50% have evidence of active H pylori infection, and 4 to
6% have celiac disease. The implications for abnormal iron absorption of celiac disease
or autoimmune gastritis are obvious. In patients with unexplained IDA and H pylori
infection, refractoriness to oral iron treatment would justify a therapeutic trial of
H pylori eradication. Stratification by age cohorts in autoimmune gastritis implies a disease presenting as IDA many years before the establishment of clinical cobalamin deficiency. It is caused by an autoimmune process likely triggered by antigenic mimicry
between H pylori epitopes and major autoantigens of the gastric mucosa. Recognition
of the respective roles of H pylori and autoimmune gastritis in the pathogenesis of iron
deficiency may have a strong impact on the diagnostic workup and management of
unexplained, or refractory iron deficiency anemia.
s a transition metal, iron plays an
essential role in life by its ability to
accept and donate electrons. Some
of the most important functions of iron
proteins are oxygen transport, mitochondrial oxidative energy production, inactivation of drugs and toxins, and DNA synthesis. The solubility of iron in its stable,
oxidised form is extremely low and,
although iron is one of the most abundant
elements in nature paradoxically, iron deficiency is one of the most common nutritional problems of the human race.1
Consequently, evolution has provided
efficient mechanisms of iron acquisition
and storage but no mechanisms at all for
excreting excess iron.
A
Mechanisms of iron regulation
Basic regulation
In order to gain access to the duodenal
enterocyte (Figure 1) ferric iron in the
intestinal lumen is first reduced to ferrous
iron by duodenal cytochrome b reductase
1 (DCYTB)2 and subsequently transported
through the duodenal brush border membrane by divalent metal transporter 1
(DMT1), a proton transporter requiring
low pH for efficient functioning.3,4 Heme
iron is transported by a different mechanism5 by heme carrier protein 1 (HCP1)
and is subsequently split by heme oxygenase to mix with the pool of intracellular
iron. The intracellular transport of lowmolecular iron in the enterocyte is not
well characterized. In principle, it can be
utilized for the de novo production of functional iron proteins, stored in ferritin, or
exported through the basolateral membrane of the enterocyte. Iron stored in
enterocyte ferritin has a low turnover rate
and is lost by sloughing of intestinal enterocytes within a few days. Iron export
through the basolateral membrane is performed by the basolateral iron transport
protein ferroportin 1 (FPN, IREG1).6,7
Hematology Education: the education program for the annual congress of the European Hematology Association | 2007; 1(1) | 1 |
12th Congress of the European Hematology Association
Brush border
Fe(III)
Basolater
DM T1
Fe reductase
Fe(II)
Fe(II)
Fe(II)
Fe(III)
Fe(II)
Fe(II)
Fe(II)
Labile Iron
Pool
Fe(II)
Ferroportin
Hephaestin
Fe(II)
HCP 1
Fe(III)
Fe(III)
Fe(III)
ferritin
Fe(II)
Fe(II)
Fe(III)
Fe(II)
Tf
Fe
heme
Fe(II)
Fe(III)
endocytic
vesicle
TfR
Fe(III)
Tf
Fe(III)
Fe(III)
Fe(III)
Figure 1. Ferric iron in the intestinal lumen is first reduced
to ferrous iron by duodenal cytochrome b2 (Fe reductase)
and subsequently transported through the duodenal brush
border membrane by divalent metal transporter 1
(DMT1).3,4 Heme iron is transported by heme carrier protein
1 (HCP1)5 and is subsequently split by heme oxygenase to
mix with the pool of intracellular iron. The intracellular
transport of low-molecular iron in the enrterocyte is not
well characterized. Iron stored in enterocyte ferritin has a
low turnover rate and is lost by sloughing of intestinal enterocytes within a few days. Iron export through the basolateral membrane is performed by the basolateral iron transport protein ferroportin (FPN, IREG1).6,7 Following its transport, ferrous iron has to be oxidized in order to bind to circulating transferrin. This function is performed by ceruloplasmin, a circulating multicopper oxidase, or hephaestin,
a cellular homolog of ceruloplasmin.8,9 Diferric transferrin
is internalized after binding to transferrin receptor TfR-1,
and following acidification and reduction crosses the vesicular membrane via the DMT1 transporter
Ferroportin is essential for the basolateral transport of
iron from enterocytes, for placental iron transfer and,
for exporting the catabolic iron derived from senescent erythrocytes from tissue macrophages. Similar
to DMT1, the export of iron by ferroportin is in the
reduced ferrous form. However following its transport, iron has to be oxidized in order to bind to circulating transferrin. This function is performed by ceruloplasmin, a circulating multicopper oxidase, or hephaestin, a cellular homolog of ceruloplasmin.8,9 The
oxidation of ferrous iron outside the basolateral
membrane creates a concentration gradient of ferrous
iron across the cell membrane, facilitating its egress
from the cell.
The above description of iron tranport based on
the interaction of a ferric reductase, two distinct
transport proteins for ferrous iron located on the duodenal brush border and the basolateral membrane
respectively and ferroxidase facilitating iron egress,
may explain basal iron handling but offers no explanation for the adaptation of iron handling to variable
physiologic needs .
Milestones in hepcidin discovery
The discovery of hepcidin and its role in iron
homeostasis represents a major advance in understanding iron regulation. The saga of hepcidin discovery is summarized in Table 1. The intensity of
Table 1. Milestones in the hepcidin saga.
Krause A et al.10
FEBS Letters
Sept 1 2000
LEAP-1 (liver expressed antimicrobial peptide) a 25 residue
peptide containing 4 disulfide bonds: identified by mass
spectrometric assay in human plasma
Park CH et al.11
JBC Epub
Dec 11 2000
Hepcidin a urinary antimicrobial peptide synthesized in the
liver: 20 and 25 amino-acid residues with 8 cysteines
connected by disulfide bonds
Pigeon C et al.12
J Biol Chem Epub
Dec 11 2000
Identification of a 83 amino acid protein with strong
homology to human hepcidin by suppressive subtractive
hybridization between iron-loaded and normal mice
implying a role in iron overload distinct from
antim icrobial activity
Nicolas G et al.13
PNAS Epub
Jul 10 2001
Lack of hepcidin gene expression in upstream stimulatory
factor 2 (USF 2) knockout mice results in severe
tissue iron overload
Nicolas G et al.14
PNAS
Apr 2 2002
Over expression of liver hepcidin in transgenic mice results
in severe iron deficiency anemia
Roetto et al.15
Nat Genet Epub
Dec 9 2002
First identification of human hepcidin mutations in 2
families with juvenile hemochromatosis
Nemeth et al.16
Science Epub
Oct 28 2004
Hepcidin regulates cellular iron efflux by binding to
ferroportin and inducing its internalization
research performed in this field is illustrated by the
rapid sequence of observations as shown by the
dates of their publication. Liver expressed antimicrobial peptide (LEAP-1), a 25 residue peptide containing
4 disulfide bonds was identified by mass spectrometric assay in human plasma, and first described by
Krause et al. in September 1 2000.10 These authors
considered their discovery as an extention of the
known families of mammalian peptides with antimicrobial activity, characterized by a unique disulfide
motif and distinct expression pattern.10 A few months
later, 2 studies were published back-to-back in the
Journal of Biological Chemistry.11,12 Park et al.11
described hepcidin, a urinary antimicrobial peptide
synthesized in the liver consisting of 20 and 25
amino-acid residues (practically identical with LEAP1) with 8 cysteines connected by disulfide bonds.
The liver was the predominant site of mRNA expression and the encoded propeptide contained 84 amino
acids. Simultaneously, in a search for abnormally
expressed hepatic genes under conditions of iron
excess, employing suppressive subtractive hybridization between iron-loaded and normal mice, Pigeon et
al.12 described the overexpression of a 83 amino acid
protein with strong homology to human hepcidin
controlled by a gene located in close proximity to the
upstream stimulatory factor 2 (USF2) gene. Pigeon et
al also recognized that hepcidin expression is
| 2 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2007; 1(1)
Vienna, Austria, June 7-10, 2007
enhanced under the effect of lipopolysaccharide, a
known inducer of inflammatory response. They correctly concluded that these observation imply a role
of hepcidin in iron overload distinct from its antimicrobial activity. These landmark studies were reinforced and extended by the report that fortuitous
inactivation of the hepcidin gene in USF2 knockout
mice results in severe tissue iron overload identical in
phenotype with HFE knockout mice suggesting that
hepcidin may function in the same regulatory pathway as HFE.13 The same group has also shown that
overexpression of liver hepcidin in transgenic mice
results in severe iron deficiency anemia.14 That hepcidin mutations are responsible for severe (juvenile)
hereditary hemochromatosis in humans was first
described in 2002 by Roetto et al.15 Finally, the mechanism of hepcidin interference with cellular iron
transport at two main sites, tissue macrophages and
the intestinal epithelium, by its binding to ferroportin
resulting is its internalization and degradation was
disovered by Nemeth et al., in 2004.16
Rate of hepcidin response
In vivo observations in humans and experimental
animals indicate that hepcidin expression is increased
in response to serum iron, iron overload and inflammation, and is suppressed by iron deficiency, hypoxia and increased erythropoietic activity.17,18 The hepcidin response is remarkably rapid. In man, iron
ingestion at a dose of 65 mg/d results in a sharp
increase of urinary hepcidin excretion within 24
hours of starting treatment.19 Likewise, infusion of
recombinant IL-6, a known mediator of hepcidin
response in inflammation, results in significant
increase in urinary hepcidin, decreased serum iron
and transferrin saturation within 2 hours of infusion.19 These observations imply that hepcidin
expression is directly controlled by serum iron and
IL-6 and not through a long-term gradual accumulation of iron in tissues.
Control of hepcidin response
The regulation of hepcidin expression is transcriptional. Two signal transduction patways modulate
the binding of transcription factors to the hepcidin
promoter: Under basal conditions, hepcidin expression depends upon signalling through the bone morphogenetic protein BMP/SMAD pathway.20,21
Hemojuvelin participates in this patway as a BMP
coreceptor. The second type of transcriptional hepcidin regulation occurs in inflammation: Here interleukin 6 IL-6 induces transcription of the hepcidin
gene by activating signal transducer and activator of
transcription 3 (STAT3) and its binding to a regulatory element in the hepcidin promoter.22
Mutations of five unrelated genes are know to
result in genetic hemochromatosis in man:23 the classic hereditary hemochromatosis HFE, transferrin
receptor 2 TFR-2, the iron transporter ferroportin
FPN1, hemojuvelin HJV and hepcidin HAMP. All
forms of genetic hemochromatosis are characterized
by decreased hepcidin production or activity. Since
HFE, TFR-2 and HJV are all expressed on the surface
of hepatocytes, it was reasonable to expect that they
all may participate in the control of hepcidin expression. Recent studies have indeed confirmed the existence of such a mechanism.24,25 It is based on the competition of transferrin receptor 1 (TFR-1) and TFR-2
for binding the HFE protein. During low or basal
serum iron conditions, HFE and TFR-1 exist as a complex at the plasma membrane and TFR-1 serves to
sequester HFE to silence its activity. Diferric serum
transferrin (Fe2-TF) competes with HFE for the binding of TFR-1. Increased serum transferrin saturation
results in the dissociation of HFE from TFR-1. Acting
as an iron sensor, HFE then binds TFR-2 and thus
conveys the Fe2-TF status to an iron sensor and signal
transduction effector complex consisting of TFR-2,
HFE and HJV for downstream transduction of hepcidin production.
These elegant studies imply that diferric transferrin
is the physiologic regulator of hepcidin and support
the implications of clinical observations in short term
oral iron administration that hepcidin expression is
directly controlled by serum iron and not by the
long-term gradual accumulation of iron in tissues.19
However, the decreased hepcidin expression associated with accelerated erythropiesis and the role of
the putative erythroid regulator in iron homeostasis
remain at present unexplained.26
Mechanisms of iron deficiency
Despite the carefully orchestrated mechanism of
normal iron homeostasis, iron deficiency is still a
major health problem. Its development is the result
of an interplay between three distinct risk factors:
increased host requirements, limited external supply,
and increased blood loss. Iron deficiency is associated with serious health risks including abnormal mental and motor development in infancy; impaired
work capacity; increased risk of premature delivery;
and increased maternal and infant mortality in severe
anemia.1
Increased requirements are the outcome of
increased physiologic needs associated with normal
development. This category of iron deficiency is
often designated physiologic, or nutritional.
Increased physiologic needs are associated with
periods of life characterized by accelerated growth
rates such as early infancy, adolescence and pregnancy. In healthy females normal menstruation represents an additional physiologic burden.27 Although
Table 2. Main diagnostic categories and coexistent findings in 300 IDA patients referred for hematologic evaluation.
Diagnosis (N)
Autoimmune atrophic gastritis
(77) 26%
H. pylori*
(57) 19%
Menorrhagia
(96) 32%
Gastrointestinal lesions
(31) 10%
Celiac
(18) 6%
Negative
(21) 7%
Age
41±16
37±19
39±10
60±14
39±14
33±13
Gender M/F
14/63
17/40
0/96
13/18
3/15
2/21
Main diagnosis alone
26
57
39
21
15
21
H.pilory
39
–
57
10
2
0
Menorrhagia
11
0
–
0
1
0
Gastrointestinal lesions
1
0
0
–
0
0
Aspirin or NSAID
9
3
1
7
0
1
69%
68%
38%
47%
100%
10%
% Refractory to oral iron
*165 total H pylori. Use of aspirin or NSAID is listed as additional information but is not counted separately in the total number of subjects. Under the heading celiac
are also included 4 patients with gastroplasty.
normal gastrointestinal iron absorption is regulated
by effective mechanisms adapting for increased
requirements by enhanced absorption, the magnitude of iron absorption is limited by caloric intake
and the quality of food. These, in turn, are often
determined by socioeconomic conditions. The prevalence of iron deficiency and iron deficiency anemia in
the world is a reflection of the interaction of these
variables. Unfavorable socioeconomic conditions are
associated as a rule with a high prevalence of iron
deficiency and the major victims are infants and fertile women i.e. those with the highest physiologic
needs.27 This dismal global situation has not shown
any indication of improvement over recent years.
Nevertheless, successful targeted food fortification
programs among high risk subpopulations demonstrate28-30 that, major improvements are possible if
resources are available in collaboration with local
health authorities or the food industry.
By contrast, pathologic iron deficiency is most often
the result of gastrointestinal disease associated with
abnormal blood loss or malabsorption. Consequently, in grown males and post-menopausal
females, complete gastroenterologic investigation is
recommended to identify pathological lesions
responsible for abnormal blood loss. However, conventional endoscopic and radiographic methods fail
to identify a probable source of gastrointestinal blood
loss in about one third of males and post-menopausal
females and in most young women with iron deficiency anemia.31-33
Obscure or refractory iron deficiency
In recent years, there is an increasing awareness of
subtle, non-bleeding gastrointestinal conditions that
may result in abnormal iron absorption leading to
IDA in the absence of gastrointestinal symptoms.
Thus, the importance of celiac disease as a possible
cause of IDA refractory to oral iron treatment, without other apparent manifestations of malabsorption
syndrome34 is increasingly recognized. In addition,
Helicobacter pylori has been implicated in several
recent studies as a cause of IDA refractory to oral iron
treatment, with a favorable response to H pylori eradication.35,36 Likewise, autoimmune atrophic gastritis, a
condition associated with chronic idiopathic iron
deficiency, has been shown to be responsible for
refractory IDA in over 20% of patients with no evidence of gastrointestinal blood loss.37,38
The recent availability of convenient, non-invasive
screening methods for identifying celiac disease
(endomysial, and gliadin antibodies) autoimmune
atrophic gastritis (serum gastrin, parietal cell antibodies) and H pylori infection (antibody screening and
urease breath test) greatly facilitated the recognition
of patients with these entities, resulting in an
increased awareness of these conditions and their
possible role in the causation of IDA.
In a prospective study, we have screened 300 consecutive IDA patients referred to a hematology outpatient clinic, employing the above methods for
identifying non-bleeding GI conditions including celiac disease , autoimmune atrophic gastritis and H pylori
gastritis (Table 2) The mean age of all subjects was
39±18 y, and 251 of the 300 (84%) were women of
reproductive age. We identified 18 new cases of adult
celiac disease (6%). Seventy-seven IDA patients (26%)
had autoimmune atrophic gastritis of whom 39 (51%)
had coexistent H pylori infection. H pylori infection
was the only finding in 57 patients (19%), but was a
common coexisting finding in 165 (55%) of the entire
group. Refractoriness to oral iron treatment was
found in 100% of patients with celiac disease, 69%
with autoimmune atrophic gastritis, 68% with H
pylori infection, but only 10% of subjects with no
underlying abnormality. In the following we wish to
discuss the implications of the above findings to the
pathogenesis and management of unexplained iron
deficiency anemia.
Autoimmune atrophic gastritis
The concept of gastric atrophy as a cause of iron
deficiency anemia is not new. Achylia gastrica associated with iron deficiency anemia has been
described as a clinical entity by Faber as early as
1909 39 and achlorhydric gastric atrophy, a synonym for
the same entity, has long been recognized as a
major cause of iron deficiency anemia40 but largely
forgotten, and completely ignored in subsequent
major surveys of gastrointestinal causes of iron deficiency anemia. More recently, achlorhydric gastric
atrophy has been rediscovered by Dickey et al.,38
and implicated in 20% of iron deficiency anemia
patients with no evidence of gastrointestinal blood
loss. This observation was confirmed and greatly
extended in a series of important studies by
Annibale et al 37 who found 27% of patients with
refractory iron deficiency anemia without gastrointestinal symptoms to have atrophic body gastritis ,
a percentage identical with the proportion of subjects with autoimmune atrophic body gastritis
found in our previous 41 and present studies.
Impaired iron absorption in pernicious anemia is
corrected by normal, but not by neutralized gastric
juice, indicating that lack of gastric acidity is the key
factor in abnormal iron absorption.42 Other studies
have also shown that iron absorption is heavily
dependent on normal gastric secretion and acidity
for solubilizing and reducing dietary iron.43,44
Although atrophic gastritis may impair both B12 and
iron absorption simultaneously, in young women in
whom menstruation represents an added strain on
iron requirements, iron deficiency will develop
many years before the depletion of vitamin B12
stores. It is, however, the crucial development of
anti-intrinsic factor antibodies with subsequent loss
of the remaining gastric intrinsic factor that determines the prevalence of pernicious anemia.
Helicobacter pylori gastritis
The role of H pylori in the causation of IDA is at
present unsettled as H pylori infection is very common in the normal population. Major population surveys involving thousands of subjects45 conducted
over diverse geographic areas all indicate that H pylori
positivity is associated with a slight and significant
decrease in serum ferritin levels implying diminished
iron stores, but there was no evidence of a high
prevalence of iron deficiency anemia associated with
H pylori seropositivity in the populations at large.
Nevertheless, in a subset of patients, a cause-andeffect relation between H pylori and serious gastrointestinal pathology including duodenal ulcer, atrophy
of the gastric body predisposing to gastric ulcer and
cancer, or the formation of mucosa-associated lymphoid tissue (MALT) lymphoma has been established
and strongly supported by the beneficial effects of H
pylori eradication in these conditions.46 Consequently,
in a search of evidence for a cause-and-effect relation
between H pylori and IDA, it could be more rewarding to focus on the possible beneficial effects of H
pylori eradication on refractory IDA.
Our previous observations, indicating that failure
to respond to oral iron treatment in H pylori positive
patients was more than twice as common as in the H
pylori negative group, and that successful H pylori
eradication resulted in an increase in hemoglobin levels indistinguishable form that in previously responsive IDA patients,41 are in agreement with a number
of previous studies.36,47,48 Most of these reports
involved young females refractory to oral iron treatment, and improvement has been observed following H pylori eradication even in the absence of continued iron administration .
Because menstrual blood loss is a serious compounding factor in evaluating alternative causes of
IDA, in a recent study we have focussed on 29 male
IDA patients with negative gastorintestinal workup
distinguished by their poor initial response to oral
iron treatment, and high prevalence of H pylori infection (25 of 29) with (10) or without (15) coexistent
autoimmune gastritis.49 Following H pylori eradication,
all patients achieved normal hemoglobin levels with
follow-up periods ranging from 4 to 69 months
(38±15 months mean± 1SD). This was accompanied
by a significant decrease in H pylori IgG antibodies
and in serum gastrin levels. Sixteen patients discontinued iron treatment, maintaining normal hemoglobin and ferritin and may be considered cured.
Remarkably, 4 of the 16 achieved normal hemoglobin without ever having received oral iron after H
pylori eradication.
A number of possible mechanisms have been
invoked to explain the relation between H pylori gastritis and IDA including occult gastrointestinal bleeding and competition for dietary iron by the bacteria .
However, the most likely explanation is the effect of
H pylori on the composition of gastric juice. Studies
by Annibale and others50 have shown that gastric
acidity and ascorbate content, both of which are critical for normal iron absorption, are adversely effected by H pylori infection and, that H pylori eradication
results in normalization of intragastric pH and ascorbate content.
B2 pg/mL; Gastrin; U/mL; H. pylory % positive
1000
900
800
700
600
500
400
300
200
100
0
<20y
21-40y
41-60y
>61y
B12
gastrin
Hp/1000
Figure 2. Effect of age on autoimmune gastritis:
Stratification by age cohorts of autoimmune gastritis from
<20 to >60 y showed a prevalence of coexistant H pylori
infection of 87.5% at age <20 y, 47% at 20-40 y, 37.5 % at
41-60 y and 12.5% at age > 60y. With ages increasing
from <20 to >60 y, there was a regular and progressive
increase in gastrin from 349±247 to 800±627 u/mL , and
a decrease in cobalamin from 392±179 to 108±65
pg/mL.
Possible role of H pylori in the pathogenesis of
autoimmune gastritis
In order to define the relation between IDA associated with autoimmune gastritis and pernicious anemia, we have studied 160 patients with autoimmune
gastritis of whom 83 presented with IDA, 48 with
autoimmune gastritis and normocytic indices, and 29
with macrocytic anemia.51 Stratification by age
cohorts of autoimmune gastritis from <20 to >60 y
showed a prevalence of coexistant H pylori infection
of 87.5% at age <20 y, 47% at 20-40 y, 37.5 % at 4160 y and 12.5% at age > 60y. With ages increasing
from <20 to >60 y, there was a regular and progressive increase in MCV from 68±9 to 95±16 fl, serum
ferritin from 4±2 to 37±41 µg/L, gastrin from
349±247 to 800±627 u/mL , and a decrease in cobalamin from 392±179 to 108±65 pg/mL (Figure 2).
The high prevalence of H pylori positivity in young
patients with autoimmune gastritis and its almost
total absence in elderly patients with pernicious anemia implies that H pylori infection in autoimmune
gastritis may represent an early phase of disease in
which an infectious process is gradually replaced by
an autoimmune disease terminating in burned-out
infection and the irreversible destruction of gastric
body mucosa. Although this question has long
intrigued investigators, the relation between H pylori
and the pathogenesis of pernicious anemia is still
unsettled.52 H pylori-infected subjects have circulat-
Figure 3. Proposed diagnostic workup of IDA in patients
with negative GI studies and in patients refractory to oral
iron treatment, involving non-invasive screening for celiac
disease (anti-endomysial antibodies) autoimmune type A
atrophic gastritis (gastrin, antiparietal antibodies) and H
pylori (IgG antibodies followed by urease breath test).
ing IgG antibodies directed against epitopes on gastric mucosal cells. Of these, the most likely target of
an autoimmune mechanism triggered by H pylori and
directed against gastric parietal cells by means of
antigenic mimicry53-60 is H+K+-ATPase, a protein that
is the most common autoantigen in pernicious anemia. Conversely, H pylori eradication in patients with
autoimmune atrophic gastritis is followed by
improved gastric acid and ascorbate secretion in
many, and complete remission of atrophic gastritis in
a variable proportion of patients.61-63 Failure to
achieve complete remission by H pylori eradication in
the majority of patients does not necessarily argue
against the role of H pylori in the pathogenesis of
autoimmune gastritis but, more likely indicates that a
point of no-return may be reached beyond which the
autoimmune process may no longer require the continued presence of the inducing pathogen.
Recommendations for the diagnostic workup of refractory
or obscure IDA
In view of the above considerations, a rapid screening for celiac disease (anti-endomysial antibodies)
autoimmune type A atrophic gastritis (gastrin,
antiparietal antibodies) and H pylori (IgG antibodies
followed by urease breath test) may provide a highsensitivity screening and an effective starting point
for further investigations . This is particularly recommended in all patients with obscure IDA and in those
refractory to oral iron treatment (Figure 3). The implications of diagnosing celiac disease or autoimmune
atrophic gastritis for abnormal iron absorption are
obvious. Interpretation of positive serology for H
pylori confirmed by positive urease breath test
requires clinical judgment as 20 to 50% of the general and largely healthy population in industrialized
countries will have such findings. In such patients,
refractoriness to oral iron treatment may justify a testand-treat approach of H pylori eradication as currently
advocated for the management of dyspeptic
patients.64 Cure of previously refractory IDA by H
pylori eradication could then be regarded as evidence
supporting a. cause-and-effect relation.
24.
25.
26.
27.
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Pathophysiology, diagnosis and treatment
of the anemia of chronic disease
A
G. Weiss
Department of General Internal
Medicine, Clinical Immunology and
Infectious Diseases, Medical
University of Innsbruck, Austria
2007;1:9-17
B
S
T
R
A
C
T
Recently, progress has been made in clarifying the patho-physiological networks
underlying ACD. ACD is an immunity driven disease and disturbances of iron homeostasis, an impaired proliferation of erythroid progenitor cells and a blunted response to erythropoietin are major factors in its development. Main regulators of body iron homeostasis, such as hepcidin or hemojuvelin, together with pro- and anti-inflammatory
cytokines, cause a diversion of iron traffic which leads to iron retention in macrophages
and an iron-restricted erythropoiesis. ACD can be diagnosed by characteristic changes
of systemic iron homeostasis such as decreased serum iron concentrations and transferrin saturation while ferritin levels are normal or increased. Nevertheless, the correct
evaluation of ACD patients with absolute iron deficiency as a consequence of chronic
bleeding episodes is still a clinical challenge since therapeutic measures differ according to iron status. Therapeutic strategies involve treatment of the underlying disease,
iron, recombinant human eythropoeitin, or blood transfusions with the appropriate therapy. It is hoped that the advent of new assays will improve diagnosis of functional versus true iron deficiency in ACD, determine predictive parameters for therapy and above
all, help choose the best therapeutic regimen in terms of quality of life, good cardiac
performance, and a favourable clinical course of the underlying disease.
nemia of chronic disease (ACD) is
the most frequent anemia in hospitalized patients, and after iron deficient anemia the second most frequent
anemia in the world.1 ACD occurs in
patients with chronic diseases which are
accompanied by acute or chronic immune
activation, and is thus also termed anemia
of inflammation.2-6 However, data on the
exact incidence of ACD are rare. Anemia
has been identified in 30-60% of patients
with small cell carcinoma,7 and the percentage of anemic cancer patients is further aggravated by therapeutic measures
such as radio- and/or chemotherapy.8 An
even higher incidence of anemia has been
found in association with advanced age
and cancer, where 77% of men and 68%
of women were anemic, and ACD was
the underlying cause of anemia in up to
77% of these patients.9 In addition, features of ACD may also contribute do the
development of anemia in the elderly.
This depends on many factors and is still
not fully understood.9
The incidence of anemia was estimated
between 20-60% in patients with autoimmune disorders, such as rheumatoid
arthritis or inflammatory bowel disease.7
Anemia is a frequent abnormality in sever-
A
al infections and in patients with human
immunodeficiency virus (HIV) infection
with incidence rates between 18% to
95%.10 As with all other causes of ACD,
the prevalence and severity of anemia is
associated with an advanced stage of disease.11,12 Most interestingly, anemia frequently emerges within a few days in
patients with severe acute infections and
sepsis. It is still not clear whether the
patho-physiology in these conditions is
the same as for ACD.13
Anemia with chronic renal failure bears
some characteristics of ACD, although the
absolute erythropoietin deficiency and the
anti-proliferative effects of accumulating
urinary excretion products form the
pathophysiolgical basis in this setting.14
Furthermore, chronic immune activation
in patients with end stage renal disease
can arise from contact activation of
immune cells by dialysis membranes
and/or frequent infection episodes14 which
result in pathophysiological changes typical of ACD.
Pathophysiology
ACD is an immune driven condition in
which cytokines and acute phase proteins
alter body iron homeostasis, erythroid
hepcidin
hepcidin
liver
Fe+2 duodenum
Fe+2
+
IL-6
LPS
monocyte
+
IL-1
TNF-α
Tf/TfR
Fe+2
ferritin
IL-10
CD3+
macrophage
IFN-γ
FP-1
+
Fe+2
Pathways for iron retention in ACD
collaboration between acute phase
proteins and cytokines
hepcidin
progenitor cell proliferation, erythropoietin production and red cell life span. The anemia can be further
aggravated by bone marrow infiltration and anti-proliferative effects exerted by tumor cells or microorganisms, accompanying bleeding episodes, deficiencies of vitamin auto-immune hemolysis, concomitant infections such as HP gastritis or chronic
renal insufficiency.6,15
Iron retention within cells of the reticuloendothelial system
Iron retention within cells of the reticuloendothelial system (RES) is characteristic of the development
of ACD leading to subsequent limitation of iron
availability for erythroid progenitor cells, and thus to
an iron-restricted erythropoiesis.
Initial studies demonstrated that injection of mice
with interleukin (IL)-1 or tumor necrosis factor (TNF)α resulted in hypoferremia, hyperferritinema and anemia.16 This can on the one hand be related to an
increased iron acquisition by cells of the RES, such as
macrophages, while on the other hand iron re-distribution from macrophages to the circulation is
reduced (Figure 1). Macrophages have multiple pathways to acquire iron which include most importantly
erythrophagocytosis,17 uptake of ferrous iron via the
transmembrane protein divalent metal transporter-1
(DMT-1),18 of transferrin bound iron via transferrin
receptors (TfR) and of hemoglobin/hemopexin-haptoglobin complexes via CD 91 or CD 163.19-21
Pro-and anti-inflammatory cytokines affect these
iron uptake pathways differently. TNF-α increases
erythrophagocytosis via stimulation of target receptor expression on macrophages and damage of erythrocytes via radical formation thereby reducing erythrocyte half life.22-24 Interferon (IFN)-γ and lipo-
Figure 1. Pathophysiological mechanisms underlying ACD. Invasion of
micro-organisms or emergence of
malignant cells lead to activation of Tcells (CD4+) and monocytes. These
cells induce immune effector mechanisms, thereby producing cytokines
such as interferon (IFN)-γ (from T-cells)
and tumor necrosis factor (TNF)-α,
interleukin (IL)-1, IL-6 or IL-10 (from
monocytes/macrophages) which affect
hepcidin synthesis in the liver and the
iron uptake mechanism in monocytes/macrophages. The latter leads to
increased macrophage iron acquisition
via induction of erythrophagocytosis,
TfR and DMT-1 mediated iron uptake
and promotion of iron storage via ferritin. In addition, the expression of the
iron exporter ferroportin (FP-1) is downregulated by the concerted action of
IFN-γ, lipopolysaccharide (LPS) and
hepcidin leading to reduced serum iron
levels (Fe+2). For more details please
refer to text.
polysaccaride (LPS) up-regulate DMT-1 expression
with increased iron uptake into activated macrophages25 while IL-4, IL-10 and IL-13 enhance TfR
mediated iron uptake into activated macrophages26
and together with TNF-α, IL-1 and IL-6 promote iron
storage within macrophages via transcriptional and
translational stimulation of ferritin expression27-29
(Figure 1). Importantly, while macrophages have
multiple pathways to acquire iron, so far only one
route has been identified for iron export from these
cells, via the transmembrane protein ferroportin1.30
Ferroportin mRNA expression in human monocytes
is down-regulated most prominently by LPS and IFNγ leading to iron retention in these cells.25,31
In addition to cytokines, a liver derived acute
phase protein is crucially involved in iron regulation
under inflammatory conditions. The expression of
hepcidin, a 25 aminoacid cyteine rich peptide, is
induced by LPS, IL-6 and TGF-β.32 Over-expression
of hepcidin results in hypoferremia33, 34 suggesting
that hepcidin may be involved in the diversion of
iron traffic occurring in ACD 35 by decreasing duodenal iron absorption and blocking the release of
iron from macrophages (Figure 1).36, 37 This was further supported by a study in patients with ACD
demonstrating an inverse relationship between
monocyte ferroportin expression and circulating
pro-hepcidin levels.38
Impairment of erythroid progenitor proliferation and
differentiation
Impairment of erythroid progenitor proliferation
and differentiation is another factor for ACD development. This can be related to pro-apoptotic effects
of IFN-γ, IFN-α, TNF-α and IL-1 towards erythroid
Cytokine effects on erythroid progenitor cell proliferation
IL-6
LPS
Molecular mechanisms:
IL-1
monocyte
TNF-α
IL-10
CD3+
IFN-αβγ
TNF-α inhibitory effect via stroma cells
IL-1 acts primarily via IFN-γ induction
IFN-α induces apotposis of CFU-e
IFN-γ caspase mediated apoptosis involving
ceramide
IFN-γ induces NO-inhibits heme synthesis
Cytokines (IFN-γ) inhibit Epo and SCF
formation and functionality
Epo
Iron restriction due to cytokines/hepdicin
kidneys
Bone Marrow
Fe+2
burst forming units (BFU-e) and colony forming units
(CFU-e).5,39 In addition to the limited availability of
iron for erythropoiesis, the cytokine mediated downregulation of erythropoietin-receptor expression on
progenitor cells, an impaired formation and activity
of erythropoietin , a reduced expression of other prohematopoietic factors, such as stem cell factor5,39, 40 as
well as direct toxic effects of cytokine inducible radicals such as nitric oxide (NO) or superoxide anion
cause this inhibition of erythroid progenitor cell proliferation41 (Figure 2).
Acute phase proteins, such as α-1 antitrypsin,
effectively bind to TfRs and inhibit TfR mediated
iron uptake into erythroid progenitor cells, thus
blocking their growth and differentiation.42 Anti-proliferative effects towards erythropoiesis have been
described for ferritin43 which can be referred to limitation of iron availability for progenitor cells.
Moreover, ACD patients may develop deficiencies
of vitamins such as cobalamin or folic acid, conditions which further impair the proliferation of
hematopoietic progenitor cells.44 Finally, in ACD in
association with cancer radio- and chemotherapeutic
interventions can aggravate anemia.45
Reduced formation and biological activity of endogenous
erythropoietin
Reduced formation and biological activity of
endogenous erythropoietin is the third factor in ACD
development. Erythropoietin levels in ACD have
been found to be inadequate for the degree of anemia
in many conditions.46,47 In addition, the biological
response of hypoxia in ACD is impaired. This cannot
be related to changes in iron homeostasis but rather
to negative effects of cytokines on erythropoietin for-
Figure 2. Pathways which
inhibit the proliferation
and differentiation of erythroid progenitors cells by
inflammatory cytokines.
For more details please
refer to the text and to the
legend of Figure 1.
mation and activity.38 IL-1 and TNF-α induce the formation of toxic radicals thereby damaging erythropoietin producing cells and inhibiting erythropoietin
formation.48 This was also observed after the injection of LPS into mice and led to reduced erythropoieitin mRNA expression in the kidneys.48 The
responsiveness of erythroid progenitor cells to erythropoietin correlates to the amount of circulating
cytokines, since in the presence of high concentrations of IFN-γ or TNF-α, much higher amounts of
erythropoietin are needed to restore CFU-e colony
formation.49 After binding to its receptor, erythropoietin stimulates members of the signal transducer and
activator of transcription (STAT) family and subsequently activates mitogen and tyrosin kinase phosphorylation, processes which are affected and modulated by inflammatory cytokines and the negative
feed back regulators they induce48,50 (Figure 3).
Diagnosis
ACD is mostly a mild to moderate normochromic
and normocytic anemia. Diagnosis is based on the
characteristic changes of body iron homeostasis to
differentiate from iron deficiency anemia (IDA).3-5,23 A
definitive diagnosis can be hampered by co-existing
chronic bleeding complications, renal insufficiency,
the effects of medications, primary disorders of iron
homeostasis or of hemoglobin synthesis.
The evaluation of ACD includes a determination of
body iron status. Ferritin is widely used as a marker
of iron storage. While serum ferritin levels are low
(<15 µg/L) in patients with IDA, ferritin levels are
normal or increased in patients with ACD.51 This is
due to two factors. Firstly, the increased ferritin levels reflect iron storage within the RES and secondly,
Cytokine effects on Epo production
IL-6
Molecular mechanisms
IL-1
TNF-α/IL-1induce NF-kB/GATA-2 with suppression of Epo gene promotor
LPS
monocyte
TNF-α
IL-10
CD3+
Cytokine mediated radical formation neg.
affects Epo producing cells in the kidney
IFN-γ
Interaction with Epo/EpoR signal transduction
(JAK2/STAT5/MAPK/PKC)
Reduction of EpoR expression on CFU-e
Epo
kidneys
Bone Marrow
Impaired Epo function because of reduced iron
available
Impaired Epo function due to impaired
erythroid progenitor proliferation
Fe+2
ferritin expression is also induced by inflammation.
Therefore ferritin levels do not relate to the level of
iron stores in inflammatory patients as is the case in
subjects without inflammation. In addition, serum
ferritin levels may also be elevated in conditions such
as hyperthyroidism, chronic liver disease, alcohol
consumption, and after administration of certain
medications.19,52 Thus a cutoff level of 30 µg/L may be
more appropriate for detecting patients with true
iron deficiency.53
As serum iron concentrations and transferrin saturation (TS), as well as zinc protoporphyrin, are low in
both IDA and ACD, these parameters cannot help to
differentiate between them. In contrast, the transferrin concentrations move in the opposite direction,38
normal or low in ACD and it is increased in iron-deficiency anemia.
The soluble transferrin receptor (sTfR) is a truncated fragment of the membrane receptor, and sTfR
levels are increased when the availability of iron for
erythropoiesis is low as in iron-deficiency anemia.54
In contrast, sTfR levels in ACD are not significantly
different from controls. This is because TfR expression is negatively affected by inflammatory
cytokines.55 The determination of sTfR in serum can
help a differential diagnosis between patients with
ACD and functional iron deficiency or ACD patients
with absolute iron deficiency.56 ACD in association
with absolute iron deficiency is found in ACD subjects who suffer from blood loss, for example due to
gastrointestinal or urogenital tumors, menstruation,
inflammatory bowel disease, or intestinal infections.
These patients present decreased serum iron levels
and TfS, low transferrin and decreased ferritin levels.
Calculation of a ratio of sTfR concentration versus
Figure 3. Pathways leading
to reduced formation and
biological activity of erythropoietin. For more details
please refer to the text and
to the legend of Figure 1.
the log of ferritin levels may help to in estimate the
demands of iron for erythropoiesis.54 A ratio of
sTfR/log ferritin <1 suggests ACD with iron-restricted erythropoiesis, whereas a ratio > 3 is associated
with absolute iron deficiency along with ACD.
These parameters are also included in a four quarter
plot which can be very helpful in the differential
diagnosis between ACD and ACD with IDA.57
Determination of the percentage of hypochromic
red blood cells or, even more importantly,
hypochromic reticulocytes, may be helpful in estimating the iron availability for erythrocyte progenitors. This may also be true for mean cellular hemoglobin and mean cellular volume, which appear to
decrease with absolute iron deficiency together with
ACD.6,38,58 It will be of interest to see whether the
determination of hepcidin or hemojuvelin in serum
is helpful in the differential diagnosis between ACD
and ACD with IDA.
Treatment
The persistence of anemia is associated with an
impaired cardiac and kidney function, a reduced systemic oxygen delivery, reduced physical activity,
fatigue, and reduced in quality of life. In a retrospective review of hemodialysis patients, levels of hemoglobin ″8.0 g/dL were associated with a two-fold
increase in the probability of death when compared
with hemoglobin ranges of 10.0 to 11.0 g/dL.59 In
patients with kidney failure on dialysis, treatment of
anemia is associated with improvements in quality
of life (QOL) based upon both Karnofsky Score and
Sickness Impact Profile.60 In cancer patients undergoing chemotherapy, a significant improvement in
QOL with anemia management was observed, with
the largest improvement occurring between hemoglobin 11 to 12 g/dL.61 These findings contributed to
the development of guidelines for the management
of anemia in cancer patients.62
When possible, the therapeutic approach toward ACD
is treatment of the underlying disease.4,5,11 When this is not
achievable, alternative strategies are necessary.
Blood transfusions
Blood transfusions are widely used as a rapid and
effective therapeutic intervention. They are particularly helpful in the context of severe anemia (Hb < 8
g/dL) or life-threatening anaemia (Hb < 6.5 g/dL) due
to ACD or in ACD aggravated by bleeding complications. Blood transfusions have been associated with
increased survival in anemic subjects with myocardial infarction.63 Whether blood transfusions induce
immuno-modulation with clinically relevant, deleterious effects, remains controversial.64 For example, in
patients who underwent surgery for esophageal cancer, the perioperative transfusion of blood was associated with an unfavorable clinical course65 and has
been linked to organ dysfunction.66 Blood transfusion
may affect the immune status of patients directly64,67
or via the donation of iron to the circulation with the
potential consequences described below including an
increased risk for infectious complications.68
Some of these studies may be biased by the fact
that a more sustained anemia is a reflection of a more
advanced disease. Thus, such patients would have a
worse prognosis per se, and the transfusion of blood
for the correction of anemia may not then account
for the negative clinical outcome. Based on these different data it is essential that future studies should
address the impact of red blood cell transfusion on
the clinical outcome of ACD patients and how leukocyte depletion of transfusions may affect the course
of the underlying disease.
Iron
Iron therapy alone, in the absence of iron-deficiency, is not helpful in patients with ACD. Oral
iron is poorly absorbed due to down-regulation of
iron absorption in the duodenum.69,70 This has been
clearly demonstrated by a clinical study in patients
suffering from anemia and active inflammatory
bowel disease.71 Moreover, iron therapy for patients
with ACD may be potentially damaging, since this
it counteracts two potential pathophysiologic
mechanisms underlying ACD development. Firstly,
iron is an essential nutrient for proliferating organisms. Thus, the withdrawal of iron from microorganisms or tumor cells into the RES is a potentially effective defense strategy to inhibit the growth of
pathogens.19,72
Secondly, iron inhibits the activity of IFN-γ,55 a
cytokine centrally involved in the co-ordination of
cell mediated immune effector mechanisms against
invading pathogens.
Accordingly, iron loaded macrophages cannot clear
infections with various intracellular micro-organisms
by IFN-γ mediated pathways.55,72
Moreover, iron therapy in a setting of chronic
immune activation promotes the formation of highly
toxic hydroxyl-radicals via the catalytic action of the
metal by the Haber-Weiss-reaction. This can cause
tissue damage, endothelial dysfunction and increase
the risk of acute cardiovascular events,73 along with
the promotion of carcinogenesis via malignant cell
transformation.72 An increased iron availability in
serum or tissues has been associated with an
increased risk of malignancy,74 and diabetes mellitus.75 Negative effects on immunity with iron therapy can also potentially increase the risk of infectious
complications or septicemia in patients with ACD
patients.76 Iron therapy in chronic hemodialysis
patients has been shown to induce neutrophil dysfunction. Consequently, these patients are unable to
phagocytose invading bacteria.77
On the other hand, due to its immune-deactivating
potential, iron therapy may have some benefit in
ACD in connection with autoimmune disorders. By
inhibiting TNF-α formation, iron may reduce disease
activity in rheumatoid arthritis or end stage renal disease.78,79 However, as iron is needed for the basic cellular metabolic process, iron must be supplemented
to ACD patients with absolute iron deficiency. This
can also develop under conditions of intense erythropoiesis80,81 during therapy with erythropoietic agents,
as patients exhibit a decrease in TfS and in ferritin to
levels 50% to 75% below baseline.80,81 Parenteral (i.v.)
iron has been shown to improve response rates to
therapy with erythropoietic agents in cancer patients
undergoing chemotherapy82 and in patients with
chronic kidney disease undergoing dialysis.83 In addition, patients with inflammatory bowel disease and
anemia respond well to parenteral iron therapy.84
Patients with ACD and absolute iron deficiency
should receive supplemental iron therapy,62,83,85 and
i.v. iron supplementation should be considered for
patients who are not responsive to therapy with erythropoietic agents and who are suspected to have
functional iron deficiency. Iron is more likely to be
absorbed and utilized by the erythron rather than by
pathogens under these conditions, as demonstrated
by an increase in hemoglobin levels without demonstrable infectious complications.82,86 Iron therapy is
currently not recommended for ACD patients with
high/normal ferritin levels (>100 µg/L) due to the
possibility of unfavorable outcomes.55,73-75,77,87 This has
been clearly confirmed by a recent prospective study
investigating the risk of bacteremia with iron therapy
in hemodialysis patients which found that subjects
with a TfS > 20% and ferritin levels > 100 µg/L at
study entry had a significantly higher risk for bacteremia.86
Importantly, the long term effects of iron therapy,
even in iron deficient subjects, on the course of the
underlying malignant or chronic infectious disease
are almost completely unknown and prospective
studies have been unable to define how to treat such
patients.4,7,23 Prospective studies must still show
whether or not the combination of recombinant
human erythropoietin (rhEpo) with iron is an efficient therapeutic approach without the potentially
negative side effects described above.
Human recombinant erythropoietin
rhEpo has been used in ACD patients with autoimmune, infectious (such as HIV) and malignant diseases with varying efficacy,61,62,88 and is currently
approved for use in cancer patients undergoing
chemotherapy, patients with chronic kidney disease,
and in patients with HIV infection undergoing
myelosuppressive therapy. It may counter-act the
anti-proliferative effects of cytokines by rhEpo23,49
and stimulate iron uptake and heme biosynthesis in
erythroid progenitor cells4. Accordingly, a poor
response to rhEpo treatment is associated with
increased levels of pro-inflammatory cytokines on
the one hand and poor iron availability on the other
hand.89,90 Currently, several rhEPO are available with
different receptor binding affinities and serum half
lives.91
The measurement of endogenous erythropoietin
levels can be useful for predicting the response to
rhEpo treatment in ACD.89 Calculation of a ratio of
observed endogenous erythropoietin levels to the
predicted ones for the given degree of anemia (O/P
ratio) may also be useful.92 ACD patients with high
erythropoietin levels (>100 U/L) and increased O/P
ratios (>0.9) have a low probability of responding to
treatment with rhEPO.89,92 This is also true of patients
with high levels of markers of inflammation, and/or
high ferritin levels as well as in patients with iron
deficiency.6
Although the positive short term effects of rhEpo
therapy with the correction of anemia are well documented,61,62,88 hardly any data are available on the
effect of rhEpo on the course of underlying disease,
particularly since erythropoietin can exert biological
effects as well as induce of erythropoiesis.
In addition to this, rhEpo also exerts an immunomodulatory effect by interfering with the signal transduction cascade of cytokines.48 In patients with end
chronic kidney disease, the long term administration
of rhEpo decreased TNF-α levels, and good responders
to rhEpo therapy had significantly higher CD28
expression on T cells and reduced IL-10, IL-12, IFN-γ
and TNF-α levels compared to poor responders.93 Such
anti-inflammatory effects may be of benefit in
rheumatoid arthritis, where combined treatment with
rhEpo and iron not only increases hemoglobin levels
but also reduces of disease activity.78
Furthermore, erythropoietin receptors (EpoR) are
found on several malignant cell lines94-96 but substantial concerns were raised concerning the specificity of
the antibodies used97 and the biological role of such
receptors.98,99 The production of EpoR and erythropoietin by breast cancer cells appears to be regulated
by hypoxia, and in clinical specimens of breast carcinoma, the highest levels of EpoR were associated
with neo-angiogenesis, tumor hypoxia and infiltrating tumors.96 A recent study investigating the effect
of rhEpo therapy on the clinical course of non-anemic
patients suffering from metastatic breast carcinoma
was discontinued because of a trend towards a higher mortality among patients receiving rhEpo. 100
However, a subsequent follow up study demonstrated that the survival-lines converged at 19 months of
follow up and subsequently survival was improved
in the rhEPo treated arm.101
This suggests that rhEpo and/or adjunct iron therapy has a short term negative effect in non-anemic
cancer patients which requires further investigation.102
This breast cancer trial was based on a study in
patients with head and neck tumors which demonstrated that an increase in hemoglobin levels upon
rhEpo therapy was associated with a favorable clinical outcome. This was traced back to an improved
tumor-oxygenation and an increased susceptibility of
the tumor to preoperative chemoradiation therapy.103
In contrast, in a subsequent double-blind prospective
study investigating whether target Hb levels (> 13
g/dL for women and > 14 g/dL for men) improved
loco-regional control of patients undergoing radiation
therapy for squamous cell carcinoma of the head or
neck, a slightly poorer prognosis was found in
patients treated with rhEpo compared to a placebo.104
A negative effect of anemia normalisation has
been observed in patients with chronic kidney disease. Hematocrit levels between 33% and 36%
were associated with the best outcomes in mortality and morbidity,105 while both over-correction of
anemia to normal hemoglobin levels and insufficient treatment were associated with unfavorable
clinical courses.6,56,105,106 Until further data are forthcoming; these observations are also applicable to
patients with ACD.
Current evidence suggests that rhEpo is safe and
effective for the corrective treatment of anemia and
to improve the quality of life in ACD patients with
malignancy. Importantly, current evidence suggests
that Hgb levels should not be normalized, and that
target levels for anemia correcting therapy should be
no greater than 12 g/dL. The evaluation of EpoR in
tumor tissue may help to select which patients
should receive rhEpo.
This leads to the as yet unsolved questions of the
clinical impact of anemia correction on the course of
the underlying disease and of defining therapeutic
ends point for therapy. We designed, large randomized, double blinded, multi-center placebo controlled
studies would help answer these questions in the
future.
New therapeutic strategies will follow our
improved knowledge of the pathophysiology of
ACD. These may include the use of iron chelators
which can induce the endogenous formation of erythropoietin, new products such as hepcidin or hemojuvelin antagonists which may overcome the retention of iron within the RES, modifiers of erythropoietin and/or EpoR sensitivity, and new hormones and
cytokines which can effectively stimulate erythropoiesis under inflammatory conditions.
With the availability of new assays, the specificity
of the diagnosis of functional versus absolute iron
deficiency in ACD, along with predictive parameters
for therapy, will produce strategies for the best therapeutic regimen of ACD for each patient. It will be
important to define therapeutic end points which are
positively associated with quality of life, improved
cardiac performance, and a favorable impact on the
underlying disease.
Acknowledgements
Supported by a grant from the Austrian Research
Funds, FWF, P-19664 and the European Union project, EUROIRON1.
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Screening hemochromatosis and iron overload
C. Camaschella
A. Pagani
E. Poggiali
L. Silvestri
Università Vita-Salute and IRCCS
San Raffaele, Milan, Italy
2007;1:18-23
A
B
S
T
A
C
T
Iron overload may develop in several genetic and acquired conditions, either associated with anemias or with normal hemoglobin levels. At present several approaches are
available to early recognize iron overload in otherwise symptomless patients. First level
tests are serum iron parameters that allow a distinction between cases with increased
transferrin saturation (± serum ferritin) and cases with an isolated increase of serum ferritin. Genetic tests for the most common HFE variants allow the detection of most
hemochromatosis patients in the preclinical state so that tissue damage due to iron
overload can be prevented by early treatment. New non-invasive techniques to directly
determine tissue iron content will become widely available in the future. The ultimate
goal of testing for iron overload is to provide treatment by phlebotomy or iron chelation
to avoid irreversible iron toxicity.
Iron overload
The term hemochromatosis was first
used in the 19th century to indicate bronze
diabetes with cirrhosis. Subsequently the
term was used for disease resulting from
excessive tissue iron deposition. At present its use is restricted to the genetic disease that leads to iron overload through a
deregulation of iron homeostasis. Iron
overload is a more general term that refers
to conditions of increased total body iron
caused by iron supply exceeding iron
requirements. Since humans lack a physiological mechanism of iron excretion,
increased total body iron may be the
result of excessive iron absorption or parenteral iron acquisition. These two modalities usually reflect genetic (primary) and
acquired (secondary) disorders, although
this distinction is not always clear. Iron
stores up to a maximum of 1.0-1.5 g is
usual in adult males while iron stores in
excess of 5 g can cause significant toxicity.
The liver is the major site of iron storage.
According to the route of iron entrance
(dietary or parenteral) accumulation
occurs prevalently in the hepatocytes or in
the macrophages.
A physiopathological classification of
conditions leading to iron overload is
shown in Table 1. Primary iron overload
refers to inborn errors of iron metabolism,
including defects of regulators of iron
homeostasis and of iron transporters. The
first group of disorders, caused by an
inappropriately high duodenal iron
absorption and release from macro-
| 18 |
R
phages, are globally called hemochromatosis. Several genetic forms of
hemochromatosis have been recognized,
most due to defective production of hepcidin, the liver peptide hormone, which is
the key regulator of iron homeostasis.
The disorders of the second group include
hypotransferrinemia and newly described
entities, such as DMT1 deficiency and
aceruloplasminemia. These are much
rarer and associated with anemia.
Secondary iron overload occurs in all
patients that are treated by multiple blood
transfusions, both for congenital and
acquired anemias. It is also prevalent in
the so called iron loading anemias, which
include mainly beta-thalassemia syndromes and congenital dyserythropoietic
anemias (CDA), disorders characterized
by a high degree of ineffective erythropoiesis.
Finally, there are causes of local iron
overload such as liver disorders, iron-related brain disorders, or sequestration (hemoglobinuria and pulmonary siderosis).
Iron toxicity is similar in primary and
secondary iron overload and relates to
the well known ability of iron to generate reactive oxygen species (ROS).
When transferrin is highly saturated
excess iron is present in plasma as NonTransferrin-Bound-Iron (NTBI), which is
easily taken up by the liver. NTBI and
especially the Labile Plasma Iron fraction, may promote the generation of
free hydroxyl radicals, known mediators
of tissue damage.1
Hematology Education: the education program for the annual congress of the European Hematology Association | 2007; 1(1)
Primary iron overload
Hereditary Hemochromatosis
Hereditary hemochromatosis results from disruption of the molecular mechanisms that regulate iron
absorption, leading to a progressive increase of total
body iron. Based on the first identified gene this condition is often classified as HFE and non-HFE
hemochromatosis. According to our present knowledge of the molecular pathogenesis of the disorder it
would be more appropriate to classify these disorders under the more general definition of disorders of
the hepcidin pathway.2
The nomenclature of hemochromatosis and the
type of inheritance of the different forms are reported in Table 2.
Type 1 hemochromatosis is the most common
form, due to mutations of the HFE gene. It is the classic adult-onset disease, prevalent in males. From 64
to >90% of these patients have the homozygous
C282Y mutation, due to a G->A substitution at
nucleotide 845 of the HFE gene. The C282Y mutation is prevalent in Northern Europe and less frequent in Southern Europe, reflecting the ancient origin of the mutation3 and its spread through founder
effect and positive selection. A minority of patients
are compound heterozygotes for both the C282Y
and the H63D mutation due to the C->G transversion at position 187 of the HFE gene or homozygotes
for the latter mutation. Compound heterozygous and
H63D homozygous patients usually have a mild,
non-progressive iron overload. These HFE genotypes
are more frequent among patients from Southern
Europe where the C282Y mutation is less frequent.4
Type 2 or juvenile hemochromatosis, due to mutations of either hemojuvelin (type 2A) or hepcidin
(type 2B) is the most severe form. It affects both
sexes and is characterized by early onset of iron overload, hypogonadism and cardiomyopathy. The rare
type 3 hemochromatosis, due to mutations of transferrin receptor 2 (TFR2) has a variable phenotype. All
these conditions show elevated saturation of transferrin and excessive iron deposition in the hepatocytes.
Type 4 hemochromatosis, a dominant disorder due
to mutations of the iron exporter ferroportin 1,
which behaves as the hepcidin receptor,5 has different genetic, biochemical, histological and clinical features and is considered a disorder distinct from
hemochromatosis, often referred to as ferroportin disease (Table 2). However, according to the mutation
present, some cases may present features which
make it indistinguishable from hemochromatosis.
Late presentation of the classic disease includes
liver fibrosis and cirrhosis, diabetes, cardiomyopathy,
hypogonadism and other endocrinopathies,
arthropathy and skin pigmentation. Cirrhotic
Table 1. Classification of Iron Overload.
Systemic forms
Primary Iron overload
Normal Hb levels
Hemochromatosis (type 1,2,3)
Ferroportin disease (type 4)
Associated with anemia
Atransferrinemia
Aceruloplasminemia
DMT1 defects
Secondary iron overload
Normal Hb levels
Inappropriate parenteral iron
Associated with anemia
Chronic blood transfusions
Beta-thalassemia syndromes
Congenital dyseritropoyetic anemias
Sideroblastic anemias
Hemolytic anemias
Uncertain conditions
Neonatal hemochromatosis
African (Bantu) iron overload
Local forms
Liver iron overload
Chronic liver diseases
Porphyria Cutanea Tarda
Dismetabolic syndrome with iron overload
Neurologic diseases
Neuroferritinopathy
Friedreich’s ataxia
Pantothenate kinase neurodegeneration
Focal sequestration of iron
Pulmonary siderosis
Renal siderosis
Table 2. Genetic types of Hemochromatosis according to
Omim (On Line Mendelian Inheritance In Man).
Disease
Gene
Type 1
Hfe
Type 2A
HJV
Type 2B HAMP
TFR2
Type 3
*Type 4 SCL40A1
Locus
Inheritance
Gene Product
Phenotype
6p21.3
1q21
19q13.1
7q22
2q32
AR
AR
AR
AR
AD
Hfe
Hemojuvelin
Hepcidin
Transferrin receptor 2
Ferroportin
classic
juvenile
juvenile
classic
atypical
AR: autosomal recessive, AD: autosomal dominant; *also called ferroportin disease (see text for details).
patients have increased susceptibility to hepatocellular carcinoma. The disease should be recognized
early (or at least in the precirrhotic phase) and treated by intensive phlebotomy to prevent all the clinical
complications. Early presentation of hemochromatosis is often difficult to recognize, because of unspecific symptoms, such as weakness, abdominal pain,
weight loss and arthralgia. Some subjects are symp-
tomless at presentation and have only altered serological iron parameters. Liver function tests may also
be normal.6 At present, the possibility of identifying
the hemochromatosis genotype makes preclinical
diagnosis easier.
Other inherited disorders of iron metabolism
These disorders are due to genetic defects of proteins involved in iron transport and for this reason are
classified as inborn errors of iron metabolism. They
cause iron overload associated with anemia and for
this reason could be also classified under Iron loading
anemias.
The prototype disorder of iron transport is genetic
hypotransferrinemia, an extremely rare recessive disease, first described in 1961. It is characterized by
severe deficiency of transferrin, which appears fully
saturated, iron-deficient anemia, high ferritin and
liver iron overload. Few patients have been described
worldwide and their survival is strictly dependent on
transferrin (or plasma) infusions.7
Aceruloplasminemia is a rare recessive disease due
to deficiency of ceruloplasmin, a multioxidase protein involved in iron export. Due to defective iron
release from stores to transferrin, serum iron and
transferrin saturation are low, serum ferritin is high
while the liver is iron overloaded.8 The full picture
occurs late in life and is dominanted by neurological
symptoms, because of iron deposition in the basal
ganglia. But atypical iron-deficient anemia predates
neurological symptoms.9
Divalent Metal Transporter 1 (DMT1) is a metal
transporter expressed at the brush border of the enterocytes and on the endosomal membrane of the erythroblasts where it transports iron to the cytosol.
Spontaneous mutations of DMT1 in rodents cause
iron-deficiency anemia at birth.10,11 Mutations of
DMT1 have been described in young patients with
microcytic anemia from birth and iron overload.12-14
Secondary iron overload
Iron loading anemias
The term iron loading anemias traditionally applies
to congenital anemias of variable severity, all characterized by ineffective erythropoiesis, with highly
increased intestinal iron absorption and significant
iron overload. Thalassemia, especially beta-thalassemia syndromes, is the prototype of these conditions. Iron absorption is mostly increased in untransfused patients with thalassemia intermedia to meet
the request of an abnormally expanded erythron,
whereas blood transfusions limit erythroid expansion and iron absorption in thalassemia major.
Congenital dyserythopoietic anemia (CDA) comprises a group of rare inherited disorders characterized
by distinct morphological erythroblast abnormalities
and a high degree of ineffective erythropoiesis.
Hereditary sideroblastic anemia is an heterogeneous
condition whose main feature is defective heme synthesis.15 The most common form results from mutations of aminolevulinic acid (ALA)-synthase 2 and a
rarer form, sideroblastic anemia with ataxia, from
mutations of ABCB7, which transports iron/sulphur
clusters from mitochondria to the cytosol. Due to the
interconnection between heme and iron sulfur cluster synthesis it is likely that in the future, inherited
sideroblastic anemia will be listed under primary iron
overload.
The preliminary observation of low levels of hepcidin in iron loading anemias, especially in thalassemia intermedia patients16,17 confirms that deregulation of the same pathway characterizes both primary and secondary iron overload. It also indicates
that the erythron has a dominant effect over iron
stores in signalling body iron needs and suppressing
hepcidin synthesis.
The development of iron overload in hemolytic
anemias occurs in sporadic cases.
Parenteral iron loading
Chronic blood transfusions, administered for only
reason other than blood loss, result in iron overload.
Each unit of transfused red cells provides about 180200 mg iron. This results in a marked increase in total
body iron in congenital (or even acquired) anemias
where there is a life-long dependence on transfusion.
Iron overload in acquired anemia may be relevant in
myelodysplastic syndromes with good prognosis. A
variable degree of iron overload may be present in
patients after therapy for leukemias or lymphoma or
in patients recovering after bone marrow transplantation. Excess iron accumulates in macrophages and
increased stores are expected to suppress duodenal
iron absorption. However, the amount of transfused
iron is usually much greater than the small reduction
in iron absorption. In addition, in most forms of congenital anemias iron loading occurs both through
increased absorption and blood transfusions.
Iron overload due to inappropriate parenteral iron
therapy is at present an extremely rare occurrence.
Inappropriate oral iron treatment, in the absence of a
genetic defect that enhances iron absorption, is
unable to induce iron overload.
Miscellanea
Moderate iron loading may occur in chronic liver
diseases such as alcoholic cirrhosis, porphyria
cutanea tarda (PCT) and liver insufficiency.
Underlying mechanisms have not yet been fully
explored at the molecular level. Preliminary data in
end-stage liver diseases suggest that hepcidin-deficiency plays a role in iron loading and that its produc-
tion correlates with hepatic dysfunction.18 Interestingly PCT is associated with HFE mutations, especially C282Y.19
Screening iron overload
Serum iron parameters as a screening tool
for hemochromatosis
Serological iron markers are the most important
tool to detect hemochromatosis and are widely
available. Transferrin saturation may be calculated
directly using serum iron and total iron binding
capacity measurements or indirectly when serum
iron (µl/L) and transferrin (g/L) are available (transferrin saturation % = serum iron ×100/transferrin×1.42) Elevated transferrin saturation (cut-offs
vary from> 45% to > 55%) and serum ferritin above
the upper normal limits (200 ng/mL in adult females
and 300 ng/mL in adult males) are the thresholds
used to screen hemochromatosis type 1, 2 and 3.
Detecting type 4 hemochromatosis requires family
studies because of the dominant inheritance and
high serum ferritin concentration, often with normal transferrin saturation. However, a tendency has
been observed towards an increase of transferrin
saturation with age.20
Serum iron, transferrin and ferritin should preferably be assessed in fasting conditions. High transferrin saturation reflects increased iron absorption and
recycling and high serum ferritin increased liver iron
stores. At least two assessments should be made
before proposing genetic testing. In the absence of
secondary causes of iron overload high iron parameters suggest hemochromatosis. Whereas the clinical
penetrance of hemochromatosis is low and variable
among different populations, the biochemical expression (increased iron parameters levels) is frequent
although age-dependent especially in males
(>70%).21,22
Alteration of serum iron parameters allows
hemochromatosis patients to be identified in the
asymptomatic stage. However, this approach identifies several other conditions that are not associated
with hemochromatosis. Clinical information should
always be available to correctly interpret iron parameters. Liver and hematological disorders, alcohol
intake and the concomitant occurrence of the iron
overload-associated metabolic syndrome, should all
be considered. Inflammatory indexes (CRP) and
serum transaminases should be available for all cases
with high serum ferritin to exclude inflammatory
conditions and liver necrosis or other alterations. In
addition, cancer screening may be indicated in older
patients with elevated serum ferritin levels.
Hyperferritinemia-cataract syndrome is a rare
dominant condition characterized by a constitutive
increase in L-ferritin synthesis, because of heterozy-
gous mutations of L-ferritin IRE promoter element
that hamper repression of L-ferritin translation. The
level of serum ferritin is high while transferrin saturation and iron stores are normal. Bilateral cataracts
or minimal lens opacities are features of the syndrome.20
Genetic testing for hemochromatosis
Genetic testing is widely used to confirm or
exclude genetic hemochromatosis. It requires the
provision of adequate information about the patient
(and the family) and informed consent before testing.
Multiple PCR-based strategies have been developed
to assess the HFE genotype. Most available tests
identify the two most common variants – C282Y and
H63D. Commercial tests are available to identify
multiple mutations both in HFE and in the other
genes. However, it must be remembered that genetic
types of hemochromatosis other than HFE are rare.
Genetic test results should always take into
account the patient clinical data and the biochemical
iron parameters. The hemochromatosis genotype
only indicates a susceptibility to iron loading and
should never be considered outside the context of the
iron status. C282Y/C282Y homozygotes are at risk
for iron loading. Staging of the disease and evaluation
of treatment needs should be made according to ferritin levels. In subjects with normal-borderline levels,
monitoring iron parameters yearly is advisable.
C282Y/H63D compound heterozygotes and H63D
homozygotes are genotypes at low risk of iron overload and monitoring of iron overload can be less
stringent. C282Y or H63D heterozygous individuals
are not at risk of iron overload unless strong environmental factors coexist. The presence of alcohol
intake, PCT or steatohepatitis increase the risk of
iron loading (and liver fibrosis) in all genotypes.
A search for other mutations in HFE or other genes
should be offered to selected cases, especially young
subjects and familial cases. These patients should be
referred to specialized centers, since second level
diagnosis is expensive and time consuming. Rare
cases of digenic inheritance with simultaneous (heterozygous) mutations in different genes have been
described.23
Molecular tests did not identify genetic haemochromatosis as the cause of an increased transferrin
saturation and serum ferritin found in the majority of
such cases identified in a large study of a racially
diverse population.24 There is therefore no indication
for extensive sequencing of multiple genes in most
isolated cases since the assays used are expensive and
time consuming.
General population vs selected cases screening
Since information is incomplete as to the natural
history of the hemochromatosis, and on the proportion of individuals with the affected genotype that
will show progression to clinical disease, genetic
screening is not cost effective and is not therefore
recommended. Most of the screening strategies are
based on transferrin saturation or are two-step, considering genetic evaluation as a second level test to be
applied only to positive phenotypes.24,25
On the other hand, evaluating iron parameters in
adult males is advisable, preferably starting at the end
of the third decade of life. In addition, family testing
of affected subjects should be implemented in order
to recognize early presenting or symptomless potential patients. All first-degree relatives of a patient
should be tested and counselled on the need of disease staging and of phlebotomy treatment.
Children should not be tested since the disease is a
late-onset. The exception is a high suspicion of juvenile hemochromatosis, a potentially fatal disorder. In
this case, children should be referred to specialized
centers for diagnosis and treatment.
Screening iron loading in secondary forms
All chronically transfused patients should undergo
periodical assessment of iron status in order to be
adequately treated by iron chelation.26 Iron overload
may be assessed by iron balance (iron introduced by
number of transfusions and iron excreted by chelation treatment), serum ferritin and tissue iron directly evaluated through liver iron concentration by invasive (liver biopsy) and/or non invasive determinations (SQUID or MRI)27,28 and more recently by cardiac iron measurement (when T2* is available).29
In untransfused thalassemia and other iron loading
anemias transferrin saturation is usually increased
and of limited value. A trend towards increased
serum ferritin is considered a reliable index of iron
accumulation.30 However, it has been observed that
serum ferritin is lower than expected for the liver iron
concentration in thalassemia intermedia, probably
due to hepcidin suppression and low macrophage
iron.31 This would indicate that serum ferritin is not a
reliable marker since it may underestimate iron overload and not recognise that these patients should
undergo regular evaluation of liver iron concentration. Other serological markers, such as NTBI or LIP
have been proposed to assess iron toxicity, but their
assay has not yet been standardized and assays are
only available in a few research laboratories.
Genetic testing for HFE mutations has been applied
in secondary iron overload. General experience seems
to suggest that severe iron loading occurs independently by HFE mutations although in mild forms and a
possible role of the HFE gene cannot be excluded.
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F, et al. Redox active plasma iron in C282Y/C282Y
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Blood 2005;106:3710-7.
3. Distante S, Robson KJ, Graham-Campbell J, Arnaiz-Villena
A, Brissot P, Worwood M. The origin and spread of the HFEC282Y haemochromatosis mutation. Hum Genet.
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Montosi G, et al. Heterogeneity of hemochromatosis in Italy.
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Ward DM, et al. Hepcidin regulates cellular iron efflux by
binding to ferroportin and inducing its internalization.
Science 2004;306:2090-3.
6. Pietrangelo A. Hereditary hemochromatosis-a new look at
an old disease. N Engl J Med 2004;350:2383-97.
7. Beutler E, Gelbart TPL, Trevino R, Fernandez MA, Fairbanks
VF. Molecular characterization of a case of atransferrinemia.
Blood 2000;96:4071-4.
8. Harris ZL, Takahashi Y, Miyajima H, Serizawa M,
MacGillivray RT, Gitlin JD. Aceruloplasminemia: molecular
characterization of this disorder of iron metabolism. Proc
Natl Acad Sci USA 1995;92:2539-43.
9. Bosio S, De Gobbi M, Roetto A, Zecchina G, Leonardo E,
Rizzetto M, et al. Anemia and iron overload due to compound heterozygosity for novel ceruloplasmin mutations.
Blood 2002;100:2246-8.
10. Fleming MD, Trenor CC 3rd, Su MA, Foernzler D, Beier DR,
Dietrich WF, et al. Microcytic anaemia mice have a mutation
in Nramp2, a candidate iron transporter gene. Nat Genet
1997;16:383-6.
11. Fleming MD, Romano MA, Su MA, Garrick LM, Garrick
MD, Andrews NC. Nramp2 is mutated in the anemic
Belgrade (b) rat: evidence of a role for Nramp2 in endosomal
iron transport. Proc Natl Acad Sci USA 1998;95:1148-53.
12. Mims MP, Guan Y, Pospisilova D, Priwitzerova M, Indrak K,
Ponka P, et al. Identification of a human mutation of DMT1
in a patient with microcytic anemia and iron overload. Blood
2005;105:1337-42.
13. Iolascon A, d’Apolito M, Servedio V, Cimmino F, Piga A,
Camaschella C. Microcytic anemia and hepatic iron overload
in a child with compound heterozygous mutations in DMT1
(SCL11A2). Blood 2006;107:349-54.
14. Beaumont C, Delaunay J, Hetet G, Grandchamp B, de
Montalembert M, Tchernia G. Two new human DMT1 gene
mutations in a patient with microcytic anemia, low ferritinemia, and liver iron overload. Blood 2006;107:4168-70.
15. Bottomley SS. Congenital sideroblastic anemias. Curr
Hematol Rep 2006;5:41-9.
16. Papanikolaou G, Tzilianos M, Christakis JI, Bogdanos D,
Tsimirika K, MacFarlane J, et al. Hepcidin in iron overload
disorders. Blood 2005;105:4103-5.
17. Kearney SL, Nemeth E, Neufeld EJ, Thapa D, Ganz T,
Weinstein DA, et al. Urinary hepcidin in congenital chronic
anemias. Pediatr Blood Cancer 2007;48:57-63.
18. Detivaud L, Nemeth E, Boudjema K, Turlin B, Troadec MB,
Leroyer P et al. Hepcidin levels in humans are correlated with
hepatic iron stores, hemoglobin levels, and hepatic function.
Blood 2005;106:746-8.
19. Bonkovsky HL, Poh-Fitzpatrick M, Pimstone N, Obando J,
Di Bisceglie A, Tattrie C, et al. Porphyria cutanea tarda, hepatitis C, and HFE gene mutations in North America.
Hepatology 1998;27:1661-9.
20. Cazzola M. Hereditary hyperferritinaemia/ cataract syndrome. Best Pract Res Clin Haematol 2002;15:385-98.
21. Beutler E, Felitti VJ, Koziol JA, Ho NJ, Gelbart T. Penetrance
of 845G→A (C282Y) HFE hereditary haemochromatosis
mutation in the USA. Lancet 2002;359:211-8.
22. Olynyk JK, Hagan SE, Cullen DJ, Beilby J, Whittall DE.
Evolution of untreated hereditary hemochromatosis in the
Busselton population: a 17-year study. Mayo Clin Proc
2004;79:309-13.
23. Merryweather-Clarke AT, Cadet E, Bomford A, Capron D,
Viprakasit V, Miller A, et al. Digenic inheritance of mutations
in HAMP and HFE results in different types of haemochromatosis. Hum Mol Genet 2003;12:2241-7.
24. Adams PC, Reboussin DM, Barton JC, McLaren CE, Eckfeldt
JH, McLaren GD, et al. Hemochromatosis and iron-overload
screening in a racially diverse population. N Engl J Med
2005;352:1769-78.
25. Gagne G, Reinharz D, Laflamme N, Adams P, Rousseau F.
Hereditary hemochromatosis screening: effect of mutation
penetrance and prevalence on cost-effectiveness of testing
algorithms. Clin Genet 2007;71:46-58.
26. Cohen AR. New advances in iron chelation therapy.
Hematology Am Soc Hematol Educ Program 2006:42-7.
27. Brittenham GM, Farrell DE, Harris JW, Feldman ES, Danish
EH, Muir WA, et al. Magnetic-susceptibility measurement of
human iron stores. N Engl J Med 1982;307:1671-5.
28. St Pierre TG, Clark PR, Chua-anusorn W, Fleming AJ, Jeffrey
GP, Olynyk JK, et al. Noninvasive measurement and imaging
of liver iron concentrations using proton magnetic resonance. Blood 2005;105:855-61.
29. Pennell DJ. T2* magnetic resonance and myocardial iron in
thalassemia. Ann N Y Acad Sci 2005;1054:373-8.
30. Rund D, Rachmilewitz E. Beta-thalassemia. N Engl J Med
2005;353:1135-46.
31. Origa R, Galanello R, Ganz T, Giagu N, Maccioni L, Faa G,
et al. Liver iron concentrations and urinary hepcidin in betathalassemia. Haematologica, in press. 2007
Hemostasis
Epidemiology of coagulation disorders
F. Peyvandi
M. Spreafico
Bianchi Bonomi Hemophilia and
Thrombosis Center, University of
Milan and Department of Medicine
and Medical Specialties, Luigi Villa
Foundation, IRCCS Maggiore
Hospital, Mangiagalli and Regina
Elena Foundation, Milan, Italy
2007;1:24-30
| 24 |
he most common congenital deficiency of clotting factors are von
Willebrand disease (VWD) and the
hemophilias. Bleeding symptoms are also
reported for other rarer bleeding disorders,
including platelet disorders which will not
be discussed in this report. Inherited deficiencies of plasma coagulation factors generally lead to lifelong bleeding disorders.
Their severity is inversely proportional to
the degree of factor deficiency.
T
Hemophilia A and B
Inherited as X-linked recessive traits,
hemophilia A and B are the most common
hereditary bleeding disorders caused by a
deficiency or dysfunction of blood coagulation factor (F)VIII and FIX. Hemophilias
are present worldwide, with a prevalence
in the general population of approximately 1:10,000 (hemophilia A) and 1:50,000
(hemophilia B), without ethnic or geographic limitations. They remain still lifethreatening and often disabling conditions
despite recent improvements in replacement therapy. Hemophilia A and B have
the same clinical presentation, consisting
predominantly of acute and chronic
hemarthroses, chronic synovitis, restriction of joint movement, and soft tissue
hematomas. Bleeding in the central nervous system is uncommon but can occur
after relatively light head injury and was
formerly the most common cause of death
in hemophilia. Gastro-intestinal bleeding,
presenting as hematemesis and/or melena,
occasionally occurs and its mechanisms
should be thoroughly investigated.1
Hemophilia A and B occur in mild, moderate and severe forms, defined by plasma
FVIII/FIX levels of 6-30%, 1-5% and less
than 1% respectively.2 From 1980
onwards, technological advances in the
field of molecular biology have meant the
gap in hemophilia treatment and diagnosis has widened significantly between the
developed and developing countries.
There are 450,000 people with hemophilia A in the world but only 20% of them
can expect diagnosis and treatment.3 Data
collected by the World Federation of
Haemophilia show that, in developing
countries, people with hemophilia rarely
live beyond childhood. This is because,
owing to the limited resources available,
hemophilia cannot be given a priority over
widespread health problems such as infections and malnutrition.4 Therefore, in
those countries where factor concentrates
for replacement therapy are scarce, carrier
detection and prenatal diagnosis remain
the key steps to prevent the birth of
hemophiliac children.5
von Willebrand disease
Von Willebrand factor is a large multimeric glycoprotein that functions as the
carrier protein for FVIII. It is also required
for normal platelet adhesion. As such, it
functions both in primary (involving
platelet adhesion) and secondary (involving FVIII) hemostasis. In primary hemostasis, von Willebrand factor attaches to
platelets by its specific receptor glycoprotein Ib on the platelet surface and acts as
an adhesive bridge between the platelets
and damaged subendothelium at the site
of vascular injury. In secondary hemostasis, von Willebrand factor protects FVIII
from degradation and delivers it to the site
of injury.6,7 von Willebrand disease, due to
deficiencies or dysfunction of von
Willebrand factor, is the most common
hereditary bleeding disorder with, according to epidemiological studies, an estimated prevalence worldwide as high as 1 to
2% in the general population.8-10 In contrast, estimates based on referral for symptoms of bleeding suggest a prevalence of
30 to 100 cases per million, similar to that
of hemophilia A.6 Neither ethnic origin
nor gender influence the prevalence of von
Willebrand disease. Von Willebrand disease is characterized by a lifelong bleeding
tendency mainly in mucosal tracts, with
easy bruising, frequent epistaxis, and
menorrhagia.11 Von Willebrand disease
displays both dominant and recessive
inheritance. Mutations previously identified in the
von Willebrand factor gene are reported on an international database http://www.shef.ac.uk/vwf/.11,12
Von Willebrand disease can be classified into 3 main
types. Type 1 (70-80% of cases) is characterized by a
partial quantitative decrease of qualitatively normal
von Willebrand factor and FVIII. An individual with
type 1 von Willebrand disease generally has mild
clinical symptoms and the disease is usually inherited as an autosomal dominant trait. However, penetrance may vary remarkably in each family.6,13 In
addition, clinical and laboratory findings may vary in
the same patient on different occasions. Typically, a
proportional reduction in von Willebrand factor
activity, von Willebrand factor antigen, and FVIII
exists in type 1 disease.6,13 Diagnostic criteria for Type
1 von Willebrand disease were published on behalf
of the International Society on Thrombosis and
Haemostasis Scientific and Standardization
Committee von Willebrand factor subcommittee
(ISTH SSC on VWF)14 and recently updated.11 Indeed,
diagnosis of type 1 may be difficult, especially in
mild cases. This is because patients who, due to von
Willebrand gene mutations, present reduced, structurally normal von Willebrand factor cannot be distinguished phenotypically from healthy individuals
with von Willebrand factor levels at the lower end of
the normal distribution.15 Furthermore, there is no
single assay that specifically diagnoses type 1 disease. Mutation analysis could potentially contribute
to diagnosis. However, until recently, a complete
gene analysis has only been conducted in a limited
number of patients.16
Fifteen to twenty percent of patients with von
Willebrand disease have type 2 defect, a variant with
primarily qualitative defects of von Willebrand factor. Type 2 can be either autosomal dominant or
recessive. Of the 4 known type 2 subtypes (ie, 2A,
2B, 2M and 2N), type 2A is the most common.14 Type
3 is the most severe form of von Willebrand disease.
In the homozygous patient, type 3 is characterized
by marked deficiencies of both von Willebrand factor
and FVIII, by the absence of von Willebrand factor in
platelets and endothelial cells, and a lack of response
to desmopressin acetate (DDAVP). Type 3 von
Willebrand disease is characterized by severe clinical
bleeding and is inherited as an autosomal recessive
trait. Consanguinity is a common cause of this type
in some populations.17 Less severe clinical and laboratory abnormalities may occasionally be present in
heterozygotes.
Together with von Willebrand disease, hemophilia A and B include 95% to 97% of all the inherited
deficiencies of coagulation factors.18 The remaining
defects (fibrinogen, prothrombin (FII), FV, combined
FV+FVIII, FVII, FX, FXI and FXIII deficiencies), are
generally transmitted as autosomal recessive traits.
These deficiencies are quite rare in most populations, with prevalence of clinically relevant forms
ranging from 1:500,000 for FVII deficiency to 1 in 2
million for prothrombin (FII) and FXIII deficiency.19
In areas where consanguineous marriages are frequent, such as Middle-Eastern countries and
Southern India, these coagulation disorders are more
frequent and are together more prevalent than
haemophilia B, representing a significant clinical
problem.20 Rare coagulation disorders are generally
less severe than hemophilias, with clinical manifestations ranging from mild to severe.2,19 Exceptions are
FX and FXIII, that are at least as severe as hemophilia A and B. They also affect women and are associated with serious obstetric and gynecological problems.2,20 These deficiencies are characterized by the
early onset of life-threatening symptoms such as
umbilical cord and central nervous system bleeding.
Central nervous system bleeding is also a common
symptom in severe FVII deficiency. Here it is reported in 15-60% of cases.21,22 It presents shortly after
birth and is associated with high morbidity and mortality.23 Other severe symptoms such as recurrent
hemoperitoneum during ovulation, as well as limbendangering hemarthroses and soft tissue
hematomas, are more frequent in patients with FII,
FX, and FXIII deficiency than in other rare coagulation disorders. Common to all rare coagulation disorders is the occurrence of excessive bleeding at the
time of invasive procedures such as circumcision and
dental extraction. Bleeding in mucosal tracts (particularly epistaxis and menorrhagia) is also a relatively
frequent feature.20,23,24 The majority of rare coagulation disorders are expressed phenotypically by a parallel reduction of plasma factors as measured by
functional assays and immunoassays (so-called type
I deficiencies). Qualitative defects, characterized by
normal, slightly reduced or increased levels of factor
antigen contrasting with much lower or undetectable functional activity (so-called type II deficiencies), are less frequent.18 Inherited as recessive
traits, rare coagulation disorders are due in most
cases to mutations in the genes that encode the corresponding coagulation factors. Exceptions are the
combined deficiencies of FV+FVIII,20 and of vitaminK-dependent proteins (FII, FVII, FIX, and FX).25 There
are caused respectively by mutations in genes
encoding proteins involved in the FV and FVIII intracellular transport,26,27 and in genes encoding enzymes
involved in post-translational modifications and in
vitamin K metabolism.28,29 Whereas hemophilia A is
due to an inversion mutation involving introns 22 or
1 of the FVIII gene in approximately half of the
patients, rare coagulation disorders are often due to
mutations which are unique to each kindred scat-
RBDs
Hemophilia A+B
von Willebrand disease
Hemophilia type unknown
Platelet disorders
Other hereditary bleeding disorders: type unknown
tered throughout the genes.18 Current treatment of
rare coagulation disorders is based on the replacement of the deficient coagulation factor by plasmaderived products. In European countries, a good
quality of life is assured to patients with rare coagulation disorders, both in terms of availability and
safety of coagulation factors. By contrast, in developing countries, economic constraints, limitated laboratory resources and the scarce availability of therapeutic products make the provision of an acceptable level of care and quality of life impossible.
Thus, molecular characterization and prenatal diagnosis remain the key steps for the prevention of the
birth of children affected by rare coagulation disorders in developing countries. Indeed, patients with
these deficiencies rarely live beyond childhood
because management is still largely inadequate.5
Epidemiology can be defined as the study of the
frequency and distribution of diseases in specific
populations. The distribution of coagulation disorders in different part of the world is often unknown.
It is essential, therefore, to increase the knowledge of
the clinical and therapeutic aspects of each disorder
and to establish in which region and population
intervention is needed. Such intervention could
include genetic prevention as well as the development of drugs, particularly for those deficiencies
with no available therapeutic concentrate. Data on
the distribution of hemophilia A and B are quite well
established and described in literature. This is mainly due to the higher prevalence and the severity of
symptoms. Instead, data on epidemiology of the
Figure 1. Distribution of bleeding
disorders from World Federation
of Haemophilia global survey.
other coagulation disorders, are limited, particularly
in developing countries. This is also because the biologic heterogeneity and variable presentation of
these diseases make an accurate diagnosis difficult.
Low incidence means few centres have the possibility to follow and manage a consistent number of
patients and scientific reports in literature are usually
limited to small groups of affected patients. In recent
years, however, there has been an attempt to collect
more information with the creation of national registries [France, www.francecoag.org; Switzerland,
www.aekreg.ch; North-America;24 England, UKHCDO; www.ukhcdo.org, ] and international registries
[www.rbdd.org].
In 2002 to improve understanding of the distribution of coagulation deficiencies, a report compared
the number of patients affected by coagulation disorders in the Islamic Republic of Iran with those registered in the UK by the Hemophilia Centre Directors
Organization (UKHCDO), and in Italy by the Istituto
Superiore di Sanità and the Associazione Italiana
Centri Emofilia (AICE).19 This comparison was possible due to their similar general populations (approximately 60 million) and the availability of National
Registries of inherited bleeding disorders. In Iran, rare
coagulation disorders were clearly more prevalent
than in Italy and the UK (15% vs 7% UK and 5%
Italy), whereas in all the three countries hemophilia
A (Iran: 65%; UK: 77%; Italy: 80%) was the most frequent bleeding disorder, followed by hemophilia B
(Iran: 20%; UK: 16%; Italy: 15%).19 This survery confirmed that in populations where the practice of con-
A
World Federation of Hemophilia survey
40
35
% of affected patients
35
30
24
25
20
15
10
11
9
5
9
6
3
7
0
FIBRINOGEN
FII
FV
FV-FVIII
FVII
FX
FXI
FXI
deficiency
Rare Bleeding Disorder Database survey
B
30
% of affected patients
25
24
25
20
16
15
11
10
8
5
7
6
2
0
FIBRINOGEN
FII
FV
FV-FVIII
FVII
FX
FXI
FXI
deficiency
Figure 2. Prevalence of each RBD in the WFH global survey [23] (A) and in the RBDD survey [www.rbdd.org] (B)
sanguineous marriage is widespread the frequency of
rare congenital coagulation disorders is higher.
However, these data only related to three countries.
More detailed information and comparable data can
only be obtained from a global survey.
The World Federation of Haemophilia represents
the most important organization. That, since 1998,
annually performs a worldwide survey on people
with hemophilia, von Willebrand disease and rare
coagulation disorders. The last global survey of the
World Federation of Haemophilia, which collected
data for the years 2004 and 2005, was published in
2006 [ref. #30, http://www.wfh.org/2/7/7_0_Link7_
GlobalSurvey2005.htm]. The survey includes information from 98 participating countries, covering
88% of the world population. Of these countries, 49
used national registries to report information about
186,089 patients with hemophilia, von Willebrand
disease and other bleeding disorders throughout the
world. Of these, 123,942 were affected by hemophilia A and B (66,7%), 44,471 by von Willebrand disease
(23,9%), 6,934 (3,7%) by rare bleeding disorders,
2,648 (1,4%) by platelet disorders and 8,094 (4,3%)
by unknown type of hemophilia or rare disorders.
Figure 1 shows the prevalence of hemophilia, von
Willebrand disease, rare coagulation disorders and
platelet disorders, calculated on the total number of
affected patients reported by countries responding to
the World Federation of Haemophilia global survey.
As expected, hemophilia A and B, followed by von
Willebrand disease, are the most frequent bleeding
disorders. Frequencies differ around the world. The
reported prevalence of hemophilia A and B seems to
be higher in South America, Africa, the Middle East
and Asia (approximately 80%) than in North
America, Europe and Oceania (approximately 55%).
von Willebrand disease was reported to be more frequent in North America, Europe and Oceania (32 to
39%) than in South America, Africa, the Middle East
and Asia (6 to 10%). This is probably due to underdiagnosis of von Willebrand disease in countries with
low economic resources. However, a previous survey31 reported that this is not always the case in the
developing world. Infact, there is significant support
from the government for the management of these
patients in southeast Asia, South America and Africa.
Rare coagulation disorders seem to have the same
prevalence in North America, Europe, the Middle
East and Africa. These data contrast with previous
reports of a higher frequency of the recessive rare
coagulation disorders in countries where the practice
of consanguineous marriage is widespread, particularly in Middle Eastern countries.32,33 As far as the
hemophilias are concerned, 93 of the 96 responding
countries (97%) were able to provide data on the
number of affected patients. In contrast, only 36 of
the 96 responding countries (37%) provided specific
information on the number of patients affected by
rare coagulation disorders, and 41 countries gave statistics referring to patients affected by other hereditary
bleeding disorders: type unknown, which could include
both rare coagulation disorders, von Willebrand disease and platelet disorders without being able to distinguish them. Furthermore, only 10 of the 96
responding countries were from Africa and only 3 of
them gave information on the number of patients
affected by rare coagulation disorders. No detailed
information came from the Middle East, as only 3 of
the 11 responding countries supplied the number of
patients affected by rare coagulation disorders. To
sum up, the World Federation of Haemophilia global
survey gives an indication of the variety but not the
real world distribution of bleeding disorders, in particular von Willebrand disease and rare coagulation
disorders. Perhaps, the lack of comprehensive information and detailed responses might be due to the
limited number of reliable national registries for
these disorders, particularly in developing countries
where political, social and economic situations often
mean affected patients are not properly diagnosed
and managed. Indeed, without participation and
funding from governments and insurance agencies, it
is impossible to contemplate a basic level of care for
all affected people with hereditary bleeding disorders. However, the World Federation of Haemophilia
is making many effort to improve the global survey
and it is hoped that more comprehensive data will be
Rare coagulation distribution according to the World Federation of Hemophilia global survey
Fibrinogen
FII
FV
FV+FVIII
FVII
FX
FXI
FXIII
Rare coagulation disorders distribution according to the RBDD suvey
Fibrinogen
FII
FV
FV+FVIII
obtained in the future.
A further indication of the world-wide prevalence
of rare coagulation disorders can also be obtained by
comparing the frequency of rare coagulation disorders collected by the World Federation of Haemophilia global survey with that of the International
Rare Bleeding Disorders Database (RBDD, www.
rbdd.org) survey.34 The Rare Bleeding Disorders
Database collected data from 61 responding treatment centres all over the world, for a total of 2,916
patients affected by rare coagulation disorders,
FVII
FX
FXI
FXIII
Figure 3. Prevalence of each
RBD in different world
regions according to the WFH
global survey [23] (A) and the
RBDD survey [www.rbdd.org]
(B).
including severe, moderate or mild deficiencies. As
shown in Figures 2 and 3, the prevalence of each rare
coagulation disorder is similar in the two surveys,
with two major exceptions: combined FV+FVIII deficiency and FXI deficiency. The high frequency of
FV+FVIII deficiency reported in the Rare Bleeding
Disorders Database survey probably indicates the
need for a more detailed revision of patient diagnosis
to exclude misdiagnosis of FV deficiency associated
with mild hemophilia.
The majority of cases of FXI deficiency, reported in
literature are of Ashkenazi Jewish origin, the frequency of heterozygosity for FXI deficiency being as
high as 8%.19 Many Ashkenazi Jews reside in Israel,
but large populations live in Western Europe and the
United States. The higher frequency of FXI deficiency observed in the World Federation of Haemophilia
survey might be derived from data provided from
countries not included in the Rare Bleeding Disorders
Database with large Jewish populations.
In conclusion, recents years have seen efforts to
increase our knowledge of the epidemiology of coagulation disorders, particularly of those which, due to
their frequency, are considered orphan diseases.
However, obtaining specific and global information
is still problematic, principally because carrying out
comprehensive studies in all countries is difficult.
This is particularly true in developing countries that
are often those with higher incidence rates.
Furthermore, in these countries, the priority is not
given to these life-threatening and often disabling
condition due to of limited resources. Affected individuals therefore, often do not survive childhood, or
are not diagnosed and treated at all. So national registries must be improved and integrated into a single
international registry. This instrument is an essential
tool to improve knowledge on the prevalence of
patients affected by each coagulation disorder in different regions of the world.
A comprehensive analysis of patient distribution
will help to identify and define intervention at both
national and international levels to improve the
access to care for all affected patients, optimizing
diagnosis, treatment and management.
Greater knowledge of the worldwide prevalence
and distribution of coagulation disorders could
increase pharmaceutical interest in the development
and distribution of replacement products (e.g. FV, FX)
which are currently unavailable. However, product
safety and cost must always be strictly controlled,
particularly in countries with limited resources.
Acknowledgments
We are grateful to the World Federation of
Haemophilia (WFH) for its continuous effort in the
annual global survey data collection, to all those
Centres that contribute to the Rare Bleeding
Disorders Database (RBDD: http://www.rbdd.org/
alreadyjoined.htm) data collection, and to Prof. Pier
Mannuccio Mannucci who critically reviewed this
manuscript with useful criticism.
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in congenital factor VII deficiency. Thromb Haemost
2005;93:481-7.
23. Bolton-Maggs PH, Perry DJ, Chalmers EA, Parapia LA, Wilde
JT, Williams MD, et al. The rare coagulation disorders review
with guidelines for management from the United Kingdom
Haemophilia Centre Doctors’ Organisation. Haemophilia
2004;10:593-628.
24. Acharya SS, Coughlin A, Dimichele DM. North American
Rare Bleeding Disorder Study Group. Rare Bleeding Disorder
Registry: deficiencies of factors II, V, VII, X, XIII, fibrinogen
and dysfibrinogenemias. J Thromb Haemost 2004;2:248-56.
25. Zhang B, Kaufman RJ, Ginsburg D. LMAN1 and MCFD2 form
a cargo receptor complex and interact with coagulation factor
VIII in the early secretory pathway. J Biol Chem.
2005;280:25881-6.
26. Nichols WC, Seligsohn U, Zivelin A, Terry VH, Hertel CE,
Wheatley MA, Moussalli MJ, et al. Mutations in the ER-Golgi
intermediate compartment protein ERGIC-53 cause combined
deficiency of coagulation factors V and VIII. Cell 1998;93:6170.
27. Zhang B, Cunningham MA, Nichols WC, Bernat JA, Seligsohn
U, Pipe SW et al. Bleeding due to disruption of a cargo-specific ER-to-Golgi transport complex. Nat Genet 2003;34:220-5.
28. Brenner B. Hereditary deficiency of all vitamin K-dependent
coagulation factors. Thromb Haemost 2000;84:935-6.
29. Rost S, Fregin A, Ivaskevicius V, Conzelmann E, Hortnagel K,
Pelz HJ, Lappegard K, et al. Mutations in VKORC1 cause war-
30.
31.
32.
33.
34.
farin resistance and multiple coagulation factor deficiency
type 2. Nature 2004;427:537-41.
World Federation of Hemophilia Report on the annual Global
survey 2005.
Srivastava A, Rodeghiero F. Epidemiology of von Willebrand
disease in developing countries. Semin Thromb Hemost
2005;31:569-76.
Peyvandi F, Asselta R, Mannucci PM. Autosomal recessive
deficiencies of coagulation factors. Rev Clin Exp Hematol.
2001;5:369-88.
Bauduer F, Ducout L, Dutour O, Degioanni A. Is there a
‘Basque’ profile regarding autosomal recessive deficiencies of
coagulation factors? Haemophilia 2004;10:276-9.
Peyvandi F, Kaufman RJ, Seligsohn U, Salomon O, BoltonMaggs PH, Spreafico M, et al. Rare bleeding disorders.
Haemophilia 2006;12 Suppl 3:137-42.
Hemostasis
Recent advances in hemophilia management
C. Négrier1-2
Y. Dargaud1-2
J-L Plantier1
1
EA3735, IFR62, Université Lyon 1,
Faculté de Médecine R. Laennec,
Lyon;
2
Hospices Civils de Lyon, Service
d’Hématologie Biologique,
Centre de traitement
de l’hémophilie, Hôpital Edouard
Herriot, Lyon, France
2007;1:31-38
A
B
S
T
R
A
C
T
In the last few decades, there have been dramatic improvements in the management
of hemophilia. Hemophilia has moved from a fatal or disabling disease to a hereditary
disorder and treatments that can improve clinical outcome have become available. The
safety of anti-hemophilic clotting factor concentrates has been dramatically improved
and no major infection transmitted by plasma-derived or recombinant products has
been recorded since the late ’80s. The development of virally safe factor concentrates
through a combination of improved donor selection and screening, effective virucidal
technologies, and the exploitation of biotechnology-engineered recombinant proteins
have provided an impressive safety record with regard to pathogen transmission. Despite
concerns about the potential risk of prion transmission, the major complication of treatment is currently represented by the development of inhibitory antibodies following
clotting factor administration, mainly during childhood. The progressive development of
long-term prophylaxis in developed countries, and surgical correction of disabilities,
have markedly improved the quality of life of hemophiliacs. Current efforts are mainly
focused on improving the safety of plasma-derived products and bioengineering recombinant proteins that have been modified to enhance pharmacokinetic properties and/or
reduce immunogenicity. In addition, several preclinical and clinical studies are currently being carried out for optimizing and individually designing therapeutic regimens using
recently developed or revisited coagulation assays. Some attempts to cure hemophilia
through gene therapy have been made without significant clinical efficacy in humans,
but, from a clinical perspective, this represents the ultimate goal.
emophilia is a sex-linked genetic
disorder resulting from a deficiency in factor VIII (hemophilia A) or
factor IX (hemophilia B) coagulant activity. In most patients, the plasma level of
factor VIII (FVIII)/factor IX (FIX) predicts
the clinical severity of the disease. Severe
hemophilia patients are subject to frequent joint and intramuscular bleeds, and
those who are not on prophylaxis have an
average of 20 to 30 episodes of spontaneous or trauma-related bleeds per year.1
Treatment usually consists of replacing
the missing coagulation factor from
exogenous sources. Modern and effective
management of hemophilia only became
possible with the development of concentrated forms of plasma-derived coagulation factors in the late 1960s. Plasma from
multiple donors was pooled, but this practice was a major contributor to the transmission of blood-borne infectious agents
such hepatitis B, hepatitis C and HIV.2-4
The subsequent evolution of coagulation
factor replacement therapy focused on
maximizing viral safety through the
H
expansion of donor selection and screening tests, and the implementation of chromatographic purification and viral inactivation steps (Figure 1). However, there is
still the risk of parvovirus, hepatitis A
(HAV) and emerging pathogens such as
prions.5 Recombinant FVIII (rFVIII)/FIX
concentrates, with very remote risk of
infection, were developed in the late
1980s following the cloning and sequencing of FVIII and FIX.6-8 Human rFVIII can
only be produced using mammalian cellculture systems due to the complex glycosylation and other post-translational modifications required for its full co-factor
activity. While culture media formerly
contained human or animal-derived proteins, more recent media utilize chemically synthesized or genetically engineered
molecules. Impurities derived from the
medium and cultured cells are removed
through various chromatographic steps.
All currently available rFVIII products are
purified using immunoaffinity chromatography employing a murine monoclonal
antibody directed against human FVIII.
Subfractional-O
Low purity
pdFVIII
Intermediate-purity
concentrates
rFIX
High-purity
concentrates
Cryo-precipitate
Late Mid Early Late Early
1950s 1960s 1970s 1970s 1980s
Donor/plasma
screening for
HBV
Plasma
fractionation
rFVIII
Mid
1980s
Late
1980s
Heat-treated
concentrates
widely
Heat treatment
of pdFVIII
Early
1990s
Late
1990s
Early
2000s
HIV/HCV
screening
Immunoaffinity,
S/D, iron exchange
Qualification of donors
inventory hold, NAT;
nanofiltration
rFVIII, recombinant FVIII; rFIX,
recombinant FIX; pdFVIII, plasma
derived FVIII; HBV, hepatitis B;
HCV, hepatitis C; S/D, solvent detergent; NAT, nucleid acid testing
Figure 1. Evolution of FVIII/FIX concentrates in the last 50 years.
Although no evidence of viral transmission has been
recorded with any rFVIII product, a remote theoretical risk of transmitting a human-derived infectious
agent still remains in the first generation products,
since human and animal proteins were not completely eliminated from the production process. Later generations of recombinant clotting factors have therefore been developed with a progressive elimination
of animal or human-derived raw materials.
Therapeutic usage of clotting factor concentrates
Propylaxis or “on demand” therapy?
Severe hemophilia patients (FVIII/FIX<1 IU/dL)
suffer from repeated bleeding episodes which result
in chronic painful joint disease and deformity known
as haemophilic arthropathy.9 The conventional treatment approach is episodic on demand therapy, where
the missing factor concentrate is administered as
soon as possible after the onset of a bleeding event.
Alternatively, the missing factor may be administered at regular intervals to prevent bleeding, in the
so-called prophylactic therapy regime to prevent lifethreatening hemorrhages and musculo-articular
bleedings.10 The rationale for prophylactic treatment
is to maintain clotting factor activity levels above 1%
and therefore to convert the bleeding phenotype of
patients with severe hemophilia to a milder bleeding
pattern similar to that of patients with moderate
hemophilia.11,12 Several studies have demonstrated
that primary prophylactic therapy improves outcome13 in comparison with on demand treatment
strategies. Primary prophylaxis in hemophilia has
recently been evaluated in a prospective randomized
controlled trial conducted in the United States.14
Prophylaxis should be started at an early age (usually
before the age of 2) either before or after the first
joint bleed. The 25-year Malmö experience indicates
that treatment is most effective when administered
in relatively large doses (25-40 IU/kg) at least 3 times
per week,15 although less demanding treatment
modalities have also been described. One of the
advantages of these dose-escalating regimes is to
avoid the insertion of a central venous access device
which is associated with a significant risk of infectious or/and thrombotic complications.16,17 These regimens begin with a weekly injection via peripheral
veins. Infusion therapy is increased in either frequency or quantity of factor unit per kg if breakthrough
bleedings occur. The total annual consumption of
factor on prophylaxis regimens varies considerably,
and there is evidence that the lower doses used in the
Netherlands have been as effective in protecting the
joints as the higher doses used in Sweden.18 A wider
application of prophylaxis therapy is also limited by
its high cost. It is, however, generally agreed that prophylaxis is the method of choice for treating severe
hemophilia patients since regular clinical and radiological evaluation of joints and quality of life assessments have demonstrated good clinical outcome.19
Development of inhibitors and management of bleeds in
inhibitor patients
As clotting factors have become safer with a
reduced risk of transmission of blood-borne
pathogens, the development of inhibitory antibodies
to the transfused clotting factor has become the most
serious treatment complication, with a cumulative
incidence up to ~30% in previously untreated
patients with severe hemophilia A with first generation and second generation rFVIII.20-23 While the risk
of inhibitor development is partly determined by the
specific underlying mutation and severity of the deficiency, concerns remain about the relative immunogenicity of various types of concentrates. In Europe,
two well-documented outbreaks of inhibitors in low
risk patients followed the introduction of modified
plasma-derived FVIII (pdFVIII) concentrates24-25 and a
recent retrospective study continues to raise the
question that pdFVIII may be less likely to lead to
inhibitor development than rFVIII.26
Inhibitors are more likely to develop in severe
hemophilia patients. Several variables appear to
affect inhibitor development including age, ethnic
origin, family history and mutation type (associated
most commonly with large deletions, non sense
point mutations, and intron 22 inversion). Inhibitors
are classified as low titre if the level is <5 Bethesda
Units (BU) and high titre if ≥5 BU. Patients with low
titre and low responding inhibitors can be treated
with higher doses of FVIII/FIX concentrate to saturate existing antibodies and provide available
FVIII/FIX to achieve hemostasis. FVIII replacement
therapy is usually ineffective in patients whose
inhibitor titres exceed 5-10 BU. For these patients,
bypassing agents, for example, activated prothrombin complex concentrates (APCC) or recombinant
activated FVII (rFVIIa), represent effective treatment
strategies for the treatment or prevention of hemorrhages. Several investigations are being carried out to
further investigate the clinical usefulness of prophylactic administration of bypassing agents.
Bypassing therapies
FEIBA® (Baxter Healthcare, Westlake, CA, USA) is
currently the only activated prothrombin complex
concentrate (APCC) still available on the market. It
contains activated factor X, prothrombin, factor IX,
factor VII, protein C, activated factor VII, and trace
amounts of FVIII.27 FEIBA® has been reported to successfully control approximately 80% of joint and
soft tissue bleeds in patients with inhibitors.28-29
FEIBA® has also been shown to be effective in surgery.30 Rare thrombotic events including acute
myocardial infarction have been reported with
FEIBA® treatment. Because of the thrombotic risk, it
has been recommended that the maximum daily
dose of FEIBA® should not exceed 200 U/kg.31
Recombinant activated factor VII (rFVIIa)
(NovoSeven®, NovoNordisk, Bagsvaerd, Denmark),
which is structurally similar to human plasma FVIIa,
has a complex mechanism of action which includes
tissue factor-dependant and independent triggering
of coagulation. NovoSeven® can bind to the surface
of activated platelets and directly activates FX, leading to improved generation of thrombin.32
Recombinant FVIIa is effective in achieving hemo-
stasis in hemophilia patients with inhibitor in ~80%
of cases using 2 or 3 injections.33 Elective surgery in
hemophiliacs was safely undertaken with rFVIIa
therapy.34 As for APCC, infrequent thrombotic
adverse events including venous thromboembolism,
myocardial infarction were reported.35 Recent studies evaluating the efficacy of a single megadose of
rFVIIa (>200 µg/kg) reported a significantly higher
efficacy of the megadose with lower product consumption.36 A major concern for APCC and rFVIIa is
the absence of a routine laboratory test for monitoring efficacy and potential thrombogenicity.
Recently, global hemostasis tests such as thromboelastography and thrombin generation testing have
been proposed for monitoring NovoSeven® and
FEIBA® treatment.
Surrogate markers of hemostatic efficacy
Since inhibitor by-passing therapies control bleeding without any influence on the plasma level of
FVIII-FIX, FVIII-FIX clotting activity cannot be used
to monitor the response to these treatments.
Thrombin generation is essential for clot formation
and this thrombin generation is defective in hemophilia. Therefore, direct or indirect methods for
assessing thrombin generation can theoretically be
used as surrogate markers for monitoring hemophilia therapies. Recently, several groups reported the
potential usefulness of thromboelastography and
thrombin generation test in the evaluation of the
hemostatic response to anti-hemophilic treatments.
Recent developments and automation of thromboelastography facilitated its use in clinics, operating
theatres and clinical laboratories. It provides a graphic representation of clot formation and fibrinolysis
within 30 minutes. The main advantage of thromboelastography is that it includes the interactions
between all components of blood, platelets, coagulation proteases and inhibitors, red and white cells. As
blood sample clots, significant viscoelastic changes
occur and resistance against movements are transmitted to the detector system and continuously registered. A trace is generated as a function of time to
produce a thromboelastography curve. Sorensen et
al.37 introduced 3 novel parameters using the first
derivative (velocity profile) of the initial thromboelastography curve, the maximum velocity, the time
to maximum velocity and the area under the velocity curve. Using a very low tissue factor concentration (~0.35 pM) and the velocity profile, they
showed that thromboelastography could detect
hypocoagulability in patients with severe hemophilia and demonstrated variation between patients.38
Some patients had particularly low clot firmness
whereas others had results similar to patients with
moderate hemophilia A. Thromboelastography has
also been shown to reflect the clinical efficacy of
activated prothrombin complex concentrate39 and
recombinant activated factor VII40 in hemophiliacs
with inhibitors. In the presence of rFVIIa, the
dynamics of blood coagulation was usually
improved but wide inter-individual variations were
observed in response to the same dose of rFVIIa. In
a case report, Hayashi et al.41 showed that thromboelastography could be useful for detecting clinical
resistance to by-passing therapies. These data suggest that a preadministration of different dose levels
of by-passing agents could be helpful in defining the
type and dosing of the drug for each patient. This
concept of individualising by-passing therapy was
recently confirmed by Young et al.42 who showed
that thromboelastography was useful for testing the
individual response of patients to APCC or rFVIIa
and also for determining the minimal effective dose
of the selected product.
Although the principle was described many years
ago43 thrombin generation assay has been recently
adapted for use with a slow reacting fluorogenic
substrate (Z-GGR-AMC) specific for thrombin. This
allows the automatic measurement of thrombin generation in plasma containing fibrinogen/fibrin and in
platelet-rich plasma.44-45 This made the method more
practical and suitable for use in clinical laboratories
and significantly improved accuracy. The most
important parameters that can be derived from
thrombin generation test (TGT) are (i) the lag time
(minutes) corresponding to the initiation phase of
coagulation; (ii) the Endogenous Thrombin Potential
(ETP, nM.min) which quantifies the total thrombin
enzymatic activity; (iii) the thrombin peak (nM)
which corresponds to the maximal amount of
thrombin that can be generated by the plasma sample during the thrombin burst; (iiii) the time to peak
(minutes) which corresponds to the time course of
the thrombin generation curve up to the maximal
thrombin peak.
A statistically significant correlation has been
shown between plasma FVIII concentrations and
ETP measured by TGT.46 Thes result obtained in PPP
was later confirmed in PRP47 and in a cell based
model system.48 The values of TGT parameters in
severe, moderate and mild hemophiliacs using a low
tissue factor concentration (1 pM) were recently
published.49 A statistically significant correlation
between plasmatic FVIII-FIX levels and ETP, peak
and time to peak was demonstrated. In addition,
independently of the FVIII-FIX plasma level, a correlation was found between severe clinical bleeding
phenotype and ETP, suggesting that TGT could be
used to evaluate clinical bleeding phenotype in
patients with hemophilia. In vitro and ex vivo experiments demonstrated that TGT could be used for
monitoring FVIII-FIX replacement therapy. It could
also possibly be used for designing individual prophylactic regimens as well as for adapting clotting
factor infusions in surgery.
In vitro and ex vivo studies also showed that the
addition or infusion of Feiba® or Novoseven® dosedependently increased TG capacity (ETP and peak)
but this increase could not reach normal values.50-51
These results strongly suggest that TGT could be
used for monitoring pharmacodynamics of by-passing agents and for optimising the infusion schedule.
Therefore, the test might make a major contribution
to the decision-making process of the most adapted
by-passing therapy for the treatment of high risk
severe hemophilia patients with inhibitor.52
Thromboelastography and TGT seem very promising, and might represent a tool for a novel
approach to the management of hemophilia based
on individual regimen design rather than a standard
approach for all. Well-designed prospective multicentre studies are now required to confirm these
early findings and further define correlations with
clinical outcome.
The development of hemophilia services –
comprehensive care
Comprehensive care addresses the treatment and
prevention of bleeding, the long-term management
of hemophilic arthropathy and other bleeding complications, the management of significant treatment
complications (development of inhibitors and transfusion transmitted infections), and the psychosocial
support and education required to manage the
bleeding disorder. Laboratory services should support factor assays and inhibitor detection, and
Comprehensive Care Centres should offer 24-hr
access to both medical and laboratory expertise.
Improved survival of hemophiliacs can be
achieved with access to specialist care. Soucie et al.53
demonstrated that, in the USA, hemophiliacs who
received their care in hemophilia centres had a lower
hospital admission rate and a lower mortality than
those who accessed their care outside. This data is a
reminder of the importance of continued specialist
services for this group of patients. With the improvement in treatment in developed countries, hospital
attendances and admissions for hemophiliacs have
reduced substantially over the past decades. This has
led to concerns about the future provision of care
since hemophilia centres have problems recruiting
adequately trained physicians.54 Wider networking
has been provided by a pediatric hemophilia network (PedNet) across Europe55 and a proposal has
recently been made to establish a European co-operative group on hemophilia and other allied disorders.56
Bio-engineering of improved FVIII – FIX molecules
The production of coagulation factors by recombinant technology, combined with the disappointingly
slow progress of gene replacement for hemophilia, has
prompted the development of bio-engineered products that have been mutated to overcome their
remaining therapeutic limitations. The proteins of
interest are usually modified to enhance their pharmacokinetic properties and/or reduce immunogenicity.
Improvement of production
Improving the production of coagulation factors
offers the theoretical an interest of increasing the
availability of the molecules and diminishing production costs. The improvement of FVIII production
was an attractive challenge since this molecule is
particularly poorly processed compared to FIX and
FVII.57 One of the early modifications was to produce
a FVIII on the basis of a cDNA coding for a FVIII
devoid of its B-domain sequence. The deletion of
about 35% of the full-length cDNA increased the
FVIII mRNA amounts leading to a higher secretion
of the molecule.58
Transgene modifications were also realized by
introducing regulatory elements. The insertion of
introns 5’ of the coding sequence or within the coding sequence allowed a higher mRNA synthesis that
was generally accompanied by an increase in protein
secretion.59-61 Coagulation factors are complex molecules which need specific intracellular processing,
and a fully active molecule requires crucial posttranslational steps. Some modifications within the
molecule were introduced to facilitate this processing. A point mutation in factor VIII (F309S) facilitates
the secretion of the molecule probably in relation to
a modification of its interaction with the Bip chaperone.62 The reintroduction of a portion of the Bdomain in FVIII, which is the support of most of the
glycosylations, was also able to favor secretion in vitro
and in vivo.63
Improvement of circulating properties
Once injected, the clotting factor molecules interact with their respective partners, resulting in relatively short circulating half-lives. An increase in this
biological parameter would lower the frequency of
infusion and at the same time improve patient quality of life. The introduction of disulfide bonds
between A2 and A3 (C662–C1828 and C664–
C1826) appeared to moderately improve VWFbinding affinity.64 The use of pegylated -liposomes
was also proposed as factor VIII binds with a relatively high affinity to such modified compounds,
resulting in prolonged bleeding-free intervals upon
administration.65
Various pre-clinical approaches have supported the
potential therapeutic value of FVIII modified by
other means (such as polysialylation) to enhance its
circulating half-life, or mutated to improve its resistance to degradation or clearance.66 The members of
the LRP family were shown to be responsible for
FVIII and FIX degradation.67-68 Several studies
described the respective domains implicated in the
interaction between the receptor and its agonists.69-70
The use of antagonists which could reproduce these
domains would reduce the clearance of both molecules and prolong half-life. However, a major problem will be to specifically inhibit the clearance of factor VIII and/or factor IX without affecting the other
functions of LRP.71
Improvement of the hemostatic potential
Attempts to improve hemostatic potential have
pursued three objectives, improve stability following
activation, improve factor X activation capacity, and
increase resistance to degradation. The mutant molecules will consequently possess an increased coagulant activity even at low concentrations.
Soon after activation, factor VIII rapidly loses its
procoagulant activity due to the dissociation of its
A2 domain. Disulfide bridges were introduced
between A2 and A3 domains, resulting in an
increased stability following activation by thrombin.72 In whole blood clotting assays, only 10% of
the factor VIII wild-type dose is needed to correct
hemophilia A.73 A genetically engineered factor VIII
molecule (IR8) in which the cleavage between A2
and A3 was abolished by modification of the protein sequence, also induced a prolongation of factor
VIII clotting activity.74
Several mutants of FVII(a) were also created to generate a highly active bypassing agent. Two of these
(K337A and M298Q) were able to correct bleeding in
hemophilic mice with inhibitors at much lower
doses.75 The introduction of 4 point substitutions
(V158D/E296V/M298Q/K337A) in factor VIIa accelerated procoagulant and antifibrinolytic activities
through enhancement of TAFI activation.76 Finally,
specific antibodies were shown to improve factor Xa
generation by favoring the binding of FVIIIa to FIXa
and enhancing the catalytic efficiency of the tenase
complex.77 A novel approach to improve coagulation
involves heparin-like sulfated polysaccharides.
Fucoidan was shown to inhibit TFPI, improve clotting time of human hemophilia A and B plasmas as
well as hemostasis in vivo in mice with hemophilia A
or B.78 Synthetic activated protein C inhibitors may
also be considered as adjuvants for hemophilia treatment. These compounds may inhibit FVa inactivation by activated protein C and prolong FVa functional activity in the prothrombinase complex. When
evaluated in a synthetic coagulation proteome
model, one inhibitor partially compensated for the
absence of FVIII.79
Modulation of the immune response
The mechanisms underlying the immune response,
particularly those involved in the anti-factor VIII
response, are the subject of intense investigation. The
generation of an immune response requires a series
of complex interactions between antigen presenting
cells, B and T-lymphocytes.80-81 Different options can
therefore be taken to alter the immune response.
These are the decrease of factor VIII immunogenicity, the differential recognition of factor VIII molecule
by already existing antibodies, and the efficiency of
the immune response.
Different mouse models recreating the different
types of hemophilia (CRM+ and CRM-) have been
generated with the aim of studying the immune
response.82-84 The interest of such models is reinforced
by the fact that the mouse immune response against
factor VIII seems relatively close to that of humans.
The most direct and simple way to modulate this
response is to induce tolerance to factor VIII, using
oral or nasal routes of administration.85 This
approach would nevertheless require large amounts
of antigen for an efficient induction of tolerance in
humans. The depletion of specific B-cells or T-cells
can also be envisaged, and the use of rituximab, a
humanized monoclonal antibodies directed against
CD20, gave promising results in small series of
patients suffering from acquired hemophilia. The
potential usefulness of this drug in congenital hemophilia still needs further investigation. T-cells are
another target of choice, and by blocking the crosstalk mediated by the CD40/CD40L, several groups
were able to induce a short term but significant
reduction in the primary immune response to human
factor VIII in mouse models.86-88
Alteration of factor VIII immunogenicity
Considering that the porcine factor VIII was less
immunogenic than its human counterpart, a group of
investigators initiated a systematic substitution of
functional sequences of human factor VIII by porcine
counterparts, located mainly in the A2 and C2
domains.89-90 Porcine FVIII is also a potentially useful
therapeutic agent because of its low crossreactivity
with many inhibitors. Recombinant porcine FVIII is
undergoing clinical trials in inhibitor patients.
Adjuvants protecting factor VIII
As the domains recognized by the antibodies are
now partially identified, the co-injection of factor
VIII in the presence of mimetic peptides can also be
envisaged, though those peptides probably have to
be modified to enhance their in vivo capacities.80
Another approach used anti-idiotypic antibodies
raised against a potent anti-factor VIII antibody
(mAbBO2C11). These anti-idiotypic antibodies were
shown to inhibit the anti-FVIII inhibitory effect in
hemophilia mice.91
Conclusion
In the last 30 years, hemophilia therapy has
improved dramatically while complications of this
crippling disease have significantly decreased
through the development of safe clotting factor concentrates, prophylaxis therapy and corrective surgical interventions. Bio-engineered recombinant factor
concentrates with longer half-life, higher potency
and less immunogenicity will probably be available
earlier than gene therapy. These achievements will
significantly improve overall compliance therapy,
while in the meantime, optimization of the current
treatment options towards a more individualised
therapy will probably decrease costs and improve
patient quality of life. However, these technological
achievements should not hide the disparity in the
availability of coagulation factor concentrates worldwide since it is estimated that > 75% of the world
population receives either inadequate treatment or
no treatment at all. One potential solution could
come from transgenic animals. The mammary
glands of livestock can generate a very high concentration of secreted proteins. Transgenic pigs can generate recombinant FIX in milk. Calculations suggest
that 60 pigs could supply enough prophylaxis for all
the FIX deficient patients (estimated at 3,000) in the
USA.92 It is thought that the cost of these clotting
factors could be much lower than that of current
therapeutic molecules.
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Haemophilic factors produced by transgenic livestock: abundance that can enable alternative therapies worldwide.
Haemophilia 2004;10 (Suppl. 4):70-6.
Hemostasis
Gene therapy for hemophilia
M.K.L. Chuah
T. VandenDriessche
Center for Transgene Technology and
Gene Therapy, Flanders Institute of
Biotechnology (VIB) University of
Leuven, Leuven, Belgium
2007;1:39-44
t is widely expected that improved
gene therapy approaches will produce
new treatments and cures for a wide
range of diseases including those involving
cardiovascular and hemostatic systems,
cancer, diabetes, infections and autoimmune disorders. Convincing evidence continues to emerge from clinical trials that
gene therapy is effective for patients suffering from a wide range of diseases.1-4
Nevertheless, gene therapy has also faced
a number of setbacks and there have been
concerns regarding the safety of some
gene delivery approaches.5,6 Fortunately,
these obstacles can be overcome.
Over the past 15 years, hemophilia has
become one of the most studied disease
models for gene therapy. This is because a
single gene defect is responsable and even
a slight increase in plasma factor VIII
(FVIII) or factor IX (FIX) levels can already
convert a severe to a moderate phenotype.
Although protein substitution therapy has
significantly improved patient quality of
life, it cannot be considered a cure and the
risk of spontaneous bleeds remains.
Furthermore, the implementation of prophylactic regimens is prohibitively expensive. This limits its widespread use and
further justifies the search for a cure or
novel cost-effective therapies. The development of gene therapy for hemophilia
not only constitutes an important priority
in its own right but also offers opportunities for the application of new gene therapy approaches for many different disease
targets. This review summarizes the most
recent developments in the field of gene
therapy for the treatment of these inherited bleeding disorders.
I
Lentiviral and retroviral vectors
Retroviral vectors were among the first
vectors ever to be used successfully in clinical gene therapy trials, demonstrating
effective long-term correction of severe,
potentially lethal hereditary diseases.1,2
Lentiviral vectors have only recently been
explored in the clinic for the treatment of
HIV-AIDS7 and β-thalassemia.8 The main
distinction between traditional retroviral
vectors derived from MLV (murine
leukemia virus) and lentiviral vectors
(derived from HIV) is that lentiviral vectors are capable of delivering their genetic
cargo into both dividing and non-dividing
cells, whereas non-dividing cells are
refractory to MLV transduction. There are
several advantages for using these type of
vectors for gene therapy of hereditary disorders, including the hemophilias.
Retroviral and lentiviral vectors integrate
stably into the target cell genome allowing
long-term expression of the therapeutic
product. Another advantage of these types
of vectors is that pre-existing immunity is
not a main concern since most human
subjects have typically not been preexposed to the cognate wild-type viruses
from which the vectors have been derived
(with the exception of HIV-infected
patients).
These stably integrating vectors are well
suited for gene transfer into dividing
stem/progenitor cells, particularly hematopoietic stem/progenitor cells (HSCs).
Since HSCs can both self renew and differentiate into all blood lineages, they are an
attractive potential target for gene therapy
of hemophilia. Initial attemps focused on
the use of normally expressed or inducible
promoters to drive clotting factor expression in the hematopoietic lineages.9-11
More recently, using either transgenics or
lentiviral FVIII gene delivery into HSCs, it
has been shown that FVIII can be synthesized in platelets.12-15 Since FVIII is stabilized by von Willebrand factor (vWF),
which is normally synthesized in platelets,
only small amounts of FVIII were needed
to achieve phenotypic correction.
Remarkably, the bleeding diathesis could
even be corrected in the absence of
detectable FVIII levels in the plasma. The
most important advantage of this plateletdirected FVIII delivery, is that FVIII was
therapeutic even in the presence of hightiter inhibitory antibodies to FVIII. Hence,
a protected releasable FVIII pool in platelets would
reduce the bleeding diathesis not only in patients
with hemophilia A but also in those with hemophilia A and FVIII inhibitory antibodies. While the latter
group has not previously been thought to be candidates for FVIII gene therapy, these studies suggest a
new approach that could be beneficial and may represent a unique advantage of this gene therapy
approach over protein replacement therapy.
As an alternative to megakaryocyte/platelet-directed production of clotting factors, lentiviral vectors
can also be designed to express clotting factors in the
erythroid lineage which resulted in therapeutic FIX
levels in hemophilia B mice.16 Thus, FIX is made
through the pro-normoblastic stage, and additional
safety is obtained as the red cells become terminally
differentiated and enucleated. However, not all of the
FIX molecules produced in the erythroid lineage had
undergone the necessary post-translational modifications required to generate fully functional FIX. This
may prove to be a limitation in at least some ectopic
cell types.
These HSC-based approaches could provide
patients with a self-replicating pool of stem cells that
would support lifelong clotting factor expression, at
least in the megakaryocyte or erythroid lineage.
Nevertheless, the main limitation of using HSC-based
approaches is that some level of myelo-ablative conditioning is required to facilitate stem cell engraftment. Indeed, different conditioning regimens (irradiation vs. busulfan) influenced FVIII expression levels
in hemophilia A mice that received HSC which were
stably transduced with retroviral vectors.17 Since
short-term risks of autologous transplantation are
very low and the presence of both hemophilic and
acquired inhibitors can be a difficult problem in treatment management, the risk/benefit ratio for gene
transfer in this setting may well be a favorable one.18
Other adult stem/progenitor cells that are a potentially attractive proposition for ex vivo gene therapy
are myoblasts, that avoid the need for myeloablative
conditioning. They can be readily transduced with
lentiviral vectors and engineered into retrievable
implants composed of non-dividing muscle fibers.
This technology could be adapted for FIX delivery as
a potential reversible gene therapy for hemophilia but
access to the circulation is a possible limitation.19 This
may explain the lack of sustained FVIII production in
some of the previous gene therapy clinical trials that
relied on the re-implantation of gene-engineered
fibroblasts.20
In vivo gene therapy with retroviral vectors was
restricted to the use of neonatal recipients since MLV
can only transduce dividing hepatocytes. Retroviral
delivery of FVIII or FIX genes in neonatal recipients
yielded stable therapeutic clotting factor levels in
hemophilic mice and dogs in the absence of any toxicity.21-23 This approach yielded some of the highest
stable FVIII levels obtained by gene therapy in a large
animal model. Although these findings have implications for gene therapy in pediatric subjects, the clinical implementation of this concept is not straightforward given the prevailing ethical and safety concerns
of pediatric gene therapy trials. A gene therapy clinical trial had previously been conducted based on the
in vivo gene delivery of retroviral-FVIII vectors in
adult subjects with severe hemophilia A. However,
since retroviral vectors require active cell division,
the efficacy of this approach was very limited.24
Lentiviral vectors can transduce non-dividing hepatocytes.25,26 This suggested their possible use in vivo
gene therapy of hemophilia in adult recipients.27-30
Lentiviral vectors derived from FIV (feline immune
deficiency virus) and pseudotyped with baculovirus
GP64 resulted in stable therapeutic FVIII levels without inhibitors and partial phenotypic correction.31
Systemic administration of lentiviral vectors also
resulted in efficient uptake and transduction of the
vector particles by antigen-presenting cells (APCs),25
consistent with the induction of a self-limiting proinflammatory response.30 Ectopic FIX expression in
APCs increases the risk of developing inhibitory antibodies to FIX.32 Although this risk could be reduced
by using hepatocyte-specific promoters, some residual expression in APCs still occurred resulting in
immune responses that eliminated the gene-engineered hepatocytes. However, it was possible to prevent ectopic transgene expression in APCs using
micro-RNA mediated gene silencing which prevented immune rejection of gene-engineered cells and
consequently prolonged transgene expression.33
The development of T cell leukemia in three boys
treated by ex vivo retroviral gene transfer for X-linked
SCID5 along with the emergence of tumors following
fetal gene transfer with lentiviral vectors34,35 raised
some concerns regarding the risk of insertional oncogenesis when integrating vectors are employed. In
the SCID children, insertion of the therapeutic transgene adjacent to the LMO2 transcriptional co-activator locus in the leukemic cells of all three cases has
been assumed to play an important pathogenetic role
and linked the gene transfer with the development of
the malignancy. However, over-expression of IL2Rγc
resulted in a high incidence of T-cell lymphomas in
mice, transplanted with BM stem/progenitor cells
transduced with a lentiviral vector encoding IL2Rγc.36
These recent results strongly suggest that the therapeutic gene itself contributed to the leukemiogenesis
and may explain why leukemia has been involved in
so many SCID-X1 trials compared with other trials
using similar approaches. In retrospect, this may not
be surprising, since IL2Rγc is directly implicated in
the control of cell proliferation. By contrast, FVIII or
FIX expression does not promote cell proliferation
nor is it involved in any growth control or signaling
pathways which, by default, would render gene therapy for hemophilia much safer than for some of
these primary immunodeficiencies. Furthermore, the
design of the vectors, particularly the use a self-inactivating vector configuration, cell-specific promoters
or lentiviral instead of MLV vectors greatly reduce
this potential oncogenic risk.37-40
To overcome concerns associated with random
integration and insertional oncogenesis, approaches
are being developed for in situ repair of defective
genes by synthetic zinc finger nucleases,41 or to
achieve targeted integration in defined loci in the
genome using zinc finger protein-mediated targeting
or phage integrases.42,43 Nevertheless, like all genebased systems, these approaches require efficient
gene delivery vehicles. Similarly, the possibility of
repairing the mutant mRNA in hemophilia has also
shown early potential in hemophilia A mice.44
However, the trans-splicing construct required to
repair the mutant mRNA must be delivered efficiently and expressed long-term in the cells that harbor
the mutant clotting factor gene.
Adeno-associated viral vectors
AAV remains one of the preferred approaches for
long-term cures of genetic diseases by virtue of its
potential for long-term gene expression and reduced
inflammatory properties. Encouraging preclinical
studies in hemophilic mice and dogs45,46 led to two
clinical trials for hemophilia B, based on either intramuscular48,49 or hepatic gene delivery of AAV2-FIX
vectors.49 Administration of an AAV2-FIX vector into
the skeletal muscle of hemophilia B subjects proved
safe and resulted in local gene transfer and FIX
expression for at least 3.7 years after vector injection.
However, circulating FIX levels remained less than
1% of normal. In contrast, circulating therapeutic FIX
levels (10% of normal levels) could be achieved for
several weeks following liver-directed gene therapy
with AAV2-FIX vectors.49 Though encouraging, FIX
expression eventually declined, accompanied by
transiently elevated transaminases. This was probably due to the elimination of FIX-expressing hepatocytes by an AAV2-specific cytotoxic CD8+ T-cell
response. Possibly this reflects prior exposure of the
trial subjects to wild-type AAV2 that naturally occurs
in humans. It is of interest to note that there has not
been any evidence reported of such a CD8+ T-cell
response in none of the subjects enrolled in the
AAV2-FIX muscle trial. This may be due to differences in vector doses or differential local immune
responses.47,48 Some trial subjects also had pre-existing antibodies to AAV2 which precluded efficient
gene transfer. Recent studies in mice showed that
neutralizing antibody titers of as little as 1:5 can abrogate AAV transduction.50 This is an important problem for patients with hemophilia. Since these preexisting antibodies variably cross react with AAVs
acquired during natural infection, accurate vector
dosing in individual patients will be problematic.
However, combinatorial engineering of AAV can be
used to generate novel vectors that are less efficiently neutralized by human antibodies and that could
potentially be used for the treatment of patients with
pre-existing immunity to AAV.51
This leaves gene therapists with one final obstacle
to overcome which is to prolong FIX expression at
therapeutic levels. This could potentially be accomplished by transient immunosuppression to prevent a
cytotoxic CD8+ T-cell response. This hypothesis will
be tested in a new trial, repeating the AAV2-FIX liver
delivery in patients suffering from severe hemophilia
B in conjunction with transient immune suppression.
To overcome the limitations of using AAV2, the
use of AAV vectors based on alternative serotypes
has been proposed. This has been based on the following considerations: (i) most individuals are
seropositive for AAV2 due to prior natural infections
whereas seropositivity for some of the alternative
serotypes (for example, AAV8 or AAV9) is more limited.52,53 Furthermore, antibodies directed against
AAV2 show only limited cross-reactivity with AAV8
and AAV9 and vice versa;53 (ii) unlike AAV2, AAV8
does not appear to activate T cells in non-human primates due to impaired binding to and uptake by antigen-presenting cells (APCs):54 (iii) AAV8 uncoats
much more rapidly than AAV2, suggesting that
AAV8 capsids may be less stable than AAV2.55 The
risk of inducing cytotoxic CD8+ T-cell response could
therefore be reduced in gene therapy subjects when
vectors based on alternative AAV serotypes vectors
are employed when compared to AAV2. This may in
turn increase the chances of obtaining long-term FIX
expression. However, since specific AAV2 capsid
peptides were identified as capable of tight binding
to the subject’s HLA-B haplotype, and since these
sequences are highly conserved in all primate AAVs,
it is unlikely that one can engineer around these
binding sites due to the polymorphic nature of HLA
class I. Furthermore, even AAV8 capsids may persist
for several weeks in non-human primates.56 One of
the main challenges is that no animal models available show a similar response to human subjects.56,57
So it is no certain that serotype switching would be
enough to overcome the cytotoxic CD8+ T-cell
response. This must be tested in clinical trials.
In mice, gene transfer with AAV8 or AAV9 was
more efficient in the liver than in any other organ or
tissue or with any other AAV serotype, including
AAV2. This makes them particularly suitable for
liver-directed hemophilia gene therapy.30,47,52,53,58-65
Stable supra-physiological FIX or FVIII levels could
be obtained that corrected the bleeding diathesis,
which far exceeded levels obtained using AAV2. No
neutralizing antibodies against transgene-endoded
FIX could be detected. The stable FIX or FVIII expression is in agreement with the long-term clotting factor expression following AAV8 delivery in large animal models, including non-human primates or hemophilia dogs47,66 and further supports the hypothesis
suggested several years ago that hepatic expression
of clotting factor may result in immune tolerance.67
Indeed, hepatic gene delivery of FIX using AAV vectors in mice resulted in CD4+CD25+ regulatory T
cell circuits that not only prevented anti-FIX antibody
formation following a potent immune challenge with
FIX (+ immune adjuvant) but also inhibited T-cell
responses directed against the gene-engineered cells
following adenoviral-FIX gene transfer.68, 69 By contrast, control mice that had not been treated with
gene therapy, all developed inhibitors to the FIX protein following immunization. This strongly suggests
that the risk of developing inhibitory antibodies to
FIX may be lower following hepatic gene delivery
than by protein substitution therapy.
Although, based on their superior hepatic transduction efficiencies in mice, the use of alternative AAV
serotypes has generally been proposed to achieve
higher clotting factor levels in patients, this may not
necessarily be the case in large animal models.
Indeed, in contrast to hemophilic mice, hepatic gene
transfer efficiencies and FVIII levels were similar in
hemophilia A dogs, regardless of the AAV serotype
used (AAV2, 6 or 8).56 This confirms there is no transduction advantage in using AAV8 over AAV2 or
AAV5 vectors in non-human primates.61 62 Thus, care
must be taken when translating results from rodent
species to higher or larger species, since the vectorcell surface receptor interactions and affinities remain
poorly understood. From a pratical point of view, this
confirms the need for at least some preclinical development in nonhuman primates.
Conclusions
Gene transfer technologies are improving rapidly
and vectors are being developed which have fewer
side-effects without compromising efficacy. The success of gene therapy for hemophilia still very much
depends upon the continuous development of
improved vector technologies which would hopefully and ultimately lead to a cure for patients with
these bleeding disorders.
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Thrombosis
Vitamin K epoxide reductase (VKORC1):
pharmacogenetics and oral anticoagulation
J. Oldenburg1
C.R. Müller2
M. Watzka1
1
Institute for Experimental
Hematology and Transfusion
Medicine, University Clinic Bonn,
Bonn, Germany;
2
Institute for Human Genetics,
Biocentre, University Würzburg
Würzburg, Germany;
2007;1:45-50
A
B
S
T
R
A
C
T
For decades coumarins have represented the most commonly prescribed drugs for
therapy and prevention of thromboembolic conditions. Despite the limitations of a narrow therapeutic range, the broad variation of intra- and inter-individual drug requirement, and the relatively high incidence of bleeding complications, therapy with
coumarins is rising due to aging populations in industrialised countries. The identification of the molecular target of coumarins, VKORC1, has greatly improved the understanding of coumarin treatment and introduced new perspectives for individualised
and safe oral anticoagulation therapy. Mutations as well as SNPs within the VKORC1
gene have been shown to cause coumarin sensitivity and also coumarin resistance.
Besides the known CYP2C9 variants that affect coumarin metabolism, the haplotype
VKORC1*2 has now been recognised as a major cause of coumarin sensitivity. A frequent SNP within the VKORC1 promotor is reducing the VKORC1 enzyme activity to
50% compared with the wild type. Patients who are homozygous carriers of the
VKORC1*2 allele are strongly predisposed to coumarin sensitivity. Individualized dose
adaptation, can lead to a significant reduction in bleeding complications, especially in
the initial coumarin saturation phase. Meanwhile, VKORC1 and CYP2C9 genotypes in
combination with other known dose influencing factors like age, gender, and weight
were introduced into clinical dosing algorithms. Furthermore, concomitant application
of low dose vitamin K may significantly reduce intra-individual coumarin dose variation, thus improving the safety of oral anticoagulation practice.
or decades, coumarins have been the
most often prescribed drugs for therapy and prevention of thromboembolic conditions, all over the world. Their
oral application and low cost promote
their wide use. However, clinical use of
coumarins is complicated by their narrow
therapeutic range and wide variations in
individual drug requirements result in a
relatively high incidence of bleeding complications or rethrombosis.1
Nevertheless, vitamin K antagonists
remain the therapy of choice in the short
and long term anticoagulation treatment
necessary to prevent venous thrombosis,
stroke, myocardial infarction, and other
thromboembolic events.2,3 Adverse effects
of coumarins are potentially serious (for
example, intracerebral bleeding). The risk
of haemorrhagic events exceeds 10-17%,
especially in the first 90 days of treatment, including 2-5% major bleedings.4
To minimise this risk, known influencing
factors like weight, gender, age, and race
have been used for dose determination.5,6
Furthermore, variants of CYP2C9 have
F
been shown to affect the warfarin metabolism.7-10 In 2004, the molecular target of
coumarins, vitamin K epoxide reductase
(VKORC1) was cloned and now is accessible for molecular investigation.11-12 Apart
from rare mutations in this gene,
VKORC1 was shown to determine
coumarin dose by haplotype specific
mRNA expression levels.11,13-19
Recently, new dosing algorithms have
been developed which consider VKORC1
as an independent pharmacogenetic factor influencing coumarin therapy.20-24
Determining VKORC1 variants will lead
to an improved prediction of the required
coumarin dose and allow identification of
patients prone to over-anticoagulation.
Pharmacodynamics of coumarins
Inhibition of vitamin K epoxide reductase activity by coumarins, including
acenocoumarol, phenprocoumon, and
warfarin, is the most widely-used
approach to anticoagulant therapy. The
four procoagulatory factors (FII, FVII, FIX
and FX) require post-translational gamma
carboxylation on glutamic residues to reach full
activity. Gamma carboxylation is performed by
microsomal gamma carboxylase (GGCX). This
enzyme requires CO2, O2, and vitamin K-hydroquinone to generate carboxy glutamic residues capable of chelating calcium ions to bind on phospholipid surfaces. The process of gamma carboxylation
generates the functional clotting factors and also
vitamin K epoxide. As vitamin K is needed in high
molar excess compared to the modified proteins, a
highly efficient and simple recycling mechanism is
needed. Recently, Chu et al.25 showed that purified
and reconstituted VKORC1 protein is sufficient to
accomplish both the reduction of vitamin K-epoxide
to vitamin K and further vitamin K to vitamin Khydroquinone. This enzyme was previously cloned
independently by our laboratory and D. Stafford’s
group in Chapel Hill, NC, USA.11,12 In accordance
with its hydrophobic amino acid composition and
enzymatic properties, a topology of three transmembrane domains connected by a large cytoplasmatic and a small lumenal loop has been proposed.26,27 A previously identified warfarin binding
motif (TYA) is located within the membrane directly neighboured to the proposed redox centre
(CXXC) and represents the molecular structure targeted by coumarins.28-30 Obviously, binding of
coumarins close to the active centre is the mechanism of inhibition of vitamin K recycling.
Shortly after cloning VKORC1, D'Andrea et al.18
showed that the median coumarin dose was greatly
influenced by a single intronic polymorphism of the
VKORC1 gene. By establishing complete VKORC1
haplotypes, Rieder et al. and Geisen et al.13,19 were
able to demonstrate that individual haplotype composition is responsible for interindividual and
interethnical coumarin dose variations. One single
haplotype (VKORC1*2) was shown to determine
30-50% of coumarin dose variation. Due to a promoter SNP (VKORC1 c.-1639 G>A, rs9923231)
affecting a transcription factor binding site, mRNA
expression and subsequent total VKOR enzyme
activity of haplotype VKORC1*2 is reduced in vitro
and in vivo by approximately 30-50%.13,14 Given the
type of action of this genetic variation, an almost linear dose-response relationship could be expected.
This is reflected by a dose reduction of 25 and 50%
when comparing homozygous VKORC1 wt, heterozygous
VKORC1*2,
and
homozygous
VKORC1*2 genotypes respectively. This coherence
has been confirmed by several other studies and
even interethnical differences in coumarin dosage
can be explained by VKORC1 haplotypes.13,14,19
While dose-reducing haplotype VKORC1*2 is highly prevalent in patients of Asian origin (frequency up
to 95%), VKORC1*2 represents approximately 15%
of alleles in African populations. In European
cohorts, VKORC1*2 shows a prevalence of about
40%. This population-specific distribution is reflected by low coumarin requirements in Asian, intermediate doses in European and high doses in African
populations.
Pharmacokinetics of coumarin metabolism
Coumarins are administered as a racemic mixture
of two enantiomers, for example, R-warfarin and Swarfarin. The S-enantiomer has approximately a 35-fold higher anticoagulant effect than the R-enantiomer,31,32 but generally has a more rapid clearance.
Coumarins are well absorbed with a bioavailability
of over 95% and a high fraction bound to plasma
albumin. Elimination of coumarins is performed
mainly in the liver. Warfarin and acenocoumarol are
metabolised by CYP2C9 (S-enantiomers) and other
CYP enzymes (R-enantiomers).33,34 Phenprocoumon
metabolism is not decisively affected by this pathway being mainly eliminated via unchanged renal
excretion35,36 and the following observations are
therefore only relevant in part.
Despite its lower potency, R-warfarin is thought to
exhibit around half of the overall anticoagulant
effect of racemic warfarin. This is due to the faster
clearance of the more potent S-warfarin by CYP2C9
leading to twice as high R-warfarin plasma levels
compared to S-warfarin.32 Because of its even more
rapid metabolisation by CYP2C9, S-acenocoumarol
does not contribute much to the anticoagulant effect
of racemic acenocoumarol.37,38
In CYP2C9, which is the most abundant enzyme
of the CYP2C family,39 several polymorphism have
been associated with impaired enzyme activity
(homepage of the Human Cytochrome P450 (CYP)
Allele Nomenclature Committee: http://www.
cypalleles.ki.se; accessed 20/01/2007). Of these, alleles CYP2C9*2 (Arg144Cys) and CYP2C9*3
(Ile359Leu) have the most clinical relevance. With a
prevalence of around 15% (CYP2C9*2) and 7%
(CYP2C9*3) in Caucasians, a rate of 2.5% of
homozygous or compound heterozygous for these
alleles can be calculated. Among other ethnic
groups, the prevalence of CYP2C9*2 and CYP2C9*3
is considerably lower.40-42 Due to their reduced enzymatic activity, these alleles are associated with
increased plasma levels of the S-enantiomers, resulting in a shift in R-/S-warfarin ratio. As the S-enantiomer is a more potent anticoagulant,31,32 even small
changes in plasma ratio will result in pronounced
effects. For example, weekly warfarin maintenance
doses in patients homozygous for defective
CYP2C9*3 allele were reduced to 25% of the normal
dose.43 Because of its high fraction of unchanged
renal excretion, phenprocoumon has a less decisive
effect.10 Polymorphisms in CYP2C9 other than *2 or
*3 have also been described to reduce coumarin
dose. The rare variant CYP2C9*11 (prevalence of
2% in Africans, 0.7% in Caucasians, and probably
absent in Japanese43-45 has been shown to cause warfarin and acenocoumarol sensitivity.
As mentioned above, the R-enantiomers are
metabolised by different enzymes of different
cytochrome P450 families, for example, CYP1A2,
CYP2C8, CYP3A4, or CYP3A5.34,46,47 Although several polymorphisms are known in these enzymes, a
significant effect on coumarin dose has yet not been
observed. However, a number of polymorphisms in
genes related to vitamin K transport, vitamin K
cycle, and gamma carboxylation have been shown
to influence coumarin dose.48,49 Compared to the
effects of VKORC1 haplotypes and variant CYP2C9
enzymes, those of ApoE, calumenin, gamma carboxylase, and microsomal epoxid hydrolase are of
minor importance for coumarin dose.
Coumarin resistance
Partial coumarin resistance is mainly caused by
homozygous combination of wt alleles in VKORC1
and CYP2C9.13,19,20 The frequency of this combination is approximately 20% in patients of Caucasian
origin. Compared to the average coumarin dose,
patients bearing this genetic pattern will require a
mean dose elevation of approximately 50%. These
patients correspond to the upper quarter of the normal range of the coumarin dose. Due to the wide
difference in distribution of wt VKORC1 haplotypes
among different ethnic groups, partial coumarin
resistance is much less frequent in patients of Asian
origin but more prevalent in Africans.13,18,19,43,50
In some cases, dose elevation is more pronounced,
and may even show a complete coumarin resistance.
These rare cases are explained by mutations in
VKORC1.11,15-17,51,52 When comparing mutations in
VKORC1 known to lead to coumarin resistance, two
main mutation clusters can be identified. The first
region is the third transmembrane domain which
harbours the warfarin binding motif TYA at amino
acids 138-14028 and the redox-motif CXXC.29,30 By
changing this motif, warfarin binding is impaired
and drug action is completely compromised. The
second mutation cluster is within the first extramembranous loop (amino acids 31 to 100). In this
loop, three highly conserved amino acid residues
were shown to be essential for VKORC1 activity
(Cys43, Cys51, Ser57). As the observed mutations
are distant from the proposed warfarin binding site
at aa 138-140 and the redox centre at aa 132-135, an
simultaneous interaction of the first loop with the
reactive centre and the warfarin binding site might
be hypothesised.
Recently, mutation Asp36Tyr was found to be
common in Jewish ethnic groups of Ethiopian and
European origin and associated with an increased
coumarin requirement. Here, a prevalence of 15%
and 4% respectively in the normal population can be
observed.53 In other Israeli Jewish populations originating from North Africa and Yemen, a frequency of
0.5% was much lower.53 In recent years, sequencing
projects have suggested that Asp36Tyr is present in
Caucasians at an even lower level. This mutation
has not yet been observed in several 400 alleles
derived from the normal population. It was only
observed in a single case of a partial coumarin resistant patient.13,19,51 Furthermore, Asp36Tyr is connected
to the supposed ancestral wt haplotype
VKORC1*1,53 which is almost absent in Caucasians
and Asians (Geisen 2005).
Coumarin sensitivity
Partial coumarin sensitivity is a phenomenon
observed in about 15% of patients of Caucasian origin. This number corresponds well with the
homozygosity rate of the low expression haplotype
VKORC1*2.19,20 Combined with the defective
CYP2C9 alleles *2, *3, or *11, an even more pronounced effect on coumarin dose can been observed
for warfarin and acenocoumarol but not phenprocoumon.20 Patients with the VKORC1*2 genotype
are predisposed to low coumarin dose and are at an
elevated risk of severe over-anticoagulation and subsequent bleeding complications.54-56 In particular,
patients with combined CYP2C9 polymorphisms
and VKORC1*2 haplotype were prone to the
adverse effects of coumarins.56 Indeed, CYP2C9*3
has been identified as a major risk factor in warfarin/acenocoumarol treatment, as rapid overdosing
is due to the impaired metabolism, with subsequent
accumulation of the more effective S-enantiomer.38,57,58
In infrequent cases, dramatically increased coumarin sensitivity is due to mutations in exon 2 of F9.
These mutations in FIX propeptide (F9: c.109G>A
p.Ala37Thr and c.110C>T p.Ala37Val; nomenclature
according to Human Genome Variation Society,
http://www.hgvs.org/mutnomen, accessed on
20/01/2007) cause a reduced affinity of the gamma
carboxylase to the factor IX precursor, leading to an
isolated and dramatic decrease of FIX activity when
receiving coumarins.59,60 Generally, in the absence of
coumarins, the phenotype of these patients is completely normal.
New dosing algorithms
Meanwhile, the genotype of both VKORC1 and
CYP2C9, together with other known dose influencing factors like age, gender, and weight, were intro-
duced into clinical dosing algorithms and prospective studies.20-24 Similarly, all studies report on
VKORC1 as the major predictor of coumarin dose,
while the influence of CYP2C9 genotype on dose is
lower. The combination of polymorphisms in both
genes is a strong indicator for a high risk of severe
over-anticoagulation.56 Therefore, patients with
combined polymorphisms in particular will benefit
substantially from genotype-adapted coumarin dosing, especially in the initial saturation phase.8,9,54,56
Individualised coumarin dosing may lead to a significant reduction of related complications in the initiation phase.9,56
Concomitant administration of coumarins and vitamin K
A recent development which might lead to a
new principle of oral anticoagulation is the concomitant administration of coumarins and vitamin
K.61 While at first, application of agonist and antagonist at the same time might appear contradictory,
this is a reasoned approach in oral anticoagulation.
The main complications of coumarin therapy are
high INRs above the therapeutical range with consecutive bleeding complications. Difficulties in
controling coumarin therapy is due to many factors. There are variable vitamin K intake from
food, lack of vitamin K storage, autocatalytical
dependence of gamma carboxylase enzyme from
vitamin K, and VKOR activity limiting the rate of
vitamin K recycling.
Two recently published articles strongly support
this view. Schurgers et al.62 were able to demonstrate that substitution of 150 µg per day of vitamin
K did not significantly change INR in oral anticoagulation in healthy volunteers. Sconce et al.63 reported on a lower daily vitamin K intake in patients
with instable INR compared to patients with stable
INR course. Given these observations, one could
speculate that a low dose vitamin K substitution,
for example 80 µg per day, could stabilise INR values within the therapeutical range and thus reduce
the risk of bleeding complications under oral anticoagulation. With an incidence of 0.25% of fatal
bleeding complications per treatment year, it is
worthwhile to examine this hypothesis by prospective randomised studies. Very recently, first experiences with this new approach have been published
by Sconce et al.64 These first data suggest improved
anticoagulation stability, but cohorts were too
small to draw final conclusions. Cloning of
VKORC1 and a better understanding of the vitamin
K cycle will eventually lead to new principles of
oral anticoagulation therapy.
Acknowledgements
The work of J.O. was supported by grants from the
Deutsche Forschungsgemeinschaft (DFG - OL 100/3-1),
the Bundesministerium für Bildung und Forschung Forschungszentrum Jülich (BMBF/PTJ - 0312708E), the
National Genome Research Net Cardiovascular Diseases
(BMBF/DLR-01GS0424/NHK-S12T21) and Baxter
Germany.
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Thrombosis
Do arterial and venous thrombosis share common risk
factors?
G. Lowe
Division of Cardiovascular and
Medical Sciences, University of
Glasgow, UK
2007;1:51-55
raditionally, arterial and venous
thrombosis have been considered
as separate diseases. Standard texts
emphasise differences in epidemiology
and risk factors, pathology, prevention
and treatment. For example, recent UK
advice on prescribing stresses the importance of antiplatelet drugs in the management of arterial thrombosis, and anticoagulant drugs in venous thrombosis.1 This
ignores a large body of recent evidence
that anticoagulant drugs have an important role in the prevention and management of arterial thrombosis, and
antiplatelet drugs in the prevention of
venous thrombosis.2 Such evidence
strongly supports the concept that both
platelets and coagulation play important
and complementary roles in both arterial
and venous thrombosis. This is logical,
given that thrombosis is haemostasis in the
wrong place;3 and haematologists have long
known that platelets and coagulation play
important and complementary roles in
haemostasis. For example, it has been
known for many years that the
antiplatelet drug aspirin increases the risk
of bleeding in patients with both congential (haemophilias) and acquired (for
example, anticoagulant therapy) coagulation defects.
As pathological and therapeutic studies
are increasingly emphasising the similarities rather than the differences between
arterial and venous thrombosis, so too are
epidemiological studies emphasising their
similarities in risk factors. Similarities in
risk factors probably explain a large part of
the association between arterial and
venous thrombosis in epidemiological
studies.2,4 This brief review highlights
recent evidence from epidemiological
studies on common risk factors for arterial
venous thrombosis (Table 1), and suggests
that clinical management of thrombosis
should address the overall thrombotic risk,
arterial and venous, of the individual
patient.
T
Time
At present, there is a global epidemic of
thrombotic cardiovascular disease, both
arterial and venous.5-7 The epidemic of
coronary heart disease (CHD) was evident
in industrialized countries by the midtwentieth century when a sequential
study of routine necropsies in London
showed that its pathological basis was not
an increasing prevalence of atherosclerosis, but rather an increasing prevalence of
arterial thrombosis occurring upon atherosclerotic lesions.8 The causes of this global
epidemic of arterial thrombosis are mainly
environmental, not genetic, as shown by
the classic Framingham Study.9 This,
together with other prospective epidemiological studies, established that the major
risk factors are tobacco smoke exposure,
arterial blood pressure and serum cholesterol. These have each now been shown
to disturb arterial endothelium and predispose to atherogenesis and thrombogenesis. Other risk factors are obesity and diabetes mellitus. Applying this knowledge
of lifestyle risk factors in public health and
education helped to reverse the exponential increase in CHD incidence observed in
industrialized countries during the later
part of the twentieth century.10 Metaanalyses of both observational studies and
randomised trials on the reduction in
blood pressure or serum cholesterol, have
established the causality of these classical
risk factors.11 Socio-economic changes12
have increased the prevalence of classic
CHD risk factors around the world.13 This
has led to a recent expansion of the global
epidemic of CHD to developing countries.
CHD will continue therefore to be a major
international cause of morbidity and mortality through the twenty-first century.
Furthermore, the decline of CHD in developed countries is partially offset by the
increasing number of arterial thrombotic
events in the brain and leg.14
Along with the continuing global epidemic of arterial thrombosis, a global epidemic of venous thromboembolism was
Table 1. Risk factors for arterial and venous thrombosis.
Time
Exponential increases in risk 1950-1980 in developed
countries; 1980-2005 in developing countries
Age
Increasing life expectancy, due to reduced risk of
premature death from infection and undernutrition
Lack of regular exercise; increased periods of immobility
Epidemic in developed and developing countries,
due to decreased regular exercise and high fat diet
Immobility
Obesity, metabolic
syndrome and type
2 diabetes
Cancer
Increasing overall risk due to increased life expectancy,
obesity, and (in developing countries) smoking
Increased survival due to earlier detection and more
effective treatment
Prothrombotic effects of chemotherapy
Pregnancy
Oral oestrogens
COC, HRT
Infections
Common acute infections, e.g. respiratory, urinary
Chronic infections, e.g. HIV?
Trauma
Surgery
Vascular catheterisation
Intravenous drug self-use
Haematological
disorders
Thrombophilias
Factor V Leiden
Prothrombin G20120A mutation
Lupus anticoagulant
Homocysteine
Polycythaemias
Smoking, blood
pressure, cholesterol?
observed in the second half of the twentieth century.5
This shows no signs of declining despite the
increased use of effective antithrombotic prophylaxis in hospitalised patients at increased risk.7 This
combined epidemic of arterial and venous thrombosis suggests a global increase in thrombotic tendency
over time. Let us now examine the increase in common risk factors for arterial and venous thrombosis
over the last fifty years.
Age
One likely reason for the large increase in arterial
and venous thrombosis in both developed and developing countries over the last fifty years is the median
age of the population. Increased life expectancy, is
the main factor due largely to the reduced risk of premature death from infection and undernutrition. This
is in turn largely due to a reduction in poverty and to
public health measures.12 While increased life
expectancy is a positive health benefit, it results in an
increasing percentage of the population aged over 60
years in whom the majority of arterial and venous
thrombotic events occur. There is an exponential
increase in the risk of both arterial and venous
thrombotic events with age.5,7,9,10 Since age is not necessarily a direct causal risk factor, possible mechanisms for this association include: cumulative effects
of causal risk factors on the arterial wall (for example,
tobacco-smoking, blood pressure and cholesterol);
less regular exercise and increasing periods of immobility which increase venous stasis in the lower limb
and hence the risk of venous thrombosis; and systemic hypercoagulability.
Epidemiological studies have shown significant
changes in circulating levels of coagulation factors,
inhibitors and activation markers, as well as inflammatory markers, in the age range 25-74 years.15-18 In particular, between ages 60 and 79 years, there are
marked increases in circulating markers of coagulation
activation (fibrin D-dimer) and inflammation (C-reactive protein), which are not attributable to increases in
classic cardiovascular risk factors.19 Further studies are
required to establish the causes of this exponential
increase in markers of systemic hypercoagulability
and inflammation with increasing age.
Immobility
Another likely factor promoting arterial and
venous thrombosis which has seen a large increase
in both developed and developing countries over the
last fifty years is the major decline in regular physical
activity in the general population.12 Industrialisation
and socio-economic changes promote immobility
through the use of private transport, the long time
spent sitting watching television or sitting in front of
personal computer screens, and reduced regular
leisure-time activity. Epidemiological studies have
shown that the latter is strongly related to both the
risk of arterial thrombosis, and systemic hypercoagulability and inflammation.20 As noted above, immobility also increases venous stasis in the lower limb,
increasing the risk of venous thrombosis.
Obesity, metabolic syndrome and type 2 diabetes
The combination of decreased regular exercise, and
increased commercial promotion of a high fat diet
(for example, fast food) has led to a global epidemic of
obesity, the associated features of the metabolic syndrome (hypertension, dyslipidemia, hyperglycemia,
hyperinsulinemia), and type 2 diabetes mellitus.21
There is strong and consistent evidence from epidemiological studies that obesity, metabolic syndrome and diabetes increase the risk of arterial
thrombosis.21,22 This is most probably due to their
many adverse effects on the arterial wall, as well as
their systemic effects on low grade inflammation,
hypercoagulability and hypofibrinolysis.15-18,21-26 Many
epidemiological studies have documented associations between obesity and venous thrombosis.4,7,27-31
Recently some studies have reported that metabolic
syndrome or type 2 diabetes may increase the risk of
venous thrombosis, even after adjustment for measures of obesity such as body mass index.31-33 This
association may again be a consequence of their systemic effects or hypercoagulability.
Cancer
Cancer has long been recognised as a risk factor for
both arterial and venous thrombosis.34 Possible
mechanisms include local effects of solid tumours on
vessels (compression, invasion), immobility for
venous thrombosis, and systemic hypercoagulability
induced by the tumour or by treatments such as
chemotherapy.34 The impact of cancer on thrombosis
is increasing globally, due to increased life expectancy, obesity and, in developing countries, smoking,
increased survival after diagnosis due to earlier detection and more effective treatment, and the increased
use of chemotherapy.
Pregrancy
Pregnancy increases the risks of arterial and venous
thrombosis.35 Possible mechanisms include the
effects of the pregnant uterus on vessels, immobility
for venous thrombosis, and systemic hypercoagulability (especially in women with thrombophilias).
These also increase the risk of other pregnancy complications, perhaps due to microthrombosis.35
However, screening for thrombophilias in early pregnancy does not seem to be cost-effective.36
Combined oral contraceptives
Combined oral contraceptives (COC) increase the
risks of arterial and venous thrombosis.35,37 This could
be due to systemic hypercoagulability. As in pregnancy, this increases the risk of thrombosis particularly in women with thrombophilias, such as factor V
Leiden. This may partly explain an early increase in
risk, the so-called starter effect.35,37 However, as in pregnancy screening for thrombophilias, prior to prescription of COC does not seen to be cost-effective.36
Hormone replacement therapy
Oral hormone replacement therapy (HRT) increases the risks of both arterial and venous thrombosis.35,37-39 The relative risks are similar to those of
COC. However, the absolute risks are higher due to
the higher age of women starting HRT.38,39 Again, the
most plausible mechanism is systemic hypercoagulability, which as in pregnancy and COC use, increases the risk of thrombosis particularly in women with
thrombophilias, which may partly explain the starter
effect.38,39 Risk increases with age and obesity, and
may differ with the type of preparation.38,39
Transdermal HRT may carry a lower risk of venous
thrombosis than oral HRT.38,39 While a recent analysis
suggests that screening for thrombophilias prior to
prescription of oral HRT may be cost-effective,36 the
risks and benefits of such screening require further
evaluation.39,40
Infections
Acute and chronic infections increase the risk of
both arterial and venous thrombosis.7,41,42 Possible
mechanisms include systemic hypercoagulability,
and, for venous thrombosis, immobility. Recent epidemiological studies have shown that common acute
infections (for example, respiratory, urinary) increase
the risk of both arterial and venous thrombosis.41,42
Also, recent reports suggest that the current global
epidemic of chronic HIV infection may also be associated with an increased risk of arterial and venous
thrombosis, with or without combination antiretroviral therapy.43 Further studies are required to assess
these risks, and the possible antithrombotic roles of
immunisations and antithrombotic therapies.
Trauma and surgery
Trauma and surgery are well-established risk factors for venous thrombosis7 due to immobility and
systemic hypercoagulability. National guidelines
emphasise the importance of prophylaxis with
mechanical methods, aspirin and/or anticoagulant
drugs.44,45 Thrombophilias increase the risk, but further studies are required to establish the benefits and
risks of screening prior to major surgery (for example,
orthopaedic).36 There is growing interest in the
increased risk of arterial thrombosis following surgery, especially in patients with clinical evidence of
arterial disease.46 This can be reduced by careful
assessment of patients and their medications, including aspirin, prior to surgery.46
Vascular catheterisation is also a well-recognised
risk factor for both arterial and venous thrombosis.
Recent reports have highlighted the global epidemic
of personal intravenous drug as a risk factor for both
venous thrombosis47 and arterial thrombosis when
arteries are accidentally punctured.
Haematological disorders
Congenital thrombophilias are established risk factors for venous thrombosis,40 especially during periods of increased risk such as pregnancy, COC use,
HRT use and surgery (as discussed above). There has
been growing interest in their association with arterial thrombosis.40,48-50 While further studies are
required, recent meta-analyses suggest that the two
common prothrombotic genetic mutations (factor V
Leiden and the prothrombin G20120A mutation) are
associated with increased arterial thrombotic risk.49,50
However, these associations are about tenfold weak-
er than their associations with risk of venous thrombosis (odds ratio about 1.2-1.3, compared to 2-3).40
These genetic mutations are associated with the phenotype of resistance to activated protein C, which
has recently been associated with risk of arterial
thrombosis.48 Acquired thrombophilias include lupus
anticoagulants,51 hyperhomocystinemia,40 and polycythemias including polycythemia vera.52 In a recent
report from the European Collaboration on Lowdose Aspirin in Polycythaemia vera prospective
study, neither haematocrit (below 50%, achieved in
over 90% of patients) nor platelet count was associated with thrombotic events or mortality.52This study
also confirmed low-dose aspirin was effective and
safe in reducing the risk of thrombotic events.53
Smoking, blood pressure and serum cholesterol
These are the three classic, causal risk factors for
arterial thrombosis, as established in prospective epidemiological studies.9-11 Evidence-based national
guidelines continue to stress the importance of smoking habit, blood pressure, and cholesterol in both risk
assessment and prevention of coronary heart disease
and stroke.54
The roles of smoking, blood pressure and cholesterol on the risk of arterial thrombosis are usually
considered to be due to their adverse effects on the
arterial wall, promoting atherosclerosis, plaque rupture and superadded arterial thrombosis. Despite epidemiological evidence that tobacco smoking, like
lack of exercise, obesity, metabolic syndrome, and
type 2 diabetes, is associated with a reversible hypercoagulable state,55 there is conflicting evidence from
epidemiological studies on the association between
smoking and risk of venous thrombosis.7,27-31 Age and
obesity, major risk factors for venous thrombosis as
noted above, are major potential confounders,
because smokers are overall younger with an
increased risk of premature death and less obese than
non-smokers.27
Increased arterial blood pressure is associated with
obesity and is part of the metabolic syndrome as
noted above. However, in multivariate analyses
including age and obesity, major factors regarding
blood pressure, there is little evidence that blood
pressure is associated with risk of venous thrombosis
7,27-31
or systemic hypercoagulability.23,26 The effect of
reduced blood pressure reduction on the risk of
venous thrombosis has not been evaluated in randomised trials.
There is also little evidence that serum cholesterol is
associated with the risk of venous thrombosis 7,21,31 or
systemic hypercoagulability.23,26 Recently, a review of
observational studies suggested that use of statins,
which lower LDL and total cholesterol, was associated with a reduction in the risk of venous thrombo-
sis.56 There is little evidence that statins reduce systemic hypercoagulability57 and the postulated effect of
statins on the risk of venous thrombosis must still be
assessed in analyses of randomised controlled trials.2
Conclusions
There is increasing evidence that arterial and
venous thrombosis share several cardiovascular risk
factors. Furthermore, global changes in population
age, immobility and obesity are increasing the likelihood that risk factors are shared. The clinical message for hematologists is that patients with arterial or
venous thrombosis increasingly share risk factors.
This means that clinical management of thrombosis
should address the overall thrombotic risk, arterial
and venous, of the individual patient. This should be
considered when evaluating secondary prevention
with antithrombotic therapies in discussion with the
patient.
Acknowledgements
The author thanks the British Heart Foundation, Medical
Research Council (UK), and Chief Scientist Office, Scottish
Executive Health Department, for research support; and
Helen Mosson for preparing the manuscript.
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Thrombosis
Venous thromboembolism in medical patients:
stratification and prevention
A
P. Prandoni
Department of Medical and
Surgical Sciences
Thromboembolism Unit
University of Padua, Italy
2007;1:56-59
B
S
T
A
C
T
Acute venous thromboembolism (VTE) is a serious and potentially fatal disorder. It
often complicates the course of hospitalized patients but may also affect ambulatory
and otherwise healthy people. While the introduction of thromboprophylactic measures
has probably had an impact on the occurrence of postoperative VTE, there is an increasing awareness of the importance of medical conditions in determining thromboembolic
events. Simple and clinically relevant risk assessment models are available to facilitate
VTE risk assessment in hospitalized medical patients. According to international guidelines, high-risk medical patients should receive in-hospital pharmacological thromboprophylaxis with either unfractionated or low-molecular-weight heparin, unless contraindicated. Other non-surgical factors that have been associated with an increased risk
of VTE disorders include cancer, air travel, inflammation, persistent elevation of D-dimer
and atherosclerotic disease. Recognition of the incidence and clinical importance of
thrombosis will probably encourage a more widespread use of antithrombotic prophylaxis in medical patients.
cute venous thromboembolism
(VTE) is a serious and potentially
fatal disorder. It often complicates
the course of hospitalized patients but
may also affect ambulatory and otherwise
healthy people. In 1884, Rudolph Virchow
first proposed that thrombosis was the
result of at least one of three underlying
etiologic factors, vascular endothelial
damage, stasis of blood flow, and hypercoagulability. In the last century, there was
growing recognition that all risk factors
for venous thromboembolism (VTE)
reflect these underlying pathophysiologic
processes and that VTE does not usually
develop in their absence.1 In a review of
12,31 consecutive patients treated for
VTE, 96% had at least one recognized risk
factor.2 Furthermore, there is convincing
evidence that risk increases in proportion
to the number of predisposing factors presented.1,3
According to recent epidemiological
data collected in two Swedish cities, VTE
is to be expected in 1.6-1.8 per 1,000
inhabitants per year.4,5
Classic risk factors for VTE include cancer, surgery, prolonged immobilization,
fractures, puerperium, paralysis, use of
oral contraceptives, and the antiphospholipid antibody syndrome.1,3 These not only
predispose apparently normal patients to
thrombosis, but are also likely to lead to
A
| 56 |
R
this condition in people with inherited
thrombophilic abnormalities.1,3 Combined
genetic defects, as well as the combination
of a genetic defect with one or more
acquired risk factors, and the combination
of two acquired risk factors, result in a risk
of VTE that exceeds the sum of the separate effects of the single factors.3 This is
the case of the combination of highly
prevalent defects, such as factor V Leiden
and prothrombin mutation, with even
small risk factors such as the oral contraceptive pill, or the combination of the oral
contraceptive pill with even minor surgery
or injury.
Most clinically recognized instances of
VTE are suspected because of typical signs
and symptoms in individuals who come
to an outpatient clinic or hospital emergency department.1 Hospita-lization for
surgery and for medical illnesses account
for similar proportions of cases.6
VTE often affects ambulant and otherwise healthy individuals.7
This review focuses on old and new
acquired hypercoagulable states that can
be responsible of VTE disorders in both
hospitalized and ambulant medical
patients.
Medically ill patients
While the introduction of thromboprophylactic measures has probably had an
Restricted mobility plus at
least 1 risk factor of VTE*
Contraindications to
pharmacological
thromboprophylaxis?
NO
Enoxaparin 4000 U,
Dalteparin 5000 U,
Fondaparinux 2.5 mg o.i.d
or UFH 5000 U t.i.d
YES
Mechanical measures
(IPC, GCS)
* Age older than 70, active cancer, previous thromboembolism, already known
thrombophilia, recent trauma or surgery, heart and/or respiratory failure, acute
infectious disease, rheumatic disease, obesity, hormonal treatment; IPC: intermittent pneumatic compression; GCS: graduated compression stockings.
Figure 1. Algorithm for risk stratification and implementation of thromboprophylaxis in high risk medical patients
impact on the present occurrence of postoperative
VTE, there is an increasing awareness of the importance of medical conditions in determining thromboembolic events, since as appropriate thromboprophylaxis is rarely administered to patients on medical wards.
Three large-scale prevention studies involving over
5,500 medically ill patients have shown that 11-15%
will have VTE and 4-5% will have proximal-vein
thrombosis as identified by screening studies in the
absence of prophylaxis.8-10 Also, the national DVT
Free Registry found that 60% of patients diagnosed
with an acute DVT were in the peri-hospitalization
period. Approximately 60% of the cases occurred in
non-surgical patients.11
Current literature highlights numerous risk factors
for VTE in the medically ill patient. These clinical risk
factors include increasing age, acute respiratory failure, congestive heart failure, prolonged immobility,
stroke or paralysis, previous VTE, cancer and its
treatment, acute infection, dehydration, hormonal
treatment, varicose veins, acute inflammatory bowel
disease, rheumatologic disease, and nephrotic syndrome.12,13 Patients with mostly asymptomatic proximal-vein thrombosis may carry an unexpectedly high
risk of in-hospital death.14 Recent data suggest that
current practice is associated with serious uncertainty leading to both the overuse and underuse of
thromboprophylaxis in patients on medical wards.15
VTE can affect apparently healthy people. Besides
circumstantial events, the additional risk factors for
VTE that increase the thromboembolic risk in these
individuals do not substantially differ from those
accounting for VTE in the hospital setting. The most
common are old age, varicose veins, cancer, heart
failure, peripheral artery disease, and previous
VTE.16,17 In this context it is interesting that obesity,
smoking, and hypertension have been found to be
associated with an increased VTE risk.5,18
Consensus guidelines published by the American
College of Chest Physicians (ACCP) and the
International Consensus Statement (ICS) recommend
assessment of all hospitalized medical patients for
the risk of VTE and the provision of appropriate
thromboprophylaxis.19,20 Furthermore, simple and
clinically relevant risk assessment models are available to facilitate VTE risk assessment.21-23 Figure 1
provides an example of risk assessment model that
integrates appropriate thromboprophylactic strategies in the form of a management algorithm.
Installation of computer-alert programs and electronic tools can potentially increase physicians’ use of
prophylaxis,24,25 and to markedly reduce the rate of
VTE arising among hospitalized patients at risk.24
Based on available information, the recently updated ACCP and ICS guidelines give a strong recommendation for thromboprophylaxis using either
enoxaparin 4,000 U in one daily administration, dalteparin 5,000 U in one daily administration, unfractionated heparin 5,000 U in three daily injections in
hospitalized medical patients aged > 40 with congestive heart failure or severe respiratory disease, or in
medical patients who are confined to bed and have
one or more risk factors for VTE, such as active cancer, acute neurological disease, infective disease,
inflammatory bowel disease, rheumatic disease, previous VTE or sepsis.19,20 As an alternative, fondaparinux 2.5 mg once daily can be considered. In patients
with a contraindication for pharmacological thromboprophylaxis (such as those with hemorrhagic
stroke or ischemic stroke with bleeding risk), intermittent pneumatic compression, graduated elastic
stockings or both should be considered.
Whether thromboprophylaxis in high risk medical
patients should be extended beyond the period of
hospitalization remains to be demonstrated although
relevant studies are ongoing.
Other non-surgical causes of VTE
Cancer
Since Armand Trousseau’s initial observation in
1865, numerous studies have addressed the relationship between cancer and VTE. VTE is either a frequent complication in cancer patients, or sometimes
acts as an epiphenomenon of a hidden cancer. In this
way it offer opportunities for anticipated cancer diagnosis and treatment26 in patients with malignancy
VTE represents an important cause of morbidity and
mortality. It has been estimated that 1 in every 7 hospitalized cancer patients who die, do so from pulmonary embolism.27 Of these patients, 60% have
localized cancer or limited metastatic disease, which
would have allowed a longer survival in the absence
of the fatal PE. According to the Medicare Provider
Analysis and Review Record, a database that records the
primary discharge diagnosis and an additional four
discharge diagnoses in the USA, the rate of initial or
recurrent thromboembolism in patients with cancer
well exceeds that recorded in those without malignancy, and affects cancers of virtually all body systems with a similar frequency.28 Patients with cancer
have a highly increased risk of VTE in the first few
months after diagnosis and in thepresence of distant
metastases.29 This risk is further increased in the presence of inherited thrombophilic abnormalities.29
The real true rate of VTE in cancer patients is virtually unknown. This is due to the surprising lack of
information in almost all studies dealing with the
natural history of malignant diseases. However, the
majority of thrombotic episodes occur spontaneously, in the absence of triggering factors that commonly account for thromboembolic complications in subjects without cancer.29 This is confirmed by the high
frequency of patients with known malignancy
referred to clinicians for the development of VTE.30
The most common factors that put cancer patients at
a higher risk of VTE include immobilization, surgery,
chemotherapy with or without adjuvant hormone
therapy, and the insertion of central venous
catheters.26 Among factors that are associated with a
higher risk of VTE during chemotherapy are the site
of cancer (namely, upper gastrointestinal or lung),
prechemotherapy platelet count > 350/nL, the use of
white cell growth factors, hemoglobin value lower
than 10 g/dl or use of erythropoietin.31
The strong association between cancer and venous
thromboembolism is further emphasized by the high
rate of cancer development in patients with venous
thrombosis. According to the results of the most
important studies, this risk has been consistently
found to be 4-5 higher as high in patients with idiopathic rather than secondary thrombosis.26 These
data have recently found important confirmation in
four very large, retrospective, population-based studies.32-35 Interestingly, although the risk for developing
cancer was particularly high in the first six months
after the diagnosis of VTE, a significant effect persisted for up to 10 years, suggesting that either a malignant disorder can induce hypercoagulability many
years prior to its overt clinical development or that
cancer and thrombosis share common risk factors. A
recent investigation has indeed provided direct
genetic evidence for the link between oncogene activation and thrombosis.36
Air travel
the risks of VTE associated with long-duration air
travel – the so-called economy class syndrome.1 There is
a general consensus that clinically important VTE
after air travel is rare. Case reports suggest that most
cases of travel-related thrombosis affected people at
risk because of previous VTE or other predisposing
factors.
Inflammation
In a recent population-based case-control study,
van Aken and al. showed that subjects with elevated
interleukin-8 levels have an increased risk of venous
thrombosis, thus giving some support to the role of
inflammation in the pathogenesis of venous thrombosis.37 It is not surprising, therefore, that subjects
with inflammatory bowel disorders, Behcet disease,
HIV infection, and other infectious diseases exhibit
an increased risk of VTE. It should be noted, however, that in a recent prospective investigation, markers
of inflammation such as fibrinogen, C-reactive protein levels, or white cell count were not associated
with VTE.38
D-Dimer
In a recent case-control study, investigators from
the Thrombophilia Leiden Study showed an interesting association between elevated levels of DDimer and the risk of venous thrombosis.39 These
findings have been confirmed by those of a recent
prospective investigation.40 It is quite evident that Ddimer cannot by itself represent the cause of venous
thromboembolism, but should be interpreted as a
marker of hypercoagulability often detectable in disease states as well as in otherwise healthy people.
These studies open new interesting perspectives for
future research.
Atherosclerosis
Although acquired and/or inherited risk factors
potentially responsible for VTE are identifiable in
the majority of patients, the disease remains unexplained in up to 30% of patients. Recently, an unexpected association of VTE with atherosclerosis was
found.41 Also, in three cohort studies dealing with
the long term follow-up of patients with VTE, those
patients who had an idiopathic episode had a statistically significant and clinically relevant increased
risk of atherosclerotic complications and cardiovascular events compared to patients with secondary
VTE42,43 or matched control subjects.44 Although two
studies failed to show an increased risk of VTE in
patients with subclinical atherosclerosis.45,46 we suspect that either atherosclerotic disease may induce
venous thrombosis or the two conditions share common risk factors.
Recently, the popular press has drawn attention to
Conclusions
VTE is a serious and potentially fatal disorder
which complicates the course of hospitalized
patients and may also affect ambulant and otherwise
healthy people. The risk factors that predispose
patients to thrombosis are numerous. Recognition of
the incidence and clinical importance of thrombosis
in high risk medical patients during hospitalization
will probably encourage a more widespread use of
antithrombotic prophylaxis in these patients.
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Hodgkin’s Lymphoma
The role of PET in staging and response assessment
L. Specht
Dept. of Oncology and Haematology
The Finsen Centre Rigshospitalet
Copenhagen University Hospital
Copenhagen, Denmark
2007;1:60-63
ositron emission tomography (PET)
is a non-invasive, quantitative imaging technique which can visualize
biochemical, physiological, and biological
processes in vivo. It is, the most specific
and sensitive molecular imaging techniqueused today. PET with 2-[18F]fluor-2deoxyglucose (FDG-PET) has proved a
very valuable tool in the management of
malignant disease including lymphomas.
The first report of FDG-PET and lymphoma imaging was presented in 1987.1
This and other studies showed abnormal
FDG uptake in most lymphomas with a
correlation between the uptake rates and
the malignancy grade and proliferative
activity.2-8 Hodgkin lymphoma (HL) is generally FDG avid.9-16 Figure 1 shows a
PET/CT scan of a patient with classic
Hodgkin lymphoma.
P
FDG-PET in staging
Physical examination and computed
tomography (CT) combined with bone
marrow and other biopsies when required
have been the main point of reference in
the staging of lymphomas.17 In the past, gallium scintigraphy was important for
detecting lymphoma involvement. But
FDG-PET has been shown to be significantly more sensitive, and gallium scintigraphy has now largely been abandoned.1,18–24 However, the standard procedures fail to identify a considerable number of sites, particularly extranodal ones.
FDG-PET has been evaluated as a supplementary staging investigation in several studies. There are, however,
methodological problems in these studies, the most important one being the
lack of a valid reference test. Since it is
not possible to obtain biopsies from all
lymph node regions and organs of interest, a reference standard based on all
available evidence from CT, FDG-PET,
and all available clinical information
including follow-up must be used
instead. This method has serious limitations with regard to evaluating the diag| 60 |
nostic accuracy of FDG-PET, but for the
moment it is the best method available.
The general conclusion so far of studies
in (HL) and aggressive non-Hodgkin lymphoma (NHL) is that FDG-PET is more
accurate for diagnosing both nodal and
extranodal disease than CT, thus having a
strong potential impact on the staging of
(HL) and aggressive (NHL).10,12-14,16,20,25-42 In
cases with bone marrow involvement,
FDG-PET seems to be at least as sensitive
as blind bone marrow biopsy.10,25,43 In general, more patients are upstaged than
downstaged by FDG-PET. In the published series, FDG-PET changed the disease stage in 10-40% of patients. This led
to changes in treatment strategy in about
half of these patients.38 Whether the
changes in treatment strategy caused by
FDG-PET will eventually lead to improvement in treatment outcome is at present
unknown. Clearly, the fact that FDG-PET
upstages more patients than it downstages
leads to stage migration and better treatment results both in patients with localized and in patients with advanced disease. Whether treatment modification
based on FDG-PET will ultimately
improve results is at present being tested
in randomized trials.
The use of FDG-PET in the staging and
evaluation of (HL) is today considered part
of the routine management in many institutions, and funding approval has been
granted in most countries, including the
United States. The recently proposed
revised response criteria for malignant
lymphoma including PET are designed
also for HL. PET is therefore strongly recommended before treatment to better
define the extent of disease.44 However, at
the moment it is not compulsory because
of limitations of cost and availability.
FDG-PET in response evaluation
Tumor response serves as an important
substitute for other measures of clinical
benefit such as progression-free and overall survival. Tumor response also serves as
Cumulative progression-free survival
1.0
0.8
0.6
Early interim PET
Negative
Positive
0.4
0.2
p<0.0001
0.0
0
1
2
3
Time in years
Figure 2. Progression-free survival of patients with Hodgkin
lymphoma according to FDG-PET results after 2 cycles of
chemotherapy. Reprinted with permission from Blood
2006;107:52-9.
Figure 1. PET/CT scan of a patient with Hodgkin lymphoma CS IIIA with cervical, mediastinal and retroperitoneal involvement.
an important guide in decisions to continue or
change therapy. Response in lymphomas was previously assessed according to the International
Workshop Criteria (IWC) based mainly on morphological criteria, with a reduction in tumor size on CT
being the most important factor.45 However, after
completion of therapy, CT will often reveal residual
masses. By conventional methods it is very difficult
to assess whether this represents viable lymphoma
or fibrotic scar tissue. To perform a biopsy on all
these lesions would be unpractical, and even if carried out would be too inaccurate because residual
masses may contain a mixture of fibrosis and viable
lymphoma cells and false negative results could be
expected.
This difficult situation led to the introduction in the
IWF (and earlier Cotswold criteria for HL) of the problematic concept of CRu (complete remission/unconfirmed).45,46 Since changes in tissue function predate
volume changes, it is possible to assess response using
functional imaging. FDG-PET seems to be able, at
least to a large extent, to distinguish between viable
lymphoma and necrosis or fibrosis in residual masses
after treatment of HL.28,30,47-60 Based on these findings,
the International Harmonization Project has developed new recommendations for response criteria for
malignant lymphomas, incorporating FDG-PET into
the definitions of response in FDG-avid lymphomas.44 However, it is clear that a negative FDGPET scan after therapy does not exclude the presence
of microscopic disease.53 For the moment, there is little clinical data to support the new recommendations
for response criteria, and long-term follow-up of lymphoma patients evaluated by these criteria is awaited
with great interest.
Early studies in breast and colorectal cancer
showed that glucose metabolism in responding
tumors already changed markedly within the first
weeks of therapy.61-63 Several studies in HL showed
that an early FDG-PET scan, after 1-3 cycles of
chemotherapy, is a strong predictor of treatment failure.64-68 Figure 2 shows progression-free survival
curves according to FDG-PET after 2 cycles of
chemotherapy in patients with HL in all stages. In
advanced disease we have shown that the result of
an early interim FDG-PET scan makes the
International Prognostic Score insignificant.
It is not yet clear if interventions based on the
result of an early interim FDG-PET scan are called for.
Randomized trials are now being set up to test if
treatment modification based on the result of an
early interim PET scan, such as treatment reduction
in PET negative patients and treatment intensification in PET positive patients, can improve outcome
with respect to improved progression-free survival in
patients with a poor prognosis and equivalent progression-free survival with less treatment (and,
hence, a lower risk of long-term complications) in
patients with a good prognosis.
20.
21.
22.
23.
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Tailoring the treatment for early-stage
M. André1
O. Reman2
1
Centre Hospitalier Notre Dame &
Reine Fabiola, Charleroi, Belgium;
2
CHU de Caen, Caen, Belgium
2007;1:64-69
he optimal treatment strategy for
early-stage Hodgkin lymphoma is
still the subject of intense debate.1,2
The key issue is: can we further decrease
treatment without compromising the
excellent results and, while reducing
treatment, decrease devastating late toxicities? The purpose of this review is to
summarize the current achievement of
chemo-radiotherapy, chemotherapy alone
and ways to better define the treatment of
early-stage Hodgkin lymphoma patients.
T
Tailoring to histology: the nodular
lymphocyte-predominant Hodgkin lymphoma
This lymphoma is pathologically and
clinically distinct from classic Hodgkin
lymphoma (cHL). Nodular lymphocytepredominant
Hodgkin
lymphoma
(NLPHL) is characterized by atypical lymphocytic and histiocytic (L&H) or popcorn
cells. L&H cells usually express the B-cell
marker CD20 and lack expression of
CD15 and CD30,3 the characteristic markers for cHL. Compared to cHL, more
patients will present with early or intermediate stage, are male and with less B
symptoms.4 The disease is commonly limited to one site (such as groin, neck, axilla)
and involvement of the mediastinum is
infrequent at the moment. NLPHL are not
generally included in cHL trials and treatment recommendations differ from those
of cHL. NLPHL is infrequent, there are no
randomized trials and the choice of treatment is difficult. The German Hodgkin
Study Group (GHSG) recently reviewed
patients included in several successive trials and compared the different treatment
approaches, such as extended field (EFRT),
involved field (IFRT) radiation and combined modality treatment (CMT) for
LPHL stage IA patients. In terms of remission, induction IF radiotherapy for stage
IA LPHL patients is as effective as EFRT or
CMT treatment. However, a longer follow-up is needed before final conclusion
can be made about optimal therapy.5
Other treatment options include chemo| 64 |
therapy and use of rituximab since the
L&H cells express CD20.6 Recently, some
have suggested that patients in complete
remission after biopsy could benefit from
observation with no further therapy.7 The
GELA/EORTC, GHSG and NCCN recommend IFRT for NLPHL in early favourable
stages. However, there is no randomized
trial to support this recommendation. The
diversity of treatment options available
for localized NLPHL, the good survival
and risk of late toxicities emphasize the
need for an intergroup randomized trial.
Prognostic factors to tailor the treatment of
early stage cHL
The Ann Arbour staging classification
with Costwold modifications8 is shown in
Table 1 and allows the distinction
between advanced and early stages. For
patients with advanced stage, The
International Prognostic Index defined
prognostic factors that are nowadays used
in all randomized trials.9 Recently, IPS has
also been applied to patients with earlystage disease.10,11 However in these times
of chemo-radiotherapy, early stage
patients are generally separated into two
categories (favorable and unfavorable) to
define treatment. But slightly different criteria are used by different groups (Table
2). Basically, more courses or more intensive chemotherapy is recommended for
unfavorable patients. Most trials using
chemotherapy alone were limited to
favourable patients. The question
whether E lesions are prognostically significant remains controversial since there
is a wide disagreement about E lesion definition.
Biological prognostic factors
In addition to commonly used prognostic factors, some investigators have studied biologic markers that might provide
additional prognostic information to these
clinical models. Several candidate molecules, including bcl-2 P53 and human germinal centre-associated lymphoma pro-
Table 1. Modified Ann Arbor staging system for Hodgkin
lymphoma.
Stage
Involvement
I
Single lymph node region (I) or one extralymphatic
side (IE)
Two or more lymph node regions, same side of the
diaphragm (II) and local extralymphatic extension plus
one or more lymph node regions same side
of the diaphragm (IIE).
Lymph node region on both sides of the diaphragm (III)
which may be accompanied by local extralymphatic
extension (IIIE)
Diffuse involvement of one or more extralymphatic
organs or sites.
II
III
IV
Symptoms
A
No B symptoms
B
Presence of at least one of the following:
1. Unexplained weight loss >10% baseline during
6 months prior to staging
2. Recurrent unexplained fever >38°
3. Recurrent night sweats
tein, have recently been proposed.12,13 The independent predictive power of these markers is controversial and none has gained wide acceptance in clinical
trials.
Combined modality treatment
Between 1950 and 1980, treatment strategy maximized the use of extensive radiotherapy (RT)
because historically it was considered the only curative method and less toxic than MOPP. The lessons
from this period should not be limited to the awareness of the late toxic effects but the high effectiveness of RT as a single agent should also be recognised. Since then several randomized trials have
shown better results with CMT as compared to RT
alone14, 15,16 (Table 3). In the GHSG HD7 study,15 the
patients receiving CMT had a better FFTF when
compared to RT alone. In the GELA/EORTC H7F
and H8F,16,17 similarly improved results for RFS were
obtained with CMT despite the reduction of the field
of radiotherapy to the site of originally involved
nodes (involved-field radiotherapy). It is now clear
that chemotherapy plus radiotherapy not only
improves relapse-free survival, but can also replace
Table 2. Early stage cHL risk factors in treatment groups.
RF
Treatment groups
Early stage favorable
Early stage unfavourable
Advanced stage
GELA/EORTC
GHSG
NCCN
1. Large MM
2. Age ≥ 50
3. B symptoms or ESR>50
4. ≥4 involved sites
1. Large MM
2. Extranodal disease
3. B symptoms or ESR≥50
1. Large MM/any >10 cm
2. B symptoms or ESR≥50
CS I-II with no RF
CS I-II with any RF
CS III-IV
CS I-II with no RF
CS I, CS IIA with any RF, CS IIB withC/D but without A/B
CS IIB with A/B, CS III-IV
No RF
Any RF
CS III-IV
* If B symptoms, ESR should be >30. Abbreviation : GELA: Groupe d’Etudes des Lymphomes de l’Adulte; EORTC: European Organization for Reasearch and
Treatment of Cancer; GHSG: German Hodgkin Lymphoma Study Group; NCCN: National Comprehensive Cancer Network; RF: risk factors; MM: mediastinal mass;
ESR: erythrocyte sedimentation rate; CS: clinical stage.
Table 3. Early stage cHL risk factors in treatment groups.
Trial
Treatment
RFS or FFTF
OS (%)
OS (years)
GHSG HD715
(617 pts)
EF
2 ABVD + EF
75
91
p<.001
94
94
P:ns
5
EORTC H7F16
(333 pts)
STLI
6 EBVP + IF
78
88
p=0.0113
92
p:ns
10
EORTC/GELA H8F18
(543 pts)
STLI
MOPP/ABV
80
99
SWOG #913314
(326 pts)
STLI
3 AV + STLI
81
94
p<.001
4
96
98
p=ns
3
RT: radiotherapy; CMT: combined modality therapy; GELA: Groupe d’Etudes des Lymphomes de l’Adulte; EORTC: European Organization for Reasearch and
Treatment of Cancer; GHSG: German Hodgkin Lymphoma Study Group; RFS: relapse free survival; FFTF: freedom from treatment failure; OS: overall survival;
ns:not significant; EF: extended field; IF: involved field; STLI: sub-total lymphoid irradiation.
Table 4. Randomized trials comparing CMT with chemotherapy alone.
Author
Longo29
Biti30
Pavlovsky31
Strauss32
Laskar33
Nachman34
Eghbali25
Meyer35
Design
MOPP vs MOPP + EF
MOPP vs MOPP + EF
CVPP vs CVPP + IF
ABVD vs ABVD +EF or IF
ABVD vs ABVD + IF
COP-ABV vs COP-ABV + IF
EBVP + IF vs EBVP
STNI +/- ABVD vs ABVD
N
106
99
277
152
99
362
578
399
Results
No difference
No difference
No difference in the favourable group
No difference
No difference
No difference
EFS p<.001
DFS p=0.006
EF: extended field, IF: involved field, STNI: sub-total nodal irradiation
radiotherapy as adjuvant treatment for sub-clinical
disease. Several trials have demonstrated that reducing the fields of radiotherapy from subtotal lymphoid
irradiation or EFRT to IFRT have produced similar
results with what seens to be less toxic effects (Table
4).18,19,20 In a randomized trial in Milan19 it was demonstrated that ABVD followed by extended field or
involved field RT produced similar results. The HD8
trial of the GHSG gave similar results, less acute toxicity (significant improvement for nausea, leukopenia, thrombopenia, pharyngeal and gastro-intestinal
effects) and also a trend toward fewer secondary cancers (24 in the EF arm (4.5%) and 15 in the IF arm
(2.8%)). A subset analysis of this trial also suggested
that older patients experienced poorer outcome in
the extended field arm (OS, 59% vs 81%, p=0.008).20
The definition of IFRT remains unsettled. A recent
publication by Girinsky et al.21 suggested that fields
that target only the involved lymph nodes, as
defined by using modern imaging technologies, can
be used to further reduce the field size of radiation
from the current IFRT to involved-node radiation
therapy. The authors based their suggestion on the
fact that most of recurrences occur in the original
nodal sites. This was confirmed by Shahidi et al.22
from Royal Marsden, who reported in a retrospective failure analysis of 61 patients with stage I and II
Hodgkin's lymphoma treated without radiation that
83% of the recurrences developed in original sites of
disease (45% as the only site). In addition to smaller
radiation fields, the radiation dose used in modernday therapy has also been reduced from 45-54 Gy to
30 Gy or even lower in the modern randomized trials. Koontz et al.23 retrospectively compared CMT
consisting of chemotherapy and low-dose IFRT
(mean dose 25.5 Gy) to definitive radiation with a
mean dose of 37.9 Gy. The authors found the CMT
to be equally effective in curing patients with earlystage Hodgkin's lymphoma (20-year overall survival,
83% for CMT vs 70% for radiation alone) with
fewer cardiac complications and second malignancies. The GHLSG HD10 again confirmed the safety
of lowering the radiation dose. In this trial, 1,370
patients with favourable early-stage Hodgkin's lymphoma were randomly assigned to IFRT of 30 Gy or
20 Gy, and at their 4-year interim analysis, the freedom from treatment failure was similar.24 The
EORTC/GELA H9F trial also compared IFRT 36Gy
vs 20 Gy and showed no significant difference with
a follow-up of 51 months.25 However, since the final
analyses of these two trials (HD10 and H9F) have
not yet been carried out, the recommended dose
remains 30 Gy.
The temptation to omit radiotherapy
With more than 90% of patients cured by the current treatment, survival is more influenced by late
toxicity, and more precisely, by second cancers. The
currently used chemotherapy, ABVD, results in very
low toxicity with no demonstrated increase for secondary leukaemia or solid tumours. It has been
known for more than 20 years that the use of radiation therapy is associated with significant rates of
second cancers presenting 10 or more years after
treatment completion. We now know that thoracic
radiation in women treated under the age of 30 years
results in a very high rate of breast cancer of approximatly 30% at 30 years following treatment. This risk
is dose dependent and is much lower in women who
received alkylating agent chemotherapy with no hormone replacement therapy.26 We also know that
after thoracic radiation for Hodgkin lymphoma
heavy smokers have a 20 times higher risk of lung
cancers while light or non-smokers have a 7 times
higher risk.27 Higher dose and volume of radiation
increases these risks. The use of smaller and better
defined radiation volumes allowed the utilization of
more conformal radiation therapy based on
improved imaging, and when indicated, tools such as
intensity modulated RT. However, so far studies
have not demonstrated a decrease in late secondary
cancers for long-term survivors of cHL.28 In a recently
published meta-analysis, IF-RT versus EF-RT (19 trials, 3,221 patients), there was no significant difference in secondary malignancy risk (p=0.28) although
more breast cancers occurred with EF-RT (p=0.04
and OR=3.25).40 Given this increased risk for secondary toxicities, several groups have proposed to omit
RT and give chemotherapy, mostly ABVD, alone.
Several studies have published results showing
similar outcomes with chemotherapy alone compared to CMT (Table 4).29-34 Four of these trials used
chemotherapy that has been shown to be inferior to
the current standard ABVD and cannot therefore
been used to give current guidelines. Furthermore
some of these four trials were too small to detect
important differences. The CCG trial based on a paediatric population only34 showed no difference in an
intent-to-treat analysis. An analysis as-treated was
also performed and showed a 3-year EFS of 93% for
those who received RT and 85% for observation
(p=0.024).Two trials31,34 were subset analysis.
The two largest trials had the power to detect significant differences. In the EORTC/GELA H9F,25 after
6 cycles of EBVP (epirubicin, bleomycin, vinblastine,
prednisone), CR/CRu patients were randomized
between 36 Gy IF-RT, 20 Gy IF-RT and no RT. From
September 1998 to May 2004, 783 patients were
enrolled and 578 randomized after EBVP. Inclusion of
patients in the no-RT arm was stopped early because
stopping rules were met (that is > 20% of events).
The 4-year EFS were 88%, 85% and 69% for 36 Gy,
20 Gy and no RT respectively. Median follow-up is
4.2 years. In the National Cancer Institute of Canada
trial,35 in comparison with ABVD alone, 5-year freedom from disease progression is superior in patients
allocated to radiation therapy (p=0.006; 93% v 87%).
No differences in event-free survival (p=0.06; 88% v
86%) or overall survival (p=0.4; 94% v 96%) were
observed. In a subset analysis comparing patients
stratified into the unfavorable cohort, freedom from
disease progression was superior in patients allocated
to combined-modality treatment (p=0.004; 95% v
88%): No difference in overall survival was detected
(p=0.3; 92% v 95%). Of the 15 deaths observed, nine
were attributed to causes other than Hodgkin's lymphoma or acute treatment-related toxicity. In patients
with limited-stage Hodgkin's lymphoma, no difference in overall survival was observed between
patients randomly assigned to receive treatment that
includes radiation therapy or ABVD alone. Although
5-year freedom from disease progression was superior in patients receiving radiation therapy, this advantage is offset by deaths due to causes other than progressive Hodgkin's lymphoma or acute treatmentrelated toxicity.
Current trials
The ongoing GHSG HD13 in favourable early
stage randomly assigns patients to four arms: 2 cycles
of ABVD, ABV, AVD or AV followed by IFRT 30 Gy.
Enrolment is ongoing but the AVD and AV arms
were closed due to an excessive number of Hodgkin
events. In the unfavourable group of GHSG HD11
HD11, patients are randomized between 4 ABVD vs
4 BEACOPP baseline followed by IFRT 20 vs 30 Gy.
Enrolment is completed with no difference between
the arms reported so far. In North America, earlystage unfavourable patients are randomized to
receive either 6 ABVD and IFRT vs Stanford V followed by IFRT to sites ≥ 5 cm.
Using PET to tailor the treatment?
After completion or during treatment, restaging by
physical examination and CT-scan is not useful to
predic ultimate outcome. Using F-18 fluorodeoxyglucose positron emission tomography (FDG-PET) scan
in the evaluation of treatment response in HL, the
negative predictive value is high (81-100%) showing
the ability of FDG-PET to identify patients with
excellent prognosis. The positive predictive value is
more variable (25-100%). When results of FDG-PET
are interpreted in combination with clinical history
and CT-scan, the positive predictive value increases
to >85%, residual activity being strongly suggestive
of active Hodgkin residue36) Treatment-induced
tumour cell death or growth arrest reduce the FDG
uptake in non-Hodgkin’s lymphoma as early as 7
days after start of therapy. There is less data for cHL.
Three studies merit special attention with regard to
the predictive value of early response evaluation:
1. In a retrospective study by Hutchings et al. FDGPET was performed after 2-3 cycles of chemotherapy
in 85 cHL patients. The interim PET scans were negative in 63 patients, showed minimal residual uptake
in 9 patients and were positive in 13 patients. The 5year progression free survival was 92%, 88% and
42% respectively.37
2. These findings were confirmed in a prospective
study in 77 cHL patients. PET scans were performed
after 2 cycles of chemotherapy. Two out of the 61
PET-negative patients experienced treatment failure,
in comparison with 10 out of 16 PET-positive
patients. With a median follow-up of 20 months,
highly significant associations were reported
between early interim FDG-PET and progression free
survival (p<0.0001) and overall survival (p<0.01).38
3. In the Gallamini study,39 the end-point was the
predictive value of PET-2 on 2-year progression-free
survival and 2-year failure-free survival. The PET-2
was positive in 20 out of 108 patients. Seventeen progressed during therapy, one relapsed and two
remained in CR. By contrast, 85/88 (97%) patients
with a negative PET-2 remained in CR. Thus, the
positive predictive value of a PET-2 was 90% and the
negative predictive value was 97%. The sensitivity,
specificity and overall accuracy of PET-2 were 86%,
98% and 95% respectively. The 2-year probability of
2 cycles ABVD
RANDOMIZATION
STRATUM F
FAVORABLE
FDGPET
Hodgkin’s
lymphoma
stage I/II
any outcome
of PET scan
negative
2 cycles ABVD
untreated,
age 15 to 70
2 cycles ABVD
FDGPET
RANDOMIZATION
STRATUM U
UNFAVORABLE
any outcome
of PET scan
negative
2 cycles ABVD
2 cycles escalated BEACOPP
IN-RT 30 Gy
(+ boost 6 Gy residual)
2 cycles ABVD
IN-RT 30 Gy
4 cycles ABVD
FDGPET
positive
ABVD: doxorubicin (adriamycin), bleomycin, vinblastine, dacarbazine Esc. BEACOPP: bleomycin,
etoposide, doxorubicin (adriamycine), cyclophosphamide, vincristine, procarbazine, prednisone INRT - Involved-Node Radiation Therapy.
2 cycles ABVD
FDGPET
positive
No LP
nodular!
1 cycle ABVD
IN-RT 30 Gy
Second
Registration
2 cycles escalated
BEACOPP
IN-RT 30 Gy
Figure 1. The H10 GELA/EORTC trial.
failure-free survival for PET-2 negative and for PET-2
positive patients was 96% and 6% respectively (log
rank test = 116.7, p<0.01).
From these data it can be concluded that early
assessment of response to chemotherapy with FDGPET is an accurate predictor of progression free survival and overall survival in Hodgkin lymphoma.
Therefore, in the currently ongoing EORTC/GELA
H10 trial, the early response to treatment analyzed
by FDG-PET scan, is used as a guidance to early treatment adaptation.
Patients with a negative FDG-PET after 2 cycles of
ABVD constitute the good-risk group for whom
treatment burden can be reduced (Figure 1).
Conclusions
The current recommendations for the treatment of
early stage cHL is CMT, including a short course of
ABVD followed by IFRT. Given the excellent results
produced by this approach and the incidence of secondary cancers, further trials will focus on a further
reduction of treatment. This will be achieved by
attempting to tailor treatment to initial response.
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4. Nogová L, Reineke T, Eich HT et al. Lymphocyte-predominant
and classical Hodgkin's lymphoma--comparison of outcomes.
Eur J Haematol Suppl 2005;:106-10.
5. Nogová L, Reineke T, Eich HT et al. Extended field radiotherapy, combined modality treatment or involved field radiotherapy for patients with stage IA lymphocyte-predominant
Hodgkin's lymphoma: a retrospective analysis from the
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6. Ekstrand BC, Lucas JB, Horwitz SM et al. Rituximab in lymphocyte-predominant Hodgkin disease: results of a phase 2
trial. Blood 2003;10:4285-42.
7. Pellegrino B, Terrier-Lacombe MJ, Oberlin O et al.
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therapeutic abstention after initial lymph node resection--a
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8. Lister TA, Crowther D, Sutcliffe SB et al. Report of a committee convened to discuss the evaluation and staging of patients
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intergroup trial of subtotal lymphoid irradiation versus doxorubicin, vinblastine, and subtotal lymphoid irradiation for
stage IA to IIA Hodgkin's disease. J Clin Oncol. 2001;19:423844.
Sieber M, Franklin J, Tesch H et al. Two cycles of ABVD plus
extended field radiotherapy is superior to radiotherapy alone
in early stage Hodgkin’s disease: results of the German
Hodgkin’s Study Group Trial HD7. Blood 100:A341, 2002.
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therapy for clinical stage I or II Hodgkin's lymphoma: longterm results of the European Organisation for Research and
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is equally effective and less toxic compared with extendedfield radiotherapy after four cycles of chemotherapy in
patients with early-stage unfavorable Hodgkin's lymphoma:
results of the HD8 trial of the German Hodgkin's Lymphoma
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Hagenbeek A, Eghbali H, Ferme C et al. Three cycles of
MOPP/ABV hybrid and involved-field radiotherapy irradiation is more effective than subtotal nodal irradiation in
favourable supradiaphragmatic clinical satge I-II Hodgkin’s
disease: preliminary results of the EORTC-GELA H8-F randomized trial in 543 patients. Blood 2000;96, abstract 575.
Bonadonna G, Bonfante V, Viviani S et al. ABVD plus subtotal
nodal versus involved-field radiotherapy in early-stage
Hodgkin's disease: long-term results. J Clin Oncol.
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Klimm B, Eicht HT, Haverkamp H et al. Poorer outcome of
elderly patients treated with extended-field radiotherapy compared with involved-field radiotherapy after chemotherapy
for Hodgkin's lymphoma: an analysis from the German
Hodgkin Study Group. Ann Oncol 2006;2006 Oct 27;[Epub
ahead of print].
Girinski T, van der Maazen R et al. Involved-node radiotherapy (INRT) in patients with early Hodgkin lymphoma: concepts and guidelines. Radiother Oncol 2006 Jun;79:270-7.
Epub 2006 Jun 22.
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chemotherapy alone for stage I and II Hodgkin's disease.
Radiother Oncol 2006;78:1-5.
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J Clin Oncol 2006;24:605-11.
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combined modality treatment intensity in early stage
Hodgkin's lymphoma: Interim analysis of a randomized trial
of the German Hodgkin's Study Group (GHSG). J Clin Oncol
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Radiation Dose Levels after EBVP Regimen in Favorable
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Travis LB, Hill D, Dores GM, et al. Cumulative absolute breast
cancer risk for young women treated for Hodgkin lymphoma.
J Natl Cancer Inst 2005;97:1428-37.
Travis LB, Gospodarowicz M, Curtis RE, et al. Lung cancer following chemotherapy and radiotherapy for Hodgkin’s disease.
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malignant neoplasms among long-term survivors of Hodgkin's
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Biti GP, Cimino G, Cartoni C, et al. Extended-field radio-therapy is superior to MOPP chemotherapy for the treatment of
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randomized clinical trial of doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) followed by radiation therapy
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Hodgkin disease. Blood. 2004;104:3483-9.
Laskar S, Gupta T, Vimal S, et al. Consolidation radiation after
complete remission in Hodgkin’s disease following six cycles
of doxorubicin, bleomycin, vinblastine, and dacarbazine
chemotherapy: is there a need? J Clin Oncol 2004;22: 62-8.
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Meyer RM, Gospodarowicz MK, Connors JM, et al.
Randomized comparison of ABVD chemotherapy with a
strategy that includes radiation therapy in patients with limited-stage Hodgkin’s lymphoma: National Cancer Institute of
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Oncology Group. J Clin Oncol 2005;23: 4634-42.
Martin R. Weihrauch, Daniel Re, Klemens Scheidhauer et al.
Thoracic positron emission tomography using 18F-fluorodeoxyglucose for the evaluation of residual mediastinal
Hodgkin disease. Blood 2001;98: 2930-4.
NG Mikhaeel, M. Hutchings, P. A. Fields et al. FDG-PET after
two to three cycles of chemotherapy predicts progression-free
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Hutchings M, Loft A, Hansen M et al. FDG-PET after two
cycles of chemotherapy predicts treatment failure and progression-free survival in Hodgkin lymphoma. Blood 2006;107:
52-9.
Gallamini A, Rigacci L, Merli F et al. The predictive value of
positron emission tomography scanning performed after two
courses of standard therapy on treatment outcome in
advanced stage Hodgkin's disease. Haematologica 2006;91:
475-81.
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Ferme C, Eghbali H, Hagenbeek A et al. MOPP/ABV hybrid
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modalities. Preliminary results of the the EORTC/GELA H8U
randomized trial in 955 patients. Blood 2000;96:Abstract 576.
Treatment of relapsing/refractory patients with
Hodgkin lymphoma
P. Brice
Hôpital Saint Louis,
Paris, France
2007;1:70-75
espite major improvements in the
cure rate for Hodgkin’s disease
(HD), 10 to 15% of patients with
localized and 25 to 30% with disseminated classical Hodgkin lymphoma (HL) fail
to respond or relapse after primary conventional treatment with chemotherapy
alone or combined with radiotherapy.1,2
Distinctions must be made between
refractory patients or induction failure,
defined as disease regression of less than
50% after 4 to 6 cycles of anthracyclines
containing chemotherapy (CT) or disease
progression during induction treatment or
within 90 days after the end of first line
treatment. These refractory patients represent 2 to 5% of stage I/II and 5 to 10% of
stage III/IV: These rates are lower than
those in regimens like the increased-dose
BEACOPP developed by Diehl for the
GHSG.3 In all series, refractory patients
have a poorer outcome than those achieving a complete remission.2 Relapsing
patients will receive second line chemotherapy but the controversy surrounding
the optimal timing (first vs second relapse)
of autologous stem cell transplantation
(ASCT) has largely subsided despite the
benefits shown in a large randomised
study.4 Radiation therapy (RT) is often
omitted in first line treatment,5,6 on the
contrary RT may have an increase place in
relapsing patients.7 Some other patients
with refractory disease or early and
advanced relapse maintain a poor prognosis and for these patients an intensive regimen with tandem HDT and ASCT or
allogeneic transplant can be proposed.8,9
D
Prognostic factors at relapse
Since1990, many mostly retrospective
publications, have shown some adverse
prognostic factors at relapse including
advanced stage, time to relapse, B symptoms, and extranodal disease.1,10 In the
largest series to date, Josting et al. from the
GHSG developed a prognostic score for
relapsed HL based on the outcome of 471
patients who failed initial therapy.11 In
| 70 |
multivariate analysis, independent risk
factors were time to relapse (″ 12 mths. vs
> 12 mths), clinical stage at relapse (stage
III/IV) and anemia at relapse (males <12
g/L; females <10.5 g/L). FF2F were estimated at 45%, 32% and 18% for patients
with prognostic scores of 0-1, 2, and 3
respectively. We therefore used only 2
adverse prognostic factors on a French
series of patients receiving HDT and
ASCT for first relapse of HL.12 For 214
patients in first relapse, we analyzed two
significant prognostic factors, the interval
between the end of treatment and relapse
and the site of relapse (nodal versus EN).
Three subgroups were defined: 0 factor
(n=59), 1 factor (n=125), 2 factors (n=30).
Their OS rates at 4 years were respectively, 93, 59 and 43%, and differed significantly (p<0.001). Moskowitz et al., presented one of the first prospective study in
second line treatment for HL and identified 3 factors before the initiation of
relapse treatment that predicted for outcome: B symptoms, extranodal disease,
and complete remission duration of less
than one year.12 EFS rates in intention to
treat were 83% for patients with 0-1 factor, 27% for patients with 2 factors and
10% for patients with 3 factors. In 1997, a
French prospective multicentric trial was
started in 245 patients at first HL
relapse/progression. Patients were stratified according to 2 prognostic factors at
relapse (interval between the end of treatment and first progression before 12
months and stage III/IV at relapse or
relapse in previously irradiated site) in 2
groups:
- Poor prognosis risk relapse with the 2
adverse prognostic factors or primary
refractory disease;
- Intermediate risk relapse with only one
factor.
Results confirmed our prognostic model
with 2 factors. At a median follow-up of
51 months, event free survival is at 46% in
the poor risk group (n=150) and 76% in
the intermediate risk group (n=95).
1
Table 1. Salvage chemotherapy in HL.
Linch
Brice
Rodrigues
Moskowitz
Schmitz
Ferme
Josting
Regimen
Mini-BEAM
IVA
ASHAP
ICE
dexaBEAM
MINE
DHAP
No.
Year
ORR
of patients of publication
55+
43°
56
65
161+
83
57
1993
1999
1999
2001
2002
2002
2005
82%
60%
66%
88%
80%
74%
80%
EFS
53 vs 10%
45% at 2 yrs
35%
58% at 3 yrs
55% at 3 yrs*
Vs 34%
0.8
Survival Probability
Author
0.6
0.4
0.2
0
0
46% at 4 yrs
25% at 2yrs
+Randomization for HDT° refractory and early relapse; * for patients receiving
HDT and ASCT.
Grupe 1
Grupe 2
Logrank p>0.0001
20
40
60
80
1000
months
Group 1
Group 2
No. of Subjects
Event
150
53% (79)
95
25% (24)
Censored
47% (71)
75% (71)
Median Survival (95% CL)
40.8 (18.0 NA)
NA (63.5 NA)
Overall survival is at 57% versus 85%.
(Morschhauser et al. submitted for publication)
(Figure 1).
Figure 1. EFS in relapsing HL patients according to stratification risk groups (group 1: poor risk group, group 2: intermediate risk group), the French GELA/SFGM prospective
study.
Salvage chemotherapy regimens
No randomized trials exist to compared the effectiveness of salvage regimens in second line treatment
for HL. These regimens have been various according
to the initial regimen given. Most patients are now
treated with ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine) and cannot receive second
line chemotherapy with doxorubicin if the cumulative dose is at 400 mg/m2. Intensive pretransplant regimens such as mini-BEAM (carmustine, etoposide,
cytatbine, melphalan) and dexaBEAM (dexamethasone, carmustine, etoposide, cytarabine, melphalan)
have a significant hematologic toxicity and a death
rate of 2-5%, that is similar to rates seen with HDT
and ASCT.4,14 Most other centers have used platinumbased regimens such as DHAP (dexamethasone,
high-dose cytarabine, cisplatin), ASHAP (doxorubicin, methylprednisolone, high-dose cytarabine, cisplatin) or ifosfamide/etoposide-based regimens as
MINE (mitoguazone, ifosfamide, vinorelbine, etoposide), IVA (ifosfamide, etoposide, doxorubicin), ICE
(ifosfamide, carboplatin, etoposide).2,8,11,13,15 Results
with overall response rate (complete response and
partial response > 50%) of some prospective salvage
regimens are presented in Table 1. Response rates
ranged from 60 to 80%. Comparison of results is difficult due to the difference in prognostic groups with
patients having refractory disease, early, late and
multiple relapse. Response rate to salvage chemotherapy is different according to prognostic factors at
relapse. With the MINE regimen, the overall
response rate was at 60% for induction failure versus
89% for relapsing patients.2 The ideal salvage regimen should not add cumulative non hematologic
toxicity (for example, cardiac or pulmonary) because
this may lead to chemosensitivity which is always
correlated to improved event free survival. Patients
should be evaluated early after 2 cycles of pretransplant regimen and the regimen must be changed if
the response is in below partial response. Due to its
lower efficacy and higher toxicity, MOPP is not used
in first line therapy but can be considered for use at
relapse if a peripheral blood progenitor-cell collection
(PBPC) is planned.16 BEACOPP standard or increased
dose can also be useful as second line therapy.3
Radiation therapy
Josting reported the most important study in this
setting of salvage RT alone in 100 patients with primary progressive or relapsed HL.7 With a median
observation time of 52 months, 5 year FFTF and survival were 28 and 51% respectively. Involved field
RT can also be given after HDT with BEAM regimen
and ASCT. This is important for patients with
relapsed/ refractory disease and bulky mediastinal
involvement. RT can be included in the conditioning
regimen using accelerated fractionation radiotherapy
either as total lymphoid irradiation (TLI) or as an
involved field followed by high-dose chemotherapy
and bone marrow infusion.17 One hundred and fiftysix patients were treated from 1985 to 1994. At a
median follow-up of 7 years, the EFS rate is 42%, and
no relapses have occurred later than 36 months after
transplantation. This strategy was confirmed in 65
prospective patients with refractory (n=22) or
relapsed (n=43) HL.13 PBPC from responding patients
to ICE were collected, and the patients were given
accelerated fractionation involved field radiotherapy
(IFRT) followed by cyclophosphamide-etoposide and
either intensive accelerated fractionation total lymphoid irradiation or carmustine and ASCT. The EFS
rate at a median follow-up of 43 months, as analyzed
Table 2. High-dose therapy and ASCT, a selection of large
retrospective studies.
Year
Author
Status
of disease
1993
Bierman Relapse IF
Number Conditioning
EFS
FFTF
Survival
128
CBV
25% 45% at 5 yrs
Relapse
280
BEAM 60%
60% 66% at 4 yrs
1997
EBMT
Relapse
Sweetenham
139
BEAM 58%
45% 50% at 5 yrs
1999
EBMT
Sweetenham
IF
175
BEAM 47%
32% 36% at 4 yrs
1999
André
IF
86
BEAM 51%
25% 35% at 5 yrs
2001
Sureda
FirstCR
Relapse IF
494
CBV 53%
45% 54% at 5 yrs
Relapse IF
184
1997
Brice
2004
Popat
NA
29% 34% at 10 yrs
IF: Induction failure.
by intent to treat, was 58%.This prospective study
also demonstrates the efficacy and feasibility of integrating higher-dose radiotherapy into an ASCT treatment program.
High dose therapy and autologous stem cell
transplantation
Results
The use of HDT and ASCT is now considered the
standard of care for most patients with refractory and
relapsed HL. Two randomized trials in this setting
showed a significant benefit in freedom from second
failure. The first, published in 1993, was a BNLI trial
comparing relapsed HL a salvage regimen miniBEAM
alone or followed by HDT (BEAM) and ASCT in 40
patients. Despite the small numbers, there was a significantly improved 3-year EFS (53% vs 10%) and
the trial ended prematurely.14 A similar trial was conducted by the GHSG and the EBMT with more
patients. It consisted of an upfront randomisation,
dexaBEAM was used as pretransplant regimen, 2
cycles before restaging and patients received either 2
other cycles of DexaBEAM or stem cell harvest and
BEAM HDT followed by autotransplant. In this trial,
161 patients were randomised (21% with multiple
relapse) but the dexaBEAM regimen was very toxic
and only 117 chemosensitive patients could receive
the procedure and are evaluable for the randomisation. Freedom from treatment failure (FFTF) at 3
years was significantly better for patients given
BEAM-HDT (55% vs 34%) but no differences were
observed in survival even with an update at ASCO
2005 with 7-year survival rates.4 The lack of survival
difference in these two trials is difficult to interpret
and may be related to the fact that most patients
received HDT and ASCT for subsequent relapses.
Some of the most important retrospective series of
HDT in relapse/refractory HL are shown on Table 2.
Toxic deaths were always below 5%. Survival rates
ranged from 25 to 60% for EFS and from 35 to 66%
for overall survival and showed that disease status
(chemosensitivity) before HDT and ASCT is the
most important prognostic factor for final outcome.12,-14,18-22 Patients not achieving a complete remission after first line treatment (for example, induction
failure) have the worse outcome when compared to
relapse but high dose chemoradiotherapy regimen
and ASCT can be associated with an EFS at 45%.23
Toxicity
HDT and ASCT are associated with a prolonged
exposure of patients to cytotoxic agents and may
lead in young patients to an increase of late toxicities.
In a study, by André et al. 464 patients receiving
ASCT were matched with 1,164 patients receiving
conventional treatment. With a follow-up of 3 years,
results did not show an increase in secondary
MDS/leukaemia, but an excess of solid tumors in
patients receiving HDT.24 In multivariate analysis, the
most important adverse prognostic factor was
relapse and it appeared that the number of
chemotherapy lines was responsible for exposing the
patient to long term toxicity rather than the conditioning regimen itself. Another British Columbia
study examined retrospectively 1,732 patients treated in their area during a 26 years period and analysed
the occurrence of second are solid tumors, comparing
the 202 patients who received HDT to others.25 The
cumulative incidence of developing any second
malignancy 15 years after therapy was 9% and did
not differ between those receiving HDT or not. The
same team analysed the long term toxicity of the first
100 patients that underwent HDT for HL. Fifty-three
patients were still alive with a median follow-up of
11 years.26 The major cause of death was progression
(35%), followed by treatment related-toxicity (17%),
but among living patients Karnofsky was >90%.
Another 47 further patients suffered hypogonadism
(n=20), hypothyroidism (n=12), common infections
(n=10), anxiety or depression (n=7) and cardiac disease (n=5).
Tandem HDT and ASCT
A French multicentric prospective study first presented results of tandem transplantation in patients
selected for their poor prognosis (induction failure,
early and disseminated relapse). The first conditioning regimen was a CBV mitoxantrone or a BEAM and
the second included total body irradiation at 12 Gys
in 6 fractions or Busulfan 16 then 12 mg/kg and highdose cytarabine and melphalan.8 Preliminary results
on 43 patients with a median follow-up of 24 months
showed that only 74% of patients received the tandem transplant with one toxic death. On intent to
treat analysis the EFS was at 40% and the survival at
53%. Results will soon be published on 150 patients
and have been presented at the EBMT 2005. They
show a 5 year EFS of 46% (CI 37-54%) overall survival of 57% (CI 48-65%) with a 4% toxic death rate.
Allogeneic stem cell transplantation
The role of allogeneic stem cell transplantation
(allo-SCT) in patients with relapsed or refractory HL
has been highly controversial. However, several
series have suggested that allo-SCT seems to be associated with a clinically significant graft-versus-HL
effect and a lower relapse rate with respect to ASCT
retrospective analyses.27,28 Transplantation demonstrated disappointing results with allo-SCT in HL
because of an extremely high transplant-related mortality (TRM) that was mainly associated with acute
and chronic graft-versus-host disease (GVHD) and
concomitant infectious episodes. Akpek et al. reported no difference in relapse rates, EFS and survival for
53 patients undergoing allo-SCT compared to 104
patients having auto-SCT for relapsed HL.29 The
recent emphasis on reduced intensity conditioning
(RIC) with allogeneic transplant has renewed interest
in the use of allo-SCT for relapsed/refractory HL.30
Many studies are limited in number of patients with
HL and the follow-up is often short. In a recent
prospective study of 40 patients from the Spanish
group they reported with an RIC protocol (fludarabine 150 mg/m2 intravenously plus melphalan 140
mg/m2) a TRM at one year at 25%, with an EFS and
survival at 2 years respectively at 32% and 48%.31 A
graft versus-lymphoma effect has been suggested
with reports of response to DLI following disease
relapse or progression.29,31 Allo-RIC should be considered an effective therapeutic approach for patients
who have had treatment failure with a previous
autologous hematopoietic stem cell transplantation.
New agents
Monoclonal antibodies
The search for effective monoclonal antibodies for
HL has not been as successful as in B-cell lymphomas. The target antigen has been mostly the
CD30 which is expressed in all cases of classic HL
and expression on normal tissue is limited. As reported ASH 2004, clinical activity of these antibodies,
both chimeric or humanised has been disappointing.
Other approaches to increase the benefit of targeted
therapy include immunoconjugates and radiolabeled
antibodies. The German study group treated 22
patients with relapsed/refractory HL with a murine
anti-CD30 monoclonal antibody labelled with
Iodine-131. Seven patients experienced grade 4
degrees hematotoxicity 4 to 8 weeks after treatment.
Response included one complete remission and five
partial remissions.32 The antiCD20 antibody rituximab has been seen to be active in nodular lymphocytic predominance HL but its activity in classic HL
(even expressing CD20) has not yet been confirmed.33 Other areas will be explored in the use of
immunoconjugates or with the association of monoclonal antibodies and chemotherapy.
Chemotherapy agents
The good prognosis of HL doesn’t encourage the
search for new chemotherapeutic agents and patients
are exposed very late to experimental drugs. Gemcitabine has been widely used in relapsed HL. A
phase II study which enrolled 23 patients found a
response rate of 39% with 9% of complete remission
and a median duration of response at 6.7 months.34
Vinorelbine has shown efficacy in HL and is included
in the relapse protocol of the GELA group for HL
with the MINE regimen.2 Presented at the EHA 2006,
our results with eloxatine combined with ifosfamide
and etoposide showed a 66% response rate in 25
relapsed/ refractory HL patients with a low toxicity
profile. Results were, however, disappointing in
refractory patients.
Therapeutic guidelines for patients relapsing after ABVD
(alone or combined modality)
Our guidelines are based on 2 simple prognostic
factors:
- the time to relapse;
- nodal and outfield site.
Other parameters can be taken account such as Bsymptoms or low hemoglobin level to include
patients in the poor prognosis group.10,11 Response to
second line therapy should be monitored on FDGPET scans which are very efficient in HL.35
Favorable relapse
This group represents those rare and occurring at
least one year after the end of first line treatment.
These patients can be treated with conventional
doses of chemotherapy and we suggest a standard
chemotherapy like BEACOPP or MOPP/ABV rather
than second line ABVD to avoid cumulative doses of
doxorubicin > 400 mg/m2 (with particular caution for
patients who have also received mediastinal RT).3,36
The number of cycles may range from 4 to 6 according to toxicity and response and should be followed
by involved field RT (30/36 Gys).
Intermediate risk relapse
Patients with only one adverse prognosis factor
should receive HDT with ASCT due to the excellent
results of this procedure in this setting with a possi-
bility of cure with an acceptable toxicity.12,19 Pretransplant regimens should avoid cumulative doses
of doxorubicin and aim to obtain chemosensitivity
before HDT, for example, in this intermediate group
CRu with negative FDG-PET scans. All the regimens
previously described are suitable for consideration. If
an initial response is not satisfactory a second line
regimen should be given to increase response. Most
of these regimens can achieve PBPC mobilisation.
BEAM is the most widely used conditioning regimen
in relapsed lymphoma and there is no data supporting the use of TBI in this setting.37 By contrast,
involved field RT can be given after HDT in nodal
sites of relapse, generally if bulky (mediastinum).
Poor prognosis relapse
This is the largest group and includes:
- induction failure or refractory patients;
- poor prognosis relapse at least early and extranodal
or stage IIIB or with significative anemia.
For those patients who received no more than 6
cycles of ABVD, we suggest treating them with
increased-dose BEACOPP 2 to 3 cycles as second line
regimen.3 This regimen can mobilize PBSC and represent one of the most effective polychemotherapies
in advanced HL. But it is associated with significant
toxicity. For other patients second line regimens
include those previously described, such as platinumbased regimens or ifosfamide/etoposide regimens.
The aim is to achieve at least a partial response >
50% before HDT. We suggest performing tandem
HDT for patients not in CR before first HDT. In
some cases when an HLA identical donor is available,
RIC-alloSCT can be discussed.8,37 The first HDT aims
to induce a complete remission and the second HDT
to prevent relapse. But often refractory patients
progress early after HDT. In these cases, they should
proceed to experimental therapies.
Multiple relapses
Despite improvements in first and second line
treatment, some patients continue to relapse and
need further treatment. For patients relapsing less
than one year after HDT, RIC-allo could be offered
when an HLA identical donor is available and if a
good response has been achieved with third line regimen. For the remaining patients third line regimens
should use different drugs to avoid cumulative toxicity from doxorubicine or beomycin and RT can be
given in the relapse outfields. All data regarding second auto-SCT for relapsed HL are from general
observations with small series and an increased rate
of late treatment related mortality. This option
should, therefore, be limited to patients, who
achieved a long CR after their first transplant.38
Conclusions
There is an increased possibility of cure among
patients with relapsed HL this can be estimated at
approximately 50% with HDT and ASCT. All results
are improved in chemosensitive patients and late
relapses. Despite this relatively favourable prognosis
for a relapsing malignancy, further attempts to
improve the outcome of these patients should be
made. New approaches are needed for chemoresistant HL where there is a less than 30% chance of
long-term remission even with auto or allo-SCT. The
data of allo-SCT are preliminary and limited by the
chemosensitivity required before procedure before
an allogeneic effect is seen. The search for new drugs
in this setting is important but problematic and
patients with HL enter protocols very late due to the
usually good disease prognosis. Relapsing HL
patients are also more exposed to long term toxicity
for example, second malignancies and cardiac toxicity, and the objective shoul be the early identification
of poor responders.
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What’s New in T-cell non-Hodgkin’s Lymphomas?
Pathology and genetics of T-cell lymphomas
A. Chott
A-I. Schmatz
E. Kretschmer-Chott
L. Müllauer
B. Streubel
Department of Pathology, Medical
University of Vienna,
Vienna, Austria
2007;1:76-82
-cell neoplasms can be divided into
two categories depending on the
developmental stage of the lymphoma cell. Precursor T lymphoblastic
leukemia/lymphoblastic lymphoma is a
neoplasm of lymphoblasts committed to
the T-cell lineage reflecting early, usually
terminal deoxynucleotidyl transferase
(TdT)-positive thymic developmental
stages, whereas post-thymic or peripheral
T-cell lymphomas display a morphologic
appearance and phenotype consistent
with mature T-cells. Because of some phenotypic and functional overlap between
mature T-cells and natural killer (NK) cells,
the neoplasms derived from these cell
types are considered together in the WHO
classification (Table 1).1 This review will
concentrate on the more common mature,
peripheral T-cell lymphomas (PTL) but also
include some of the rare entities. In particular, the PTLs printed in bold type in Table
1 will be discussed in detail. Genetic
analysis of these malignancies is required
to identify pathogenetic genes which can
define specific subtypes of disease. The
first steps in this genetic analysis have
already been taken and are summarized
below.
T
General features of peripheral T-cell
lymphomas
Peripheral T-cell lymphomas are rare.
They account for roughly 10% of all nonHodgkin lymphomas (NHL) on a worldwide basis.2 The most common subtypes
are PTL, unspecified (PTL-u)(3.7%), and
anaplastic large cell lymphoma (ALCL)
(2.4%). In general, T-cell lymphomas are
more common in Asia with the virus
HTLV-1 representing the main risk factor
for the development of adult T-cell
leukemia/lymphoma (ATLL) in Japan.
Nasal NK/T-cell lymphomas are also
much more common in Asians than they
are in other races, accounting for 8% of
cases in Hong Kong, and show an association with Epstein-Barr virus.3 Enteropathy-type T-cell lymphoma arises against
| 76 |
the same genetic background as that predisposing to celiac disease and is therefore
more common in Northern Europe.4
Classification of peripheral T-cell lymphomas
PTLs are characterized by extreme morphologic diversity and this has compromised the formulation of a reproducible
classification for many years. In 1994, the
International Lymphoma Study group,
proposed the Revised European-American
Lymphoma (REAL) classification which
grouped the T-cell and postulated NK cell
lymphomas into clinical entities.5 The
diverse histologic lesions that did not represent specific clinical entities were
encompassed in the group of PTL-u. This
classification was further refined in the
WHO classification of PTL and NK cell
lymphomas, which were divided into the
three general groups, leukemic/disseminated, nodal, and extranodal, based on
their clinical features1 as shown in Table 1.
The characterization of entities included
in this classification has integrated morphologic, clinical, immunophenotypic and
genetic studies.
Clinical features play a defining role in many PTLs
As the cellular composition of PTLs can
range from small cells with minimal
atypia to large cells with anaplastic features, a spectrum of histologic appearances can be seen even within individual
disease entities. Therefore cytologic principles have been difficult to apply for the
classification of PTLs and their reproducibility was poor.6 Similarly, immunophenotyping has been less useful than in
B-cell lymphomas since defining markers, such as cyclinD1 for mantle cell lymphoma, or marker combinations such as
CD10/BCL6 for follicular lymphoma, are
lacking. Finally, the molecular pathogenesis for most PTLs is as yet undiscovered.
For these reasons, clinical features have
played a major role in defining many of
the specific entities included in the WHO
classification (Table 2).
Pathology of peripheral T-cell lymphoma, unspecified,
angioimmunoblastic T-cell lymphoma, and anaplastic large
cell lymphoma
Peripheral T-cell lymphoma, unspecified
Earlier studies have recognized a variety of morphologic subtypes based on cell size and cytology,
including pleomorphic T-cell lymphoma, lymphoepithelioid lymphoma (Lennert's lymphoma), and Tzone lymphoma. However, no clinical or other substantial differences among these histologic types
have emerged (Figure 1A, B). As a group these
tumors account for approximately half the PTLs in
Western countries. They usually present with
advanced stage of disease and belong to the category
of the most aggressive NHLs.2,7 Immunophenotypically, these cases express the αβ heterodimer of
the T-cell receptor, more commonly CD4 than CD8,
but a significant proportion of the cases have an aberrant phenotype (CD4–CD8–, or less commonly,
CD4+CD8+) and show loss of CD5 and/or CD7.8-10 A
very recent study has attempted to correlate PTLs
with specific developmental stages of mature reactive T-cells.11 A major subset of PTLs-u was found to
correlate with a non-effector, or central memory, Tcell population characterized by a CD45RA–
CD45RO+CD27+CD4+ phenotype. In contrast, all
angioimmunoblastic T-cell lymphomas (AITL) and
Table 1. WHO classification of mature T-cell and NK-cell
neoplasms (1).
Mainly nodal
Peripheral T-cell lymphoma, unspecified
Angioimmunoblastic T-cell lymphoma
Anaplastic large cell lymphoma
Extranodal/Cutaneous
Extranodal NK/T-cell lymphoma, nasal type
Enteropathy-type T-cell lymphoma
Hepatosplenic T-cell lymphoma
Subcutaneous panniculitis-like T-cell lymphoma
Cutaneous γδ T-cell lymphoma
Mycosis fungoides
Sezary syndrome
Primary cutaneous anaplastic large cell lymphoma
Often leukemic or disseminated
T-cell prolymphocytic leukemia
T-cell granular lymphocytic leukemia
Aggressive NK-cell leukemia
Adult T-cell lymphoma/leukemia (HTLV1+)
ALCL showed a homogeneous effector cell phenotype CD45RA–CD45RO+CD27–. Nevertheless, in
daily routine cases of PTL-u showing borderline features to AITL and to anaplastic lymphoma kinase
(ALK)-negative ALCL exist and may cause problems
that need to be solved.
Table 2. Pathological and clinical characteristics of peripheral T-cell lymphomas and NK/T-cell lymphoma.
Predominant clinical features
Sites of involvement
Predominant
immunophenotype
Prognosis
Recurrent genetic
abnormalities
Peripheral
T-cell lymphoma,
unspecified
Aggressive,
often B-symptoms,
advanced stage, IPI 3-5: 60%
Lymph nodes,
bone marrow, skin
TCRa/β,CD3+CD2+
CD4+>CD8+. Often loss
of CD5, CD7
Poor
Complex karyotypes,
Very small subset
t(5;9)(q33;q22)
Angioimmunoblastic
T-cell lymphoma
Generalized
lymphadenopathy,
hepatosplenomegaly, skin rash,
hypergammaglobulinemia
Lymph nodes, bone
marrow, skin, lung
TCRα/β,CD3+CD4+
CD10+CXCL13+CD8-
Poor,
+3,+5
5-year survival 30%
Anaplastic large cell
lymphoma
Lymphadenopathy,
B-symptoms
Lymph nodes,
skin, soft tissue, bone, lung
CD30+ALK+CD3+
Favorable for
TIA1+EMA+;
ALK+ cases
loss of many T-cell antigens
t(2;5)(q23;q35)
and variants
Extranodal NK/T-cell
lymphoma, nasal type
Aggressive
locally destructive
Nose, nasal cavity,
paranasal sinuses,
pharyngeal tissue
CD3ε+CD56+CD2+
TIA1+; CD4-CD5EBV+
Poor
del 6q
Enteropathy-type
T-cell lymphoma
Acute abdominal emergency,
small bowel perforation,
malabsorption, weight loss
Upper jejunum,
often multiple segments,
mesenteric lymph nodes
Type 1: CD56-CD3+
CD7+CD2+TIA1+
Type 2: CD56+CD8+
Very poor
Gains 9q or losses 16q;
Type 1: gains 1q, 5q
Type 2: gains 8q24
Hepatosplenic T-cell
lymphoma
Young males, B-symptoms,
hepatosplenomegaly,
thrombocytopenia
Liver, spleen,
bone marrow
TCRγδ, CD3ε+CD56+
TIA1+
CD5-CD4-CD8-
Poor
Isochromosome 7
Subcutaneous
panniculitis-like
T-cell lymphoma
Solitary or multiple not ulcerated, Subcutaneous fatty tissue
erythematous tumors s
or plaques at extremities
TCRα/β,CD3+CD2+
CD5+CD8+TIA1+CD5
CD56-
5-year survival 80%
None
AITL represents a distinctive PTL subtype with
unique clinicopathological features sometimes simulating an infectious process. It usually effects the elderly and presents with generalized lymphadenopathy although lymph node enlargement may be not be
as pronounced as in other primary nodal PTLs.
Hepatosplenomegaly, skin rash and B-symptoms are
often present as well as hypergammaglobulinemia
and elevation of both the LDH and erythrocyte sedimentation rate. Patients may show anemia and occasionally pancytopenia. A significant proportion of
patients have circulating autoantibodies and a number of autoimmune phenomena have been reported.12 Histologically, the lymph node architecture is at
least partially effaced by a polymorphic infiltrate predominantly occupying the paracortical areas.
Subcapsular sinuses may be preserved. The neoplastic T-cells have clear cytoplasm and are distributed in
a marked inflammatory background comprising
polyclonal plasma cells, eosinophils, epithelioid histiocytes, arborized high endothelial venules and
expanded follicular dendritic meshworks (Figure 2).
Extranodal disease may be present at diagnosis
including involvement of the bone marrow, spleen,
skin, and lungs. The neoplastic T-cells, which may be
a minor cell population, are CD4+ T-cells that coexpress CD10 and sometimes BCL-6 suggesting the
derivation from a unique population of T-cells, called
follicular B helper cells (TFH) which are normally specialized in B-cell help within the germinal center
microenvironment.12 More recently it was shown
that in AITL the atypical CD10+ cells express
CXCL13, a chemokine highly upregulated in TFH cells
and critically involved in lymphoid organogenesis
and B-cell migration into follicles.13,14 Finally, gene
expression profiling revealed that the AITL molecular
signature was significantly enriched for TFH cell-specific genes strongly supporting the idea that TFH cells
represent the normal counterpart of AITL.15
Another frequent finding in AITL is the presence of
EBV-infected B-cells even early in the course of the
disease which may progress to an EBV-positive lymphoproliferative disorder. This may show both T-cell
receptor and immunoglobulin heavy chain gene
rearrangements when examined by clonality studies.
Usually presents with nodal disease but may initially affect a variety of extranodal sites. The WHO
provisionally included cases of ALCL positive and
negative for ALK under the heading of ALCL. But
ALK-negative cases differ in a number of respects.
They are seen in an older age group, have a worse
prognosis, and generally show greater nuclear pleomorphism. These findings suggest that ALK-negative
Figure 1. Peripheral T-cell lymphoma, unspecified. Image A
shows a representative case of peripheral T-cell lymphoma, unspecified, infiltrating the paracortical area, sparing a centrally located follicle remnant. At higher magnification, image B depicts diffusely infiltrating atypical large
cells. Cytogenetics and molecular cytogenetics of another
case demonstrate t(5;9)(q33;q22) and subsequent identification of ITK-SYK rearrangement, as found in a small subgroup of peripheral T-cell lymphoma, unspecified (C, D).
Figure 2. Angioimmunoblastic T-cell lymphoma. Overview
showing arborized vessels surrounded by a mixture of
small, medium-sized, and a few large cells, some of which
appear as clear cells with pale cytoplasm. Staining for
CD21 reveals the typical enlarged network of proliferating
follicular dendritic cells. Loose collections of CD10+ T-cells
are present.
ALCL will ultimatively represent a different disease
entity with an independent pathogenesis.16 Several
histologic variants of ALCL have been identified
including the most frequent common type. This shows
cohesively arranged large cells with lobulated, kidney-shaped nuclei and abundant grey-blue cytoplasm sometimes referred to as hallmark cells17(Figure
3). The small cell variant may be diagnostically highly
challenging because of similarities with an inflammatory process. Identification of neoplastic cells require
immunoreactivity for CD30 and ALK particularly in
Figure 3. Anaplastic large cell lymphoma, ALK positive.
Sinusoidal infiltration of cohesively arranged anaplastic
tumor cells, demonstrating cytoplasmic and nuclear reactivity for ALK indicating t(2;5).
this ALCL variant and similarly in the lymphohistiocytic variant in which histiocytic cells may obscure
the neoplastic population.16,17 Another variant is
Hodgkin-like ALCL which most often represents an
aggressive variant of classic Hodgkin lymphoma and
shows overlap with mediastinal grey-zone lymphomas.18 Immunophenotypically ALCL is characterized by positivity for CD30, and in 60-85% of the
cases, for ALK. The frequent reactivity for T-cell
intracellular antigen (TIA-1) and epithelial membrane
antigen (EMA) is also diagnostically helpful. Loss of
several or even all T-cell associated antigens is a typical feature of ALCL. Defective expression of T-cell
receptors in all types of ALCL (ALK+, ALK–, cutaneous) separates these tumors from other PTLs and
may contribute to the dysregulation of intracellular
signaling pathways controlling T-cell activation and
survival.19
Primary cutaneous ALCL is a different disease entity belonging to the spectrum of the primary cutaneous CD30+ T-cell lymphoproliferative disorders. It
differs from the systemic forms (ALK+ and ALK–) in
its site of origin, its clinical features, and almost
invariable absence of ALK protein and EMA.16
Genetics of anaplastic large cell lymphoma, peripheral Tcell lymphoma unspecified, and angioimmunoblastic T-cell
lymphoma
From the genetic point of view, ALCL is the only
well characterized PTL as the vast majority of cases
have the t(2;5)(p23;q35) involving the ALK tyrosin
kinase and the nucleophosmin gene, resulting in overexpression of a chimeric oncogene, nucleophosmin/
anaplastic lymphoma kinase (NPM/ALK).20 NPM/ALK
activates numerous downstream signaling pathways
resulting in enhanced survival and proliferation. Over
11 variant translocations involving the ALK gene
have been described in lymphomas and a subset of
pediatric tumors, including inflammatory myofibroblastic tumor and rhabdomyosarcoma. This shows
that the transforming potential of ALK tyrosine
kinase is manifest in more than one cell type. Very
recent proteomic studies may provide the basis for
the identification of biomarkers and targets for novel
therapeutic agents.21
The molecular alterations underlying the pathogenesis of AITL and PTL- are largely unknown. The
overexpression of several TFH genes, as shown by
gene expression profiling, supports the idea that
AITL is derived from TFH cells.15 However, the spectrum of AITL may be wider than suspected, and, in
contrast to AITL and ALCL, PTL-u does not share a
single molecular profile .15,22 A comparison of the gene
expression pattern of PTL with that of normal T-cells
revealed that PTL-u display deregulation of functional programs often involved in tumorigenesis, such as
apoptosis, proliferation, cell adhesion, and matrix
remodeling. Furthermore, PTLs-u aberrantly express,
among others, PDGFRα, a tyrosine kinase receptor
whose deregulation is often related to a malignant
phenotype.23
With the notable exception of the t(2;5) and its
variants in ALCL, no recurrent chromosomal translocations were known to occur in PTLs until Streubel et
al. recently reported on the identification of a novel
recurrent t(5;9)(q33;q22) in a small subset of PTLs-u24
(Figure 1 C,D). Four of five translocation-positive
cases among a total of 30 PTLs-u examined demonstrated a follicular or perifollicular growth pattern
indicating a tropism of the lymphoma cells to lymphoid follicles. Cases of AITL and ALK-negative
ALCL were negative for the t(5;9). Molecular analysis revealed breaks within the ITK gene on chromosome 5 and SYK on chromosome 9 generating an
ITK-SYK fusion transcript. The result of the translocation is likely to be activation of SYK through overexpression, driven by the ITK promoter.
Interestingly, the neoplastic cells in three of the four
t(5;9)+ cases with follicular growth pattern were
CD4+CD10+BCL6+ and thus closely resembled the
immunophenotype described for TFH in AITL.
Pathology and genetics of extranodal NK/T-cell lymphoma,
nasal type
Extranodal NK/T-cell lymphoma, nasal type, is a
distinct clinicopathologic entity highly associated
with EBV. The disease is rare in Western countries
but more prevalent in Asia and in South and Central
America. The most common clinical presentation is a
destructive nasal or midline facial lesion resulting in
nasal obstruction or epistaxis. The lymphoma can
extend to adjoining tissues such as paranasal sinuses,
nasopharynx, palate, oropharynx, oral cavity, and
orbit. In most of the cases the disease is localized to
Type 1
Type 2
Figure 4. Enteropathy-type T-cell lymphoma. In this example Type 1 is characterized by anaplastic large cells, whereas Type 2 shows a monomorphic infiltration of a jejunal villous tip with small, CD56 positive lymphoid cells.
the upper aerodigestive tract at presentation and
bone marrow involvement is very uncommon.25
Nevertheless, the disease may disseminate and may
be complicated by a hemophagocytic syndrome.
Histologically an angiocentric growth pattern is often
present, and the cytological spectrum is very broad.
Cases predominantly composed of small cells may
mimick an inflammatory processs. Immunophenotypically NK/T-cell lymphoma is characterized by
CD2+CD56+ and cytoplasmic CD3ε-chain expression
and positivity for cytotoxic granules-associated proteins such as TIA-1, granzyme B, and perforin.26 The
expression of granzyme M identifies these cells as
belonging to the innate arm of the immune system
which is independent of specific antigen activation.27,28 Other T-cell associated antigens are usually
negative and T-cell receptor genes are not rearranged.
True NK cell lymphomas more often express NK cell
receptor molecules such as CD94/NKG2A29 and
CD94 expression has been reported to imply a better
prognosis.30 The demonstration of EBV encoded
small nuclear mRNA (EBER) by in situ hybridization
is positive in almost all cases and therefore is very
useful diagnostically.31
Various genetic alterations may occur in nasal type
extranodal NK/T-cell lymphoma. Deletions at 6q are
the most common. However their significance is as
yet unknown.32
Pathology and genetics of enteropathy-type T-cell lymphoma
Enteropathy-type T-cell lymphoma (ETTCL) is a
rare, primary intestinal lymphoma arising from
intraepithelial T-cells, usually as a consequence of
clinically unknown celiac disease. The proximal
jejunum is the most frequent site of disease, although
it may occur elsewhere in the small intestine and,
rarely, in the stomach and colon.33 About 40% of
patients present as acute abdominal emergencies due
to intestinal perforation and/or obstruction. Patients
may have a short history of malabsorption, sometimes diagnosed as adult celiac disease which is often
gluten-insensitive or, less frequently, a long history of
celiac disease lasting for years or even decades.
Histologically most cases are composed of pleomorphic medium to large cells or show features resembling anaplastic large cell lymphoma, mostly carrying
the immunophenotype CD3+CD7+CD5–CD4–CD8–
TIA1+CD56– (type 1 ETTCL). A minority of cases
(10-20%) consists of monomorphic small to medium-sized cells which often express CD8 and CD56
(type 2 ETTCL, Figure 4).33,34 Type 1 and type 2
ETTCL also differ biologically and by their genetic
profile. Type 1 is linked to celiac disease by virtue of
expressing HLADQ2 or DQ8, and is characterized by
chromosomal gains of 1q and 5q. In contrast, type 2
rarely shows the genetic background of celiac disease
but more often gains of the MYC oncogene locus at
8q24.35,36 The common genetic denominator of both
EATTCL types, and hence the genetic hallmark of
the disease present in about 70% of cases, is gain at
the long arm of chromosome 9, while, to a much
lesser extent, loss at 16q is observed.35,37
To understand the pathogenesis of ETTCL it is
nessessary to recognize the role of refractory celiac
disease as an in situ ETTCL. A small fraction of adult
celiac disease patients fail to improve after a glutenfree diet, show persisting villous atrophy and an
increase of monoclonal, immunophenotypically
unusual intraepithelial lymphocytes, usually showing loss of CD8.38,39 This group of patients is referred
to as having refractory celiac disease II (RCD II) as
opposed to RCD I who show polyclonal and
immunologically normal intraepithelial lymphocytes.40 Patients diagnosed with RCD II are thought
to suffer from cryptic intraepithelial (in situ) ETTCL
associated with a poor prognosis and high risk of
progression to overt invasive lymphoma. Interestingly, Verkarre et al. have demonstrated that intraepithelial lymphocytes of patients with RCD II carry
gains of chromosome 1q. This has also been
observed in the frequently celiac disease-associated
type 1 ETTCL.41,35 Therefore several lines of evidence
support the hypothesis that RCD II represents a precursor lesion of overt ETTCL and deserves a designation such as in situ ETTCL.
Pathology and genetics of hepatosplenic T-cell lymphoma
Hepatosplenic T-cell lymphoma (HSTCL) is an
extranodal and systemic neoplasm derived from
cytotoxic T-cells, usually the γ/δ T-cell receptor type.
It is a rare and aggressive malignancy predominantly
affecting young men.42,43 The patients present with
thrombocytopenia and isolated hepatosplenomegaly
due to marked sinusoidal infiltration of mediumsized cells with pale cytoplasm. Lymphadenopathy
or significant peripheral blood involvement are usually absent, but there is sinusoidal infiltration of the
bone marrow. Cytogenetically, isochromosome 7q is
reported to be the primary genetic abnormality.44
Pathology of subcutaneous panniculitis-like T-cell lymphoma
According to the WHO-EORTC classification for
cutaneous lymphomas, subcutaneous panniculitislike T-cell lymphoma (SPLTCL) is a cytotoxic T-cell
lymphoma with an α/β CD8+ T-cell phenotype presenting with primarily subcutaneous infiltrates of
pleomorphic T-cells and many macrophages, predominantly affecting the extremities.45 A long history
of benign panniculitis may be observed. In contrast to
prior reports indicating that SPLTCL patients have a
rapidly fatal course, recent studies suggest that many
patients run a protracted clinical course.46 Constitutional symptoms, and particularly a hemophagocytic
syndrome, are more typical for mucocutaneous γ/δ Tcell lymphoma which has to be separated from SPLTCL. No recurrent chromosomal aberration has been
reported.
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Current treatment of T-cell lymphomas:
are we making any progress?
A
A. Delmer
Hematology Department,
Hôpital Robert Debré,
Avenue du Général Koenig,
Reims, France
2007;1:83-88
B
S
T
R
A
C
T
Peripheral T-cell lymphomas represent a minority of the non-Hodgkin’s lymphomas
and include several heterogeneous entities. The more frequent subtypes, at least in
Western countries are peripheral T cell lymphoma, unspecified, anaplastic large cell lymphoma and angioimmunoblastic T cell lymphoma. The prognosis of peripheral T/NK neoplasms is still poor, with 5-year survival less than 30% in most cases. There is currently
no consensus about the optimal therapy of these uncommon disorders mainly with
regard to the standard initial regimen, whether all entities should be treated similarly,
and the place of autologous or allogeneic stem cell transplantation. Progress is likely to
come from the identification of novel agents active in relapsed patients which will be
combined with or replace usual cytotoxic agents. Collaborative efforts to set up large
multicentric trials aimed at efficiently examining these are also needed.
ature T-cell and NK-cell lymphomas account for only 12% of
all non-Hodgkin’s lymphomas
with an incidence of 1.79 per 100,000 person-years in US during the period 19922001 (compared to 26.13 for the whole Bcell malignancies).1 They encompass several well identified entities listed in the
WHO classification according to their predominant clinical feature, leukemic, cutaneous, other extranodal and nodal.2 Apart
from cutaneous T-cell lymphomas
(CTCL), the most frequent subtypes of
peripheral T-cell lymphoma (PTCL) are
peripheral T-cell lymphoma, unspecified
(PTCLU), angioimmunoblastic T-cell lymphoma (AILT) and anaplastic large cell
lymphoma (ALCL). EBV-associated extranodal NK/T-cell lymphoma, nasal and
nasal-type, is rare in Western countries
and is more prevalent in Asia and in South
and Central America whereas adult T-cell
leukemia/lymphoma (ATLL) is observed
in countries where HTLV-1 is endemic.
Patients with nodal PTCL commonly
present with advanced stage disease and
unfavourable clinical features, such as B
symptoms, elevated LDH levels, bone
marrow involvement and poor performance status. Consequently, more than a
half of these patients have an unfavorable
IPI (International Prognostic Index) score
at diagnosis.3
Although the different subtypes of noncutaneous T-cell lymphomas may have
different origins and mechanisms of
M
growth, from a therapeutic point of view
they are currently considered as one
group. With the exception of anaplastic
lymphoma kinase (ALK)-positive ALCLs,
which share a similar or even better outcome than diffuse large B-cell lymphomas
(DLBCL),4,5 PTCLs have a worse prognosis
due to both a lower response rate to
anthracycline and cyclophosphamide containing regimens and a higher incidence of
relapse, mainly in patients with the highest IPI scores.6
Among the 1883 patients with aggressive lymphoma and reviewed histologic
data enrolled in the GELA LNH-87 trials
between 1987 and 1993, 288 patients
(15%) had a T-cell lymphoma including 60
patients with ALCL. Sixty-five percent of
patients with a non ALCL T-cell lymphoma displayed 2 or 3 unfavorable
parameters of the age-adjusted IPI (aa-IPI).
Most of these patients received an intensified CHOP regimen (ACVBP). In this
cohort of patients with T-cell lymphoma,
the complete response (CR) rate after
chemotherapy was significantly lower
than in patients with DLBCL: 58% vs
63% for patients with aa-IPI 2, 35% vs
52% for patients with aa-IPI 3, and 42%
vs 56% for patients with aa-IPI score 2 or
3. The 5-year overall survival was also significantly lower in patients with T-cell
lymphoma as compared with outcome of
patients with DLBCL: 35% for the entire
cohort of non ALCL PTCL vs 52% for
DLBCL, 36% vs 53% and 23% vs 35% for
Table 1. Classification of mature T-cell and NK-cell lymphomas.2
WHO histological classification of mature T-cell and NK-cell neoplasms
Leukemic or disseminated
T-cell granular lymphocytic leukemia
Aggressive NK-cell leukaemia
Adult T-cell lymphoma leukemia
Other extranodal
Extranodal NK/T-cell lymphoma, nasal type
Enteropathy-type T-cell lymphoma
Subcutaneous panniculitis-like T-cell lymphoma
Cutaneous
Blastic NK-cell lymphoma
Mycosis fungoides/Sezary syndrome
Primary cutaneous anaplastic large cell lymphoma
Lymphomatoid papulosis
Nodal
Peripheral T-cell lymphoma, unspecified (PTL, unspecified)
Angioimmunoblastic T-cell lymphoma (AIL-T)
Anaplastic large cell lymphoma (ALCL)
patients with aa-IPI 2 and aa-IPI 3 respectively.6 Very
similar results were observed in the other large series
of PTCLs with an overall survival repeatedly close to
30-35% at 3 to 5 years.7-9
These poor results emphasize the need for more
efficient strategies in patients with T-cell lymphoma
to improve both response rate and duration of
response, whereas patients with DLCBL were shown
to benefit from the combination of chemotherapy
and immunotherapy with rituximab.
Prognostic factors
Among nodal PTCLs, histologic subtype does not
appear to have prognostic relevance, except for ALKpositive ALCL which is significantly associated with
a better outcome, and the behavior of patients is similar whether they have PTCLU, AILT or ALK-negative ALCL.5,6,10
Several studies have confirmed the usefulness of
the IPI in some of the specific PTCL subtypes such as
ALCL (ALK-positive or -negative) or PTCLU.5,6,10,11
Based strictly on the IPI the 5-year survival for
patients with any T-cell lymphoma was 74%, 49%,
21%, and 6% for the low, low-intermediate, highintermediate, and high risk groups respectively.7
However, IPI was not predictive of survival in a
recent large series of AILT.12 The applicability of IPI to
NK/T-cell lymphomas has led to variable results
while it has not been evaluated in other rare subtypes
of extranodal T-cell lymphoma. A new prognostic
model, PIT, specifically designed for PTCLU, was
recently proposed by Gallamini et al. from a large
cohort of 385 patients.11 This model includes most of
the IPI parameters (age, performance status and
LDH) in addition to bone marrow involvement
instead of stage. The 5-year survival of PTCLU
patients ranges from 62% (0 factor) to 18% (3 or 4
factors) and about half the patients fall into the highrisk categories with a 5-year survival which does not
exceed 30%. In a multivariate analysis of 475 cases of
PTCLU and AILT, the international PTCL project has
found age, performance status and platelet count
(150 K vs > 150 K) significantly correlated with the 5year survival that ranged from 42% (0 factor) to 12%
(3 factors). Comparison with the standard IPI and the
PIT index on this population demonstrated that in all
3 indices, the failure free survival and overall survival
were very poor for all patients except in those with 0
or 0/1 risk factors.13 A high expression of proliferation-associated antigen Ki-67 (≥80%) was found predictive of poor survival in PTCLU and was therefore
integrated along with age, performance status and
LDH in a prognostic score where patients with 3 or 4
factors (representing only 12% of the cohort) had a
median survival of 6 months.14
The clinical heterogeneity of PTCLUs, which represent subgroup and in most cases express a
CD4+CD8– phenotype, may be explained by variable
chemokine receptors expression. In one study, two
groups of PTCLU were identified, one expressing
either Th2 (ST2(L)) or Th1 (CCR5, CXCR3) markers
and another negative for all these markers. Patients
belonging to the former group considered as functional had a better outcome than those in the latter
group.15 The heterogeneity of PTCLUs with regard to
expression of Th1 (CXCR3) and Th2 (CCR4) markers was confirmed in a second study in which
patients with CCR4 expression displayed a poorer
outcome.16 CCR4 expression was also observed in
most cases (88%) of ATLL17 and a monoclonal antibody directed against CCR4 in this disease is currently under investigation.
The expression of cytotoxic molecules TIA-1 and
granzyme B in PTCLU has been correlated with a
higher IPI, a lower CR rate (30% vs 63%), and a
worse overall survival.18
Gene expression studies have also emphasized the
heterogeneity of PTCLU and three molecular subgroups were identified in a series of 32 cases. One
subgroup had a gene expression signature including
genes known to be associated with poor outcome in
other tumors, such as CCND2. A second subgroup
was characterized by the overexpression of genes
involved in T-cell activation and apoptosis, including
NFKB1 and BCL-2. A third subgroup was mainly
defined by overexpression of genes involved in the
IFN/JAK/STAT pathway.19 The small number of cases
in this series precluded valuable clinical correlations
but there was a trend for an inferior survival in
patients belonging to the first group.
The expression of NFkB genes was investigated in
a group of 62 PTCL (PTCLU 39, AILT 7, ALCL 7, others 9). One third of cases showed a reduced expression of NF-κB genes while other cases showed a high
expression of these genes. Both patterns were
observed independently of histologic subtype
(except for ALCL which was characterized by a
reduced expression of NF-κB genes). Signature with
reduced expression of NF-κB genes was associated
with a shorter survival.20
Other studies have shown deregulated expression
of genes involved in apoptosis, proliferation, cell
adhesion and matrix remodeling, as compared with
normal T cells. Furthermore, usually PTCLUs aberrantly express the tyrosine-kinase receptor PDGFRα
and cultured PTCL cells were shown to be sensitive
to imatinib as well as to a novel inhibitor of histone
deacetylase.21
Further confirmation of these findings may help
identify new potential targets for therapy.
First line treatment
There is no consensus about the optimal first line
and subsequent treatment for PTCL but there is general agreement on the unsatisfying results observed
with conventional therapeutic strategies.
Whether all PTCL subtypes should be managed
similarly or not may be a matter of debate. For the
time being, there is no data to support the concept
that nodal PTCLs and (PTCLU, AITL and ALK-negative ALCL) should be offered different strategies
since with current cytotoxic regimens their behavior
is similar. This also applies to the rare categories with
an even poorer prognosis such as hepatosplenic γδ Tcell lymphoma (HSTCL) or enteropathy associated
T-cell lymphoma (EATCL), unless effective innovative strategies become available. One exception is
extranodal NK/T cell lymphoma, nasal-type, for
which radiotherapy appears to be the key treatment
modality in localized stages. More favorable outcomes were observed in treatment regimens that
incorporate radiotherapy.22, 23
Over the years, PTCLs have usually been treated
with CHOP or CHOP-like regimens as the mainstay
of therapy, as were advanced stage DLBCL before
the era of rituximab. With front-line anthracyclinebased combination chemotherapy, approximately
50% of patients with PTCL achieve a complete
remission and reported 5-year survival rates range
from 25 to 45%.6-9 Very few studies, generally with a
low number of patients and including patients with
ALCL, have shown an improvement in response
rates and overall survival when a more dose-intensive induction treatment was used.24 The GELA has
evaluated intensified alternative regimens in both
young and elderly patients with T-cell lymphoma,
using intensified induction therapy with etoposide
and high-dose cytarabine in 89 patients aged less
than 60 years and ESHAP regimen in combination
with 13-cis-retinoic acid in 77 patients over 60 years.
The response rate, event-free survival and overall survival in each group were not superior to those usually achieved with CHOP or CHOP-like regimen.25,26 A
randomized study comparing alternating cycles of
VIP (etoposide, ifosfamide, platine) and ABVD regimens to CHOP in 88 PTCL patients did not show
any improvement over CHOP.27
In a trial of the Nordic Lymphoma Group, where
patients were randomized to either CHOP or
MACOP-B, patients with PTCL, although still displaying a poorer prognosis than DLBCL patients, had
a slight survival advantage from the more doseintense MACOP-B chemotherapy arm.28
Two different randomized studies performed by
the German High-Grade Non-Hodgkin's Lymphoma
Study Group indicated a survival benefit of a timeintensified CHOP regimen (CHOP14) in young
patients as well as in patients aged over 60 years with
a diagnosis of aggressive lymphoma of B- or T-cell
origin.29,30 Although these studies have been published without a specific analysis of patients with Tcell lymphoma, numerous patients with PTCL are
now receiving CHOP14 as front line regimen.
Autologous transplantation
The role of autologous stem cell transplantation
(ASCT) in PTCLs, mainly performed early in first CR
as part of up-front therapy, still remains controversial.
High dose therapy followed by ASCT has for
decades been the most widely adopted approach for
patients under 60-65 years with relapsed aggressive
lymphoma (either B or T-cell derived) and chemosensitive disease. Several series have reported the outcomes of patients with relapsed T-cell NHL after
ASCT. They mainly included patients with PTCLU
or ALCL and the 3-year survival rates ranged from
36% to 58%.31,32 Patients with ALCL (vs non ALCL)
had a better outcome with a 3-year survival rate of
79% (vs 44%). In one retrospective study, the outcome of 36 patients with relapsed or primary refractory PTCL and 97 patients with relapsed DLBCL
were compared. There was no difference in 3-year
survival between patients with PTCL or DLBCL
(48% vs 53%), suggesting that high-dose therapy
may overcome the adverse effect of T-cell pheno-
type. However, patients with PTCLU had a trend for
poorer survival.33 Patients who do not achieve a complete response after initial chemotherapy may also
benefit from ASCT.34
Some studies of high-dose chemotherapy and
ASCT performed in first CR are now available for
PTCL patients. However, some of these studies have
included a significant number of patients with ALCL,
therefore leading to an overestimate of its potential
benefit.
The results of the Spanish group have been recently updated. In a cohort of 74 patients including 23
patients (31%) with ALCL (ALK status unavailable
for most of them), the 5-year survival and progression free survival (PFS) were 68% and 63% respectively with a median follow-up of 63 months.35
Two prospective phase II studies investigating the
role of high-dose sequential chemotherapy, followed
by ASCT in 62 patients with advanced stage PTCLs
have shown that 46 patients (74%) completed the
whole programme, whereas 16 patients did not
undergo ASCT, mainly because of disease progression. With a median follow-up of 76 months, the
estimated 12-year overall, disease-free and event-free
survival were 34, 55 and 30% respectively. Results
were significantly better in the 19 patients (30%)
with ALK-positive ALCL.36 A German study has evaluated a strategy combining 4 to 6 cycles of CHOP
regimen followed by one course of Dexa-BEAM,
then high dose therapy and ASCT. In 38% of the 65
evaluable patients, progressive disease occurred prior
to transplantation and among the remaining patients,
27% relapsed at a median follow-up of 10 months
post-transplant.37 The Nordic Lymphoma Group has
tested a dose-intensified induction schedule (6 courses of CHOEP14) followed by autologous transplant
in first remission. Among the 77 patients who completed induction regimen, 66 (88%) are in CR or partial response. A total of 58 patients (75%) have
received ASCT and 9 out of 39 patients with a one
year follow-up after transplant have relapsed.
Therefore, 25% could not receive the program
because of early progression and an additional 25%
will relapse after ASCT.38
In these studies, a significant number of patients
were not able to reach ASCT because of early progression. This underlines the need for more efficient
induction treatments. Furthermore, in a retrospective analysis of all patients enrolled in the French
GELA LNH87 and LNH93 programs and who were
transplanted in first CR, after multivariate analysis
T-cell phenotype (excluding ALCL subtype) still
remained an adverse prognostic parameter significantly associated with a poorer outcome after transplantation.39
Allogeneic transplantation
Data of the results from allogeneic transplantation
in patients with T-cell lymphoma is scarce. Apart
from general observations, some smaller series have
been published. A series of 8 patients with Sezary
syndrome or tumor-stage mycosis fungoides showed
that all patients achieved a CR after transplantation.
With a median follow-up of 56 months, 6 patients
remained alive and well.40 Another series reported 11
patients with various T-cell lymphomas who had
been allografted with a reduced intensity conditioning after salvage therapy including alemtuzumab and
the ICE regimen. Six patients were alive and free of
disease at a median follow-up of 7 months.41 The
largest series has been reported by Corradini et al.
and recently updated.42,43 A total of 26 patients were
allografted for relapsed T-cell lymphoma after
reduced intensity conditioning with thiotepa, fludarabine, and cyclophosphamide. The estimated 5year overall survival and progression free survival
were 61% and 51% respectively. This study showed
that long-term disease control can be achieved by
allogeneic transplantation via a graft versus T-cell
lymphoma effect. Three out of 8 patients with
relapsed disease after transplantation responded to
donor lymphocyte infusion (DLI). These results suggest that allogeneic transplantation with reduced
intensity conditioning may be a suitable option for
young patients with relapsed disease and an available
donor. It could also be evaluated as up-front strategy
in patients with poor-risk T-cell lymphoma, possibly
following autologous transplantation.
New agents
Since most patients will fail to first line therapy,
new treatment modalities should be explored. It is
likely that significant progress will not be made from
the alternative use of classic cytotoxic agents. Some
agents have demonstrated significant activity in cutaneous T-cell lymphoma but activity in CTCL does
not mean they would also be effective in nodal or
other extranodal T-cell lymphomas. Purine analogs,
pentostatin, fludarabine and cladribine, have all been
reported to have some activity in CTCL and PTCLs
with response rates ranging from 20 to 70%.
Response rates appeared higher with pentostatin
than with other compounds.44
Fludarabine was used in combination with
cyclophosphamide, doxorubicin and alemtuzumab
in patients with various subtypes of PTCLs (excluding ALK-positive ALCL and CTCL) and led to a 73%
CR rate in previously untreated patients.45
The response rate to treatment with the pyrimidine analog gemcitabine in patients with relapsed Tcell lymphoma was around 60-70%46 but this agent
has mostly been investigated in CTCL.
Pralatrexate is a derivative of methotrexate with a
higher affinity to the reduced folate carrier. Among
the 16 evaluable patients with relapsed PTCL who
completed at least 2 courses, the response rate was
impressive with 10 patients achieving a major
response including 9 CR. Some of these were of a
long duration.47
Denileukin diftitox, a fusion protein combining the
receptor-binding domain of interleukin-2 and diphtheria toxin, is already approved for the treatment of
refractory CTCL. In 25 evaluable patients with non
cutaneous T-cell lymphoma, the response rate was
48% with 20% CR and responses were observed
CD25-positive and CD25-negative lymphomas.48
Combination of this agent with CHOP regimen
appeared feasible.49
Monoclonal antibody alemtuzumab directed
against CD52 antigen has been evaluated in a short
series of 14 patients with heavily pretreated nodal Tcell lymphoma. Alemtuzumab was programed to be
administered intravenously three times a week for 12
weeks. The response rate was quite high, with 3
patients achieving a CR and 2 patients a partial
response. However, in these poor prognosis patients
there was considerable infectious toxicity.50 Using a
lower dosage and subcutaneous route, a 60%
response rate was observed in a series of 10 patients
with CTCL and nodal PTCL.51 Experience with the
combination of alemtuzumab and chemotherapy,
mainly CHOP and CHOP-like regimens, is still limited.45,52 Alemtuzumab was usually administered for
one or 2 days along with chemotherapy either by IV
or subcutaneous route. Preliminary results showed
good tolerability and feasibility for these combinations. However, a recent immunochemistry study
has evaluated CD52 expression in various hematological malignancies and only 35% (7/20) of PTCLU
and 40% (2/5) of AITL cases were found to express
CD52 whereas this antigen is highly expressed in
normal T-cells.53 These results need to be confirmed
and suggest that patients should be assessed for
CD52 expression before being offered a treatment
with alemtuzumab.
Histone deacetylase (HDAC) inhibitor depsipeptide has been evaluated in a series of 19 patients with
PTCL leading to a 26% response rate.54 Vorinostat
(suberoylanilide hydroxamic acid, SAHA), another
HDAC inhibitor, has only been evaluated so far only
in refractory CTCL. A partial response was observed
in 8 out of 33 evaluable patients with a median duration of response of 15 weeks.55
Conclusions
Progress in the treatment of T-cell lymphomas
remains a challenging issue due to their rarity, unsatisfactory response to current chemotherapy regimens
and, for the time being, the lack of novel agents likely to be incorporated into first line treatment. With a
few exceptions, treatment modalities should not
depend on histologic subtype, and nodal PTCLs
should be treated similarly unless otherwise indicated by ongoing gene profiling or other biological studies. International collaborative efforts are essential to
make advances in the treatment of T-cell lymphomas
and already several initiatives are focusing on the
evaluation of alemtuzumab in combination with
chemotherapy, and of allogeneic stem cell transplantation performed early as part of first-line treatment.
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Novel drugs for the treatment of T-cell lymphoma
O.A. O’Connor
Director, Lymphoid Development
and Malignancy Program
Herbert Irving Comprehensive
Cancer Research Center
Chief, Lymphoma Services
College of Physicians and Surgeons
The New York Presbyterian Hospital
Columbia University
New York, USA
2007;1:89-96
diagnosis of lymphoma has long
been interpreted by patients and
doctors alike as a lucky diagnosis.
While these diseases in general are relatively more chemotherapy sensitive than
many solid tumors, many sub-types of
lymphoma carry a very poor prognosis.
Complicating matters further is the fact
that the lymphomas represent one of the
most heterogenous group of malignancies
known to medicine. Underneath the single title of lymphoma exist some of the
fastest growing cancers known to science
(Burkitt’s lymphoma, lymphoblastic lymphoma/leukemia), as well as some of the
slowest (small lymphocytic lymphoma,
follicular lymphoma, and marginal zone
lymphoma). Within this complex set of
diseases are sub-types of lymphoma that
continue to pose significant therapeutic
challenges. The T-cell lymphomas represent one of those sub-types of lymphoma
for which there has been remarkably little
progress over recent years. These diseases
not only demonstrate a worse prognosis
than their B-cell counterparts but are
extremely rare. This means little consensus can be established because of the difficulties in finding enough patients to enroll
in the appropriate clinical studies.
For this reason, there is a strong need to
develop only the most active drugs for
these diseases, and to begin studying the
most promising new drugs in combination
with historically active agents. Clearly, a
consensus must be found on a new
upfront treatment for these diseases to
offer any hope of changing their present
natural history. Furthermore, identifying
new agents with novel mechanisms of
action offers unique opportunities to palliatively treat these lymphomas by
employing agents with less cross resistance to prior lines of conventional therapy.
A
Epidemiology of T-cell lymphomas
The etiology of non-Hodgkin’s lymphoma (NHL) remains largely unknown. It
is clear, however, that since before 1950,
an epidemic of lymphoma, but not other
hematopoietic neoplasms, was documented in many populations, with an estimated 50% increase in the age-adjusted incidence from 1970-1990 in the U.S1-3 While
recent reports suggest that the steep rise in
incidence may have slowed in recent
years, caution is required in interpreting
them given the innumerable factors which
can influence these statistics, like autoimmune deficiency syndrome (AIDs), new
diagnostic techniques, and the emergence
of other etiologic factors like infections.4-8
It is interesting that industrialized nations
experience a higher incidence of NHL than
do developing countries, with the highest
incidence rate in the world being seen in
the United States (US) and Canada.6 These
statistics rank NHL as the sixth most common cancer and the sixth most common
cause of cancer death, accounting for 4%
of all cancers and 4% of cancer deaths.9
Prevalence and incidence
Based on the most recent evaluation of
SEER registries presented by Morton et al.9
it is estimated there were approximately
114,548 cases of lymphoid neoplasms
diagnosed per year in the United States
over the period from 1992-2001, including
lymphoid leukemias and Hodgkin’s
Disease. Approximately 87,666 (about
76%) of these cases were B-cell lymphoid
neoplasms, while only about 10,042 cases
were attributed to Hodgkin’s disease.
T/NK-cell malignancies constituted approximately 6,228 cases per year. In general, Tcell malignancies comprise only about 1015% of lymphoid malignancies, though
there is notable variability in the literature
on the precise contribution.8,10-13
According to the National Cancer
Institute SEER Cancer Statistics Review
1975-2002, the prevalence for all types of
sub-types of Non-Hodgkin Lymphoma
was 347,039 on January 1, 2002, while the
10-year prevalence estimate was
240,065.14 Unfortunately, the prevalence
estimate specific to T-cell NHL was not
Table 1. Prevalence of NHL by type, 2005.
Disease
Total NHL
Non T-cell NHL
T-cell NHL
Table 2. WHO T-cell lymphoma classification.
5-year
10-year
181,928
162,639
19,289
267,116
237,509
29,607
Precursor T/NK neoplasms
Precursor T lymphoblastic leukemia/lymphoma
Blastic NK lymphoma
Peripheral T/NK neoplasms
provided in this report (SEER Cancer Statistics Review,
1975-2002, National Cancer Institute. Bethesda, MD,
http://seer.cancer.gov/csr/ 1975_2002/, based on
November 2004 SEER data submission, posted to the
SEER web site 2005). Data provided in an analysis
made by the U.S. Census Bureau and the NCI’s SEER
data set (SEER*Stat 6.2) using ICD codes for all T-cell
lymphoma sub-types (an analysis performed by the
DaVinci Oncology Specialists) provided the following prevalence estimates for NHL according to by
histology, as shown in Table 1.
It is clear that the overall prevalence of T-cell NHL
is well below the 200,000 limit to be net to qualify
for orphan drug status. Our best estimate of the 10year prevalence is approximately 30,000, more than
six times below the limit. Even the most aggressive
estimate possible (15% of complete NHL prevalence
of 347,039), puts the prevalence at 52,056, which is
still almost four times fold below the limit.
Numerous published reports, as well as the prior designation of orphan status for several products in Tcell lymphoma, confirm this conclusion.5
Recently, the U.S. FDA has acknowledged the rarity of T-cell lymphomas in the United States by granting orphan drug status to at least 6 other products for
T-cell lymphoma (not counting products specified for
cutaneous T-cell lymphoma). These include 1S)-1-(9deaza-hypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-Dribitol-hydrochloride (BioCryst Pharma-ceuticals),
siplizumab (MedImmune Oncology, Inc.), suberoylanilide hydroxamic acid (Merck & Co.), AGN-30,
anti-CD30 antibody (Seattle Genetics, Inc.), and pentostatin (SuperGen). Based on admittedly preliminary data, there is a reasonable expectation that
pralatrexate could act similar to or better than these
agents now in clinical trials.
Classification of T cell lymphomas
The T cell lymphomas represent a very heterogenous array of aggressive NHLs, and though rare, they
typically account for less than 10% of all newly diagnosed cases of lymphoma in the US. Interestingly,
these proportions of lymphoma are the opposite of
those found in Far East and Caribbean, where 7080% of all NHLs are derived from T cells. According
to the World Health Organization (WHO) classification schema provided in Table 2, T cell lymphomas
are divided into either precursor T/NK-cell neo-
Predominantly leukemic/disseminated
T-cell large granular lymphocytic
NK/T-cell leukemia/lymphoma
Adult T-cell leukemia/lymphoma
Predominantly nodal
Peripheral T-cell lymphoma (Unspecified)
Predominant extranodal
Mycosis Fungoides (CTCL)
Sezary syndrome
Primary cutaneos CD30+ disorders
Lymphomatoid papulosis
Subcutaneous panniculitis T-cell
NK/T-cell lymphoma-nasal
Enteropathy-type intestinal lymphoma
Extranodal peripheral T/NK-cell lymphoma (unspecified)
plasms or peripheral T/NK-cell neoplasms. These
categories are further sub-classified into a variety of
entities.15 According to this classification scheme,
there are thought to be at least 15 distinct sub-types
of T cell lymphoma, most of which can be broadly
classified into either aggressive or indolent lymphoproliferative diseases based on their natural history.
Prognosis of T-cell lymphoma is worse than B-cell
lymphoma
All lymphomas are derived from lymphocytes, and
are classified, where possible, based according to their
cell of origin. Approximately 75% of all cases of NHL
are of B cell origin, while the remainder are of T-cell
origin. Lymphomas of T-cell origin have been long
considered to represent a worse prognosis. While it is
not entirely clear why T-cell lymphomas this is the
case several theories have been advanced, including
the observation that patients with T cell lymphomas
generally present with more high risk disease, at least
based on the International Prognostic Index (IPI), at
diagnosis.16 While other hypotheses revolve around
the fundamental biological differences between Band T-cell lymphomas with regard to their intrinsic
chemosensitivity, the IPI allows for the risk stratification of patients with all types of lymphoma, and has
been applied to patients with both indolent and
1.0
L
PROBABILITY
0.8
0.6
0.4
H/I
0.2
0
0
LH/I
H
24
48
72
96
120
MONTHS
Lopez-Guillermo et al. Ann Oncol 1998
Figure 1. Peripheral T-cell lymphoma. Overall survival by
the IPI.
aggressive lymphomas. This index is based upon 5
important prognostic factors: (1) age, (2) Eastern
Cooperative Oncology Group (ECOG) performance
status, (3) abnormal levels of lactate dehydrogenase
(LDH), and (4) number of extra nodal sites, and (5)
stage. Patients are then classified according to the
number of risk factors they have as: low risk (IPI 1),
low intermediate risk (IPI 2), intermediate risk (3),
high-intermediate risk (IPI 4), or high risk (IPI 5).
All aggressive T/NK-cell lymphomas require systemic chemotherapy from the time of diagnosis, and
usually at relapse. As noted above, there are select T
cell subsets of disease that are considered more indolent. They may not require treatment at diagnosis, or
radically different treatments, including for example:
T cell prolymphocytic leukemia (T-PLL), T cell large
granular lymphocytic leukemia, primary cutaneous
CD30+ disorders including anaplastic large cell lymphoma (ALCL) and lymphomatoid papulosis, or
mycosis fungoides (MF)/Sézary syndrome. Because
of the more indolent and favorable natural history of
these diseases, they are not included in this study
population.
In general, T/NK- cell diseases are associated with
a worse prognosis compared to their B cell counterparts. Several informative clinical series of patients
have reported very poor median survivals for
patients with T cell neoplasms, with 5-year survival
rates of less than 30% and a median survival of less
than 2 years.17-19 Incredibly, the failure-free survival
for patients with high or intermediate high risk disease ranges from 0 to less than 10%, with virtually
no long term survivors.18-20 In one such study, the
complete response (CR) rate for patients with T/NKcell lymphoma was only 43%, while nearly half of all
patients were refractory to their initial up-front chemotherapy.19
A number of important studies have tried to identify the major factors that influence survival in
patients with T cell lymphoma. In one multivariate
analysis of 125 patients with T cell lymphoma
(PTCL-NOS [PTCL not otherwise specified], anaplastic large cell lymphoma; angioimmunoblastic lymphoma), the major parameters influencing outcome
were histological subtype and the IPI21. The fiveyear overall survival with all types of T cell lymphoma was only 43%, while the 5-year relapsed-free
survival was 69%.18 As expected, not all T cell lymphomas behaved exactly the same. The 5-year overall survival was 61% for anaplastic large cell lymphoma (ALCL), 45% for PTCL not otherwise specified (PTCL NOS), and 28% for angioimmunoblastic
lymphoma (AILD). Based strictly on the IPI (Figure 1)
in one such study, the 5-year survival for patients
with any T cell lymphoma was 74%, 49%, 21%, and
6% for the low, low-intermediate, high-intermediate, and high risk groups, respectively.18 These differences in survival as a function of the IPI are demonstrated in Figure 3.4.1 These data demonstrate that
IPI is an important adverse prognostic factor in PTCL,
and that patients with PTCL generally present with
more advanced IPI than patients with B-cell lymphomas, possibly accounting for some of the adverse
prognosis associated with PTCL.
It is reasonably well accepted that T cell lymphomas are more challenging to treat than B-cell
lymphomas althought this has never really established in any prospective randomized clinical trial.
Table 3.4.2 presents some of these data based on a
publication from Gisselbrecht et al.22 For example, in
this study, the 5-year survival rate for patients with 1,
2 or 3 risk factors with B- versus T cell lymphoma
was 63% versus 60%, 53% versus 36%, and 35%
versus 23% respectively. Similar trends were also
observed for the rates of complete remission. The
poor prognosis of these patients is underlined to
some extent by a report from the International
Lymphoma Study Group (ILSG) classification,19
which categorized different overall survival rates in
lymphoma by histological subtype into 4 broad
groupings,23 including: (1) those with 5-year overall
survival rate more than 70% including follicular lymphoma, marginal zone B-cell lymphoma of mucosaassociated lymphatic tissue lymphomas (MALT
type), and anaplastic lymphoma kinase (ALK) positive anaplastic large T cell lymphoma; (2) histologic
subtypes with 5-year survival rates of 50%-70%
including small lymphocytic, lymphoplasmacytoid,
and nodal marginal zone B-cell lymphomas; (3) lymphomas with 5-year overall survival rates of 30%49% including diffuse large B-cell lymphoma, primary mediastinal large B-cell lymphoma, and the
high-grade, B-cell, Burkitt-like and Burkitt lymphomas; and finally, (4) histologic subtypes with the
worse overall prognosis and 5-year survival rates less
than 30%, including PTCL, precursor T-lymphoblas-
tic lymphoma, and mantle cell lymphoma. These
results have been more recently confirmed by others
showing that patients with PTCL have an especially
poor outcome with a 5-year overall survival rate of
only 26% following treatment with standard doxorubicin containing regimens.
Collectively, these observations strongly suggest
that patients with T cell lymphoma are in urgent
need of additional new treatment options. This is
especially true for patients with recurrent or refractory disease who typically have a limited response to
salvage therapy and an extremely poor overall survival.
First-line treatment of T/NK cell lymphomas
The diversity and rarity of T cell lymphomas
poses a challenge to the systematic study of these
malignancies and the identification of standard therapeutic strategies. Recent reviews have attempted
to characterize treatment approaches to PTCL, and
to describe new agents that have the potential to act
on a variety of these diseases.10,20 The most common
types of PTCL have been typically treated with
combination chemotherapy programs. Without
question, the overwhelming majority of patients
with PTCL are initially treated with standard regimens containing cyclophosphamide, doxorubicin,
vincristine, and prednisone (CHOP), though there is
certainly no consensus that this represents an
acceptable standard up-front treatment program for
these patients.11,19,24 The response rates to CHOP
chemotherapy for patients with PTCL have been
reported to range between 50% and 70%. A variety
of other anthracycline-based combination therapies
have also seened promising, including infusional
treatment programs such as EPOCH which contains
etoposide, doxorubicin, vincristine, prednisone and
cyclophosphamide.25 Non-alkylator based treatment
programs exploiting nucleoside analogs like pentostatin, fludarabine, gemcitabine, and cladribine, as
well as monoclonal antibodies/immunotoxins, and
high-dose chemotherapy followed by peripheral
blood stem cell transplant (PBSCT) have also been
used in patients with T cell lymphomas with varying degrees of success.26
Increasingly, more and more patients with T cell
lymphomas are being referred for PBSCT in first
remission. While early data suggest that these strategies may induce meaningful durable complete remissions, there are no randomized data available that
allow us to determine whether PBSCT is superior to
other forms of up-front conventional combination
chemotherapy programs. These studies are currently
being planned. Such treatment approaches should
therefore be considered investigational until more
definitive data become available.
Treatment of relapsed and refractory T cell lymphomas
Because T cell malignancies are so uncommon,
there are few studies that have specifically investigated optimal treatment pathways for patients with
relapsed or refractory disease. High dose therapy followed by an autologous stem cell transplant, or less
commonly, an allogeneic stem cell transplant, is a
common treatment strategy for patients with
relapsed and refractory T cell lymphomas. PBSCT
has been successful only in patients with disease
responsive to chemotherapy. Patients with refractory
disease, or those transplanted with bulky disease do
not appear to derive any benefit from high dose chemotherapy.26 A cure may be possible for a small sub-set of
patients who have chemosensitive disease and are
transplanted in states of minimal residual disease.
The remainder of patients who have either refractory disease or are not candidates for PBSCT, are destined to succumb not to survive. It is these patients in
particular who are in urgent need of additional treatment options. In order to improve the outcome in
this patient population, novel agents need to be
developed and integrated into conventional treatment programs to improve treatment outcomes.
There have been relatively few studies focusing on
identifying novel drugs in the treatment of relapsed T
cell lymphomas, although several recent studies have
clearly established potentially promising new
drugs.27-30 For example, one relatively new drug which
is very promising for patients with relapsed or refractory cutaneous T cell lymphoma is gemcitabine, a
deoxycytidine analogue. Up till now, most of the
data with gemcitabine has focused on the more indolent cutaneous T-cell lymphomas. In one clinical trial
exploring the single-agent activity of gemcitabine in
cutaneous T cell neoplasms, 10 heavily treated
patients received 1,200 mg/m2 of weekly gemcitabine, which produced responses in 6 out of 10
patients, with 2 of these achieving complete remissions.29 The lack of standard response criteria, makes
this study data to interpret, however, the median
duration of response in this trial was 13.5 months. A
similar single-agent experience with gemcitabine in
44 patients with heavily pre treated mycosis fungoides and PTCL revealed a 70% response rate, with
an 11% CR rate.30 While these trials are encouraging,
it should be emphasised that these trials primarily
included patients with cutaneous T-cell lymphoma,
primarily MF. Mycosis fungoides is a more indolent
form of T-cell NHL, and is not at all representative of
the more aggressive forms of T-cell lymphoma which
is the subject of this proposal. A detailed experience
in aggressive PTCL awaits more focused clinical
studies of gemcitabine in this population.
Another interesting drug that has been used for
decades in the treatment of many lymphoprolifera-
tive malignancies, especially T cell malignancies, is
deoxycoformycin (pentostatin). Deoxycoformycin
has been used with benefit in select patient populations. In one such study, pentostatin was shown to
produce an overall response rate of 50% with 7%
CRs in patients with relapsed T cell lymphomas
(n=14).31 Unfortunately, despite a quite promising
ORR, the median duration of response was only 6
months. In another study, pentostatin produced a
response rate of 38%, with more than half of these
seen in patients with Sézary syndrome.32 Although
deoxycoformycin is active in T cell malignancies,
response tends to be relatively short and no patients
are cured. In addition to traditional cytotoxic
chemotherapy programs, it is clear that several exciting and new biological agents appear to act promisingly on T cell malignancies. One of the first agents
to prove efficacy in this class of drugs was denileukin
diftitox, or ONTAK. ONTAK is a fusion protein that
combines the receptor-binding domain of interleukin-2 and diphtheria toxin and is approved for
cutaneous T cell lymphoma. Preliminary results from
the important study in patients with relapsed/refractory B- and T cell NHL demonstrated an overall
response rate of 21% (n=28).33 Responses were seen
in both B- and T cell lymphomas as well as CD25+
and CD25- tumors. Given the benefit observed from
the addition of rituximab to CHOP in B-cell lymphomas additional studies are now underway to
explore the merits of combining ONTAK with conventional CHOP based therapy. Another promising
agent is the monoclonal antibody alemtuzumab
(Campath), a humanized anti-CD52 monoclonal
antibody which has been seen to act in the treatment
of relapsed or refractory T cell lymphomas, particularly in T PLL and PTCL. In one clinical trial, Keating
et al.34 demonstrated an overall response rate of 51%
with an impressive 40% complete remission rate.
However, despite the overall response rate, the median time to progression (TTP) was only 4.5 months.
Based on the promising action seen in T-PLL, a
prospective single agent alemtuzumab Phase 2 trial
was conducted strictly in patients with T-PLL. Again,
an impressive overall response rate of 76% was
observed, with a CR rate of over 60%, and a median
disease-free interval of 7 months.35,36 While alemtuzumab can benefit patients with relapsed T cell
lymphomas, it is also clear that it is a drug associated
with a number of toxicities, which can include the
potentially fatal reactivation of cytomegalovirus
(CMV). In a study exploring the benefits of alemtuzumab in a wider population of T cell lymphomas,
patients were found to have an overall response rate
of approximately 35%, though the treatment related
mortality was significant at approximately 35%.
Furthermore, emerging data now appear to suggest
O
NH2
H2N
N
H
N
N
N
O
OH
OH
O
N
H
Figure 2. (RS) 10-Propargyl-10deazaaminopterin (Pralatrexate).
that the rates of CD52 expression may be lower in
patients with PTCL compared to T-PLL, potentially
making alemtuzumab a less important drug for other
T-cell lymphomas besides T-PLL. Methotrexate,
while commonly used in select situations in the treatment of lymphoma, is not routinely used in the treatment of aggressive peripheral T-cell lymphomas. In
fact, available literature does not include studies
which have systematically addressed the issue of
how active MTX is in aggressive PTCL. While it may
be integrated into the maintenance treatment of lymphoblastic lymphoma, and is occasionally used in
low doses the treatment of cutaneous T-cell disorders37 few if any patients receive MTX as a part of
their routine care because of the belief that it has limited action. In fact, one report even suggests that the
use of MTX may increase the risk of transformation
in mycosis fungoides.38 It may no longer be feasible
to conduct a suitably large study of MTX in this
patient population because of this and the low number of patients. However, it is clear that the identification of novel antifolates with superior action
against lymphoproliferative malignancies represents
a valid therapeutic option for these patients, as we
will see below.
(RS) 10-Propargyl-10deazaaminopterin (Pralatrexate)
The 10 deazaaminopterins are a class of folate analogues (Figure 2) that demonstrate greater anti tumor
effects than methotrexate against murine tumor models and human tumor xenografts in nude mice.39-41 The
improved action is due to the more effective internalization by the 1-carbon, reduced folate transporter
(RFC-1) and the subsequent accumulation in tumor
cells through the formation of polyglutamylated
metabolites.
RFC-1 is a fetal oncoprotein that is almost exclusively expressed on fetal and malignant tissue. It is
believed to be the principal means through which
pralatrexate, though not necessarily all anti-folates,
enter the cell. This carrier protein has evolved to efficiently transport reduced natural folates into highly
proliferative cells to meet the demands for purine and
pyrimidine nucelotides during DNA synthesis.
Table 3. Biochemical properties of aminopterin, methotrexate, edatrexate, and pralatrexate in CCRF-CEM cells.
Treatment
Aminopterin
Methotrexate
Edatrexate
Pralatrexate
DHFR
Inhibition
Ki (pM)
Influx
Km (µM) Vmax (pmol/min/mg protein)
4.9±1
5.4±2
5.8±1
13.4±3
1.2±0.2
4.8±1.0
1.1±0.1
0.3±0.1
3.6±1.0
4.1±1.2
3.9±0.9
3.8±1.3
FPGS Activity
Vmax/Km
Km (µM)
Vmax (pmol/min/mg protein)
Vmax/Km
3.0
0.9
3.5
12.6
5.8±1
32.2±5
6.3±1
5.9±1
117±18
70±10
65±9
137±26
20.2
2.2
10.3
23.2
Ki :inhibitory constant; Km:binding constant; mg: milligram; min:minute; pM: picomolar; pmol: picomole; Vmax: maximum rate constant; µM:micromolar.
Table 3 demonstrates some of the essential pharmacokinetic properties of pralatexate. As can be seen
from the influx Vmax/Km data, pralatrexate is far more
efficiently transported than methotrexate being
incorporated at a rate nearly 14 times greater.
Similarly, the Vmax/Km for the folylpolyglutamyl synthetase (FPGS) mediated glutamylation reactions suggest that pralatrexate is also 10 times more efficiently polyglutamylated compared to methotrexate.
These biochemical features suggest that pralatrexate
should be a more powerful antineoplastic agent in
comparison to methotrexate, and could overcome
known mechanisms of MTX resistance where downregulation of RFC-1 and/or FPGS leads to clear MTX
resistance.
The cytotoxicity of pralatrexate was also compared
with that of methotrexate in multiple lymphoma cell
lines (1 transformed follicular lymphoma, 2 diffuse
large B cell lymphoma, 1 cell Burkitt’s, and 1
Hodgkin’s disease). Pralatrexate demonstrated more
than 10 times more cytotoxicity than methotrexate
in all cell lines.42
Clinical experience with Pralatrexate in patients with
lymphoma
Pralatrexate exhibits marked action in T-cell lymphomas
Until now, approximately 20 patients with lymphoma have received pralatrexate, including 11 with
T-cell lymphoma. Only one of these patients
received pralatrexate on an every other week basis,
having received a single dose of 135 mg/m2. The
remaining were enrolled on the phase I study of
weekly pralatrexate. These data demonstrate that the
first patient, with a chemotherapy refractory peripheral T-cell lymphoma NOS, experienced a complete
remission after a single dose that was documented
by PET scan to be PET negative. This patient had a
real chemotherapy refractory disease, having
received CHOP, ICE and Campath. There had been
almost no response to any of these initial therapies.
Because he developed gram positive bacteremia from
numerous healing skin lesions, he could not be
retreated on study (as dictated by the protocol). His
response lasted 3 months, and he was the last patient
treated on the every other week schedule.
The first patient treated on the weekly schedule
was a 65 year old woman with T-cell acute lymphoblastic
leukemia/lymphoma (T-cell ALL). She had received
induction chemotherapy and had been on
methotrexate maintenance for nearly 18 months
when she developed an aggressive systemic relapse
of her disease. There was extensive involvement of
her nasal passages, parotid gland, and lacrimal gland,
and she experienced difficulty breathing, seeing and
walking. She began treatment in dose cohort 1,
receiving a dose of 30 mg/m2 weekly for 3 weeks.
She experienced a marked improvement in her disease within a week of treatment, with marked resolution of disease in her lacrimal and parotid glands
and nasal passages. She achieved a documented complete
remission by PET and CT, and had a morphologically
normal bone marrow which was more than 35%
infiltrated with ALL lymphoblasts prior to pralatrexate administration. She was maintained on treatment
for 12 months, after which she developed a bone
marrow only relapse.
A second patient treated at this dose level with
chemotherapy refractory HTLV-1 ATLL which progressed rapidly after infusional EPOCH and interferon/Combivir also experienced a complete remission to
pralatrexate. He presented with a pathologically
proven relapse in the axilla that was non-bulky, and
had also been maintained on therapy for 12 months.
He is currently in CR. The other patient in this dose
cohort with a NK/T-cell lymphoma, achieved a
mixed response, having had resolution of her prior
disease, but developing a lesion on the tibial plateau
requiring radiation therapy.
Escalation of pralatrexate to 30 mg/m2 weekly for 6
weeks was well tolerated, with no DLTs in the cohort
studied. The first patient enrolled on this dose cohort
also had real chemotherapy refractory panniculitic T-cell
lymphoma. This particular disease actually belongs to a
more aggressive and very poor prognosis sub-set of Tcell lymphomas, namely the ???-T-cell receptor
rearranged T-cell malignancies. This patient relapsed
following standard chemotherapy for this disease,
and achieved a CT, PET and pathologically confirmed
complete remission after two cycles of pralatrexate. This
response has been maintained and he now has the
option to undergo an unmatched allogeneic stem cell
transplant in CR. The only patients with T-cell lymphoma to actually progress on the study drug carried
a diagnosis of angioimmunoblastic T-cell lymphoma,
both of whom received at least two cycles of pralatrexate. The two patients who died from infectious
complications following treatment and regression of
their lymphoma were discussed above. Both experienced objective disease regression, but were not
assessable for response having per protocol not completed a cycle of therapy. At the 45 mg/m2 dose level,
another patient with blastic NK/T-cell lymphoma of the
skin and blood achieved a rapid complete remission
based on complete blood counts, clinical findings and
photodocumentation. Now 4 months after registration he continues on study without any obvious toxicities. One patient with ALK positive anaplastic large
T-cell lymphoma (ALCL) is still within the first cycle
of therapy, and has not yet been evaluated for
response.
To summarize, of the 11 patients with T-cell lymphoma treated with pralatrexate (on a Phase 1 study),
5 have experienced complete remissions, with durations of response lasting 12, 12+, 9, 3, and 4+ months
respectively. Two of the eleven patients died from
infectious complications of their disease treatment,
both experiencing objective regressions of their lymphoma. Two experienced mixed responses, and 2
patients with angioimmunoblastic lymphoma experienced disease progression. One of two patients
with diffuse large B-cell lymphoma in the ongoing
weekly pralatrexate study has recently experienced a
partial remission.
Conclusions
It is clear that there are many new drugs in clinical
practice. A wide range of novel targets effecting such
unique pathways as the Bcl-2 family members, proteasome, histone deacetylase, MAP kinase and AKT
and mTOR for example, offer exciting and unique
opportunities to change the natural history of these
diseases. Furthermore, many of these agents appear
to increase the effects of conventional cytotoxic therapy. What remains most challenging however, is the
fact that diseases like mantle cell lymphoma and Tcell lymphoma are exceedingly rare diseases. Our
new understanding of biology, has rapidly outpaced
our ability to test new hypotheses in patients with
these diseases. The most significant breakthroughs
will only appear if we can continue to collaborate as
one community with a shared interest, and put all
patients with T-cell lymphoma on clinical trials. It
will be the constant transition of strategies from the
laboratory to the ward that will make the biggest
impact on the quality of our patients’ lives.
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Myeloma
Advances in myeloma biology: basis for new therapy
N.C. Munshi
Jerome Lipper Multiple Myeloma
Center Dana-Farber Cancer Institute
Boston VA Healthcare System
Harvard Medical School
Boston, MA, USA
2007;1:97-101
espite the advances in conventional and high dose chemotherapy,
Multiple myeloma (MM) remains
incurable. To overcome resistance to current therapies and improve patient outcome, novel biologically-based treatment
approaches are being developed based
upon targeting the MM cell as well as its
BM microenvironment, and to target
mechanisms whereby MM cells grow and
survive in the bone marrow (BM).1 To
achieve this goal, we and others have
defined oncogenomic changes, at both
genomic and expression level, that characterize the myeloma cells, and developed
model systems to study growth, survival,
and drug resistance mechanisms intrinsic
to MM cells. Importantly, both in vitro systems and in vivo animal models characterize mechanisms of MM cell homing to
BM, as well as factors (MM cell-bone marrow stromal cell interactions, cytokines,
angiogenesis) promoting MM cell growth,
survival, drug resistance, and migration in
the BM microenvironment.2 These model
systems have already stimulated the
development of several promising biologically-based therapies including thalidomide (Thal) and its more potent immunomodulatory analog lenalidomide,3 as well
as proteasome inhibitor Bortezomib4
which can overcome classical drug resistance and improve patient outcome. The
identification of new targets and the
development of newer agents is now
predicated based on a further understanding of genomic studies in MM and on
understanding of role of BM microenvironment on myeloma pathobiology. We
here describe the recent advances that
have helped the development of novel
tagets and agents in MM and which form
the basis for their clinical investigation.
D
Cytogenetic and genomic studies in MM
Recent genomics and proteomics studies
in MM have improved our understanding
of myeloma pathobiology, allowed for
molecular classification, identified novel
therapeutic targets, and provided the scientific rationale combination therapies to
increase tumor cell cytotoxicity and overcome drug resistance.
Most if not all MM harbours cytogenetic abnormalities including numerical
abnormality with hyperdiploid karyotypes with infrequent translocations
(<30%) or nonhyperdiploid karyotypes
with high prevalence of translocations
involving Ig gene.5 The chief characteristic
of translocations in B cell malignancies as
well as myeloma is translocations involving chromosome 14q32.6 In one third of
the patients these translocations involve
chromosomal locus 11q13 (cyclin D1), and
lead to cyclin D1 upregulation or, indirectly through other intermediate transcriptional factors, upregulation of cyclin D2
and D3. Upregulation of cyclin D genes
may render plasma cells responsive to proliferative stimuli as well as further genomic changes.7 Cyclin D therefore has
become important target for prognostic
classification as well as therapeutic target
in myeloma.8 Another oncogene dysregulated by t(4;14) translocation is the fibroblast growth factor receptor 3 (FGFR3)
gene. Ectopic expression of FGFR3 promotes MM cell proliferation and prevents
apoptosis.9,10 It is oncogenic in a murine
model, as evidenced by transformation of
hemopoietic cells. FGFR3 has become a
focus of intense therapeutic interest with a
number of small molecular FGFR3
inhibitors being evaluated in preclinical as
well as clinical studies. This translocation
also involves a novel gene MMSET with
resultant IgH/MMSET hybrid transcripts.11
The less frequent chromosomal partner in
14q32 translocation are 8q24 (c-myc)
18q21 (bcl-2), 11q23 (MLL-1), 16q23 (cmaf) and 6p25 (IRF)11 all with potential
therapeutic interest. Clinical studies have
shown that MM patients with t(4;14), or
t(14;16) translocations or chromosome 13
or 17p deletions1,5 have a poor prognosis
with conventional or high-dose therapies.
However, recent studies using novel
Cytokines
IL-6, IGF-1,
VEGF, Il21,
TNF-α, SDF-1
Ras
JAK
Raf
MEK
p42/44 MAPK
Proliferation
Bcl-xL
Mcl-1
STAT3
MM-BMSC
Adhesion
Drug
resistance
antiapoposis
mTOR
Bad
PI3-K
Akt (PKB)
NF-κB
cyclin D
FKHR
p27
PKC
Table 1. Cell surface adhesion molecules on MM cells and
BMSCs.
MM CELL
BMSC
CD56
LFA-1
CD38
VLA-4
VLA-5
Syndecan
HSP
ICAM-1
CD31
VCAM-1
ECM
Fibronectin
Fibronectin
Type I Collagen
HSP = Heparan Sulfate Proteoglycan; ECM = Extra Cellular Matrix.
agents have demonstrated the ability of both bortezomib and Lenalidomide to overcome the adverse
effects of these genetic factors on patient outcome.12
Recently studies with high-resolution analysis of
recurrent copy number alterations (CNAs) using
array based comparative genomic hybridization
(aCGH) and its integration with expression profiling
data have identified areas of chromosomal amplifications and deletions in both MM cell lines as well as
primary patient samples. These studies have provided a DNA based classification systems for MM and
have also defined 87 discrete minimal common
regions (MCRs) within recurrent and highly focal
CNAs. In this study, integration with expression data
has generated a list of MM gene candidates within
these MCRs, improving our understanding of the disease pathogenesis, and promoting the identification
of targeted drugs.13
Transcriptomic changes in MM
Gene microarray profiling has shown that the
expression profile of monoclonal Gammopathy of
Undetermined Significance (MGUS) and MM is similar but distinct from that of normal plasma cells.
These studies have defined the changes associated
Celicycle
Migration
Figure 1. Signaling cascades
mediating growth, survival, drug
resistance and migration in MM
cells.
with the progression of normal plasma cells to MGUS
and then to multiple myeloma.14 These studies have
provided the basis for RNA based prognostic classification.8 Along with aCGH studies, expression profile
has also identified novel therapeutic targets including
potential novel targets on cell surface for monoclonal
antibody development, intracellular targets for development of small molecule inhibitors, and targets for
immune-based therapies. Expression profiling preand post-therapeutic intervention have allowed for
identification of gene expression patterns that can
predict response versus resistance to therapy. These
results are likely to lead to expression-based individualized therapy in the future.
Bone marrow microenvironment and MM cell interaction
We and others have characterized the mechanisms
whereby MM cells home to the BM and adhere to
BM stromal cells (BMSCs) and extracellular matrix
(ECM) protein.15 These studies have identified adhesion molecules mediating MM cell binding to
fibronectin and BMSCs (Table 1) and lead to MM cell
growth, survival, anti-apoptosis as well as development of drug resistance. Besides the adhesion mediated signaling, the binding leads to secretion of various growth promoting cytokines by both MM cells
as well as BMSC. BMSCs secrete cytokines, such as
interleukin-6 (IL-6),16 insulin-like growth factor-1
(IGF-1),17 vascular endothelial growth factor (VEGF),18
and stomal cell derived growth factor (SDF-1)α,19
which increase MM cell growth, survival, and the
development of drug resistance via MAPK and PI3K/Akt, Jak/STAT, and PI3-K/Akt signaling cascades,
respectively (Figure 1).2 IGF-1 has been shown to
increase MM cell growth, survival, and drug resistance.17 VEGF secreted by both MM cells and BMSCs
is upregulated by the binding of MM cells to BMSCs.
It space increases MM cell growth angiogenesis,
although reports correlate an increase in angiogenesis
with MM stage its precise pathophysiologic significance in MM BM is undefined. VEGF induces migration via PKC signaling.18 Tumor necrosis factorα
(TNFα)? does not directly alter MM cell growth and
survival. However, it induces NF-κB dependent
upregulation of adhesion molecules (ICAM-1,
VCAM-1) on both MM cells and BMSCs, resulting in
increased binding and related induction of IL-6 and
other cytokine secretions.20 The role of other
cytokine such as Hepatocyte Growth Factor (HGF),
IL-15 and IL-21 in MM cell growth and survival has
been described.21, 22 Recombinant IL-1β stimulates IL6 production and thereby MM cell proliferation
increases leading to investigation of anti- IL-1β in
MGUS. Transforming growth factor-β (TGF-β) is
secreted by MM cells and triggers IL-6 secretion in
BMSCs and paracine IL-6 mediated tumor cell
growth. TGF-β secreted by MM cells probably also
contributes to the immunodeficiency characteristic
of MM by downregulating B cells, T cells, and natural killer cells, without similarly inhibiting the
growth of MM cells.23 Macrophage inflammatory
protein-1α (MIP-1α) is an osteoclast stimulating factor in MM.24
Expression profile studies of MM cells and BMSC
following their adhesion in both in vitro and in vivo
models of human MM in mice have identified upregulation of growth, survival, and drug resistance genes
in MM cells; upregulation of adhesions molecules on
MM cells and BMSCs, and changes in cytokines in
BMSCS.25 This MM-BMSC and MM-ECM interaction confers cell adhesion mediated (CAM) drug
resistance to conventional agents with induction of
p27 and G1 growth arrest and also induces melphalan resistance.26,27 However, the novel agents including Thalidomide (Thal) and its immunomodulatory
derivative lenalidomide, as well as proteasome
inhibitor Bortezomib,3,28 target both MM cells as well
as its microenvironment and are able to overcome
CAM drug resistance to conventional agents.
These newer agents work through various mechanisms operative in the BM environment. Besides the
variable extent of activity on MM cells themselves,
they inhibit MM-BMSC interaction and the consequent local cytokine production, and they decrease
VEGF and FGF secretion which are important for
neoangiogenesis.3,28 Lenalidomide, and to some
extent thalidomide, also have significant immune
effects including improving the antigen presenting
function of dendritic cells, co-stimulation of T cells
via the B7-CD28 pathway and, by secretion of IL-2,
it upregulates T and NK cell anti-MM activity.29
Lenalidomide can also upregulate antibody dependent celluar mediated cytotoxicity.30
Molecularly based rational combination therapies
The improved understanding of the molecular
changes in MM and the effects of various agents on
gene expression as well as signaling profiles have led
to the development of molecularly-based rational
combination therapies. The molecular studies have
defined the mechanism of apoptosis by various
agents and discovered mechanisms of drug resistance. Exciting, preclinical studies suggest improved
activity when these novel agents are combined with
conventional agents or with each other. These studies have established a new treatment paradigm targeting the MM cell in its BM microenvironment to
further clarity MM pathogenesis as well as overcome
drug resistance and improve patient outcome.
Expression profiling of MM cells following their
treatment with various agents can provide the preclinical rationale for combining novel targeted therapies. For example, expression profile of MM cells following Bortezomib treatment demonstrates induction of various pathways’ upregulation of both ubiquitin/proteasome and stress response gene transcripts,28 specifically Hsp90, which plays a major role
in protein folding and ubiquitin-mediated proteasomal protein degradation. In vitro studies show that
Hsp90 inhibition by 17AAG can block the Hsp90
stress response induced by Bortezomib and thereby
increase MM cell apoptosis.31 These expression profile studies therefore provided the framework for a
clinical trial coupling Hsp90 inhibitor KOS953 with
Bortezomib to enhance MM cells cytotoxicity and
even overcome Bortezomib resistance.32
Similarly, expression as well as proteomic changes
following Bortezomib treatment suggested that
Bortezomib inhibited DNA repair.33 Subsequent in
vitro studies confirmed that combining Bortezomib
with DNA damaging agents (alkylating agents and
anthracyclines) can improve sensitivity or even overcome resistance to these agents in MM. Already clinical protocols combining Velcade with Doxil and
with melphalan are producing very promising clinical
results.
The apoptotic signaling cascades triggered in MM
cells by both conventional and novel agents have
been characterized. For example, use of lenalidomide
with Bortezomib triggers both caspase 8 and caspase
9-mediated MM cell death.34 An ongoing clinical trial
has demonstrated remarkable activity in patients
treated with combination lenalidomide and
Bortezomib even in patients resistant to either agent
alone.35
MM cell signaling studies have defined the role of
the aggresome in degrading ubiquitinated protein in
MM. Blocking the aggresome with HDAC6 inhibitor
tubacin induces a compensatory upregulation of the
proteasome while, blocking the proteasome with
Bortezomib triggers a compensatory upregulation of
the aggresome. Blocking both the proteasome and
aggresome with Bortezomib and tubacin respectively induces synergistic toxicity.36 The HDAC inhibitor
LBH596 is under clnical investigation singly and in
combination with Bortezomib.37 mTOR inhibitor
rapamycin sensitizes MM cells to both conventional
(dexamethasone) and novel therapies.38 Bortezomib
inhibits growth (MEK/ERK) and survival (Jak/STAT)
signaling, but activates Akt, providing the preclinical
rationale for combining Bortezomib with the Akt
inhibitor perifosine.39 Gene expression profiling of
patient tumor samples has also identified genes associated with lack of response to Bortezomib. Hsp27
upregulation correspond to intrinsic or acquired
Bortezomib resistance. Preclinical studies showed
that p38MAPK inhibition downregulated Hsp27
expression and restored velcade sensitivity in resistant MM cell lines providing the basis for a trial combining these two agents.40 Finally, the immunomodulatory agent Lenalidomide can markedly increase
antibody-dependent cellular cytotoxicity (ADCC)
providing the rationale to combine monoclonal antibodies with novel drugs.30
Once the in vitro potential of these novel agents is
demonstrated, they are tested for their efficacy in
murine models. Novel murine models simulating
human MM have been developed to improve our
ability to confirm novel targets and agents in MM. A
SCID mice bearing human bone41 and a conditional
tranagenic mouse model expressing xbp-1 gene that
develops MGUS and MM with characteristics similar
to human MM provides such tools.42 Thalidomide,
Lenalidomide and Bortezomib all inhibit human MM
cell growth, decrease associated angiogenesis, and
prolong host survival in animal models. These agents
have already demonstrated marked clinical anti-MM
activity confirming the usefulness of preclinical models to identify and confirm novel therapeutics and
their approval for clnical use in patients with newlydiagnosed myeloma (Thalidomide) and with
relapsed myeloma (Bortezomib and Lenalidomide).
Ultimately, it may be possible to carry out gene and
protein expression profiling on individual patient
samples to allow the selection of those agents most
likely to be effective. For example, a comparison of
gene expression profile of patient MM cells to normal
twin plasma cells showed surprisingly few significant
differences.43 This data may allow the selection of
those combinations of agents targeting these gene
products to optimize clinical response.
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Myeloma
Multiple myeloma: diagnosis, staging and
criteria of response
J. Bladé
Haematology Department, Institute
of Hematology and Oncology,
Hospital Clínic, IDIBAPS,
Barcelona, Spain
2007;1:102-107
onoclonal gammopathies are
characterized by the existence of
a plasma cell clone which produces a monoclonal protein (M-protein,
paraprotein or M-component). The clinical spectrum ranges from asymptomatic
disorders such as monoclonal gammopathy of undetermined significance (MGUS)
or smoldering multiple myeloma (SMM)
to symptomatic multiple myeloma (MM).
When the light chain is amyloidogenic the
clinical picture is that of primary systemic
amyloidosis (AL) which results from the
tissue and organ light chains deposition.
The median survival of patients with MM
is approximately 3 years. However, this
varies considerably from patient to
patient. Finally, the plasma cell clone has
been classically characterized by a high
degree of resistance to treatment, and different degrees of response must be considered. The introduction of high-dose therapy/stem cell transplantation (HDT/SCT)
and the availability of new drugs with
novel mechanisms of action has resulted
in a higher tumor reduction with a significant number of patients achieving complete remission. For this reason, new
response criteria were developed. These
have been recently revisited. In this
overview, the differential diagnosis among
the above mentioned monoclonal gammopathies with the criteria for symptomatic disease, the prognostic factors/staging systems and the criteria of response
for MM are reviewed.
M
than 30 g/L and less than 10% bone marrow clonal plasma cells with no evidence
of other B-cell lymphoproliferative disorder and no symptoms or organ or tissue
impairment attributable to the monoclonal gammopathy (Table 1).1,3,4 The transformation rate is about 1% per year with an
actuarial probability of malignant evolution at 25 years of follow-up of 30%.
When the different causes of death are
considered, the actual probability of
malignant transformation at 25 years of
follow-up is only 11%, much lower than
the actuarial prediction.5 The main factors
predicting progression are: the M-protein
size, IgA-type and abnormal free light
chain ratio,6 It is the authors’s experience
the so-called evolving type (rising M-protein during the first years of follow-up) is
the most important predictor for malignant evolution.7 When the monoclonal
protein and the proportion of bone marrow plasma cells are consistent with
MGUS but the patient has a nephrotic
syndrome, congestive heart failure,
peripheral neuropathy, orthostatic hypotension or massive hepatomegaly the
most likely diagnosis is primary systemic
amyloidosis resulting from the deposition
of amyloidogenic light chains.4 In a patient
with constitutional symptoms, lytic bone
lesions, a small M-spike and less than 10%
plasma cells in the bone marrow, the most
likely diagnosis is metastatic carcinoma
with a coincidental MGUS.
Smoldering multiple myeloma
Classification and diagnosis
The criteria for classification and diagnosis of monoclonal gammopathies has
been reviewed by the International
Myeloma Working Group.1
Monoclonal gammopathy of undetermined
significance
MGUS has a high prevalence (3.2 and
5.8 percent in people over 50 and 70 years
of age respectively).2 It is characterized by
the presence of a serum M-protein lower
| 102 |
The term smoldering multiple myeloma
was clearly defined by Kyle and Greipp as
the presence of a serum M-protein higher
than 30 g/L and a proportion of bone marrow plasma cells equal or greater than
10% in the absence of lytic bone lesions or
clinical manifestations due to the monoclonal gammopathy.8 More recently, the
International Myeloma Working Group
considered that the term asymptomatic
myeloma might be more appropriate than
smoldering multiple myeloma. This condition
Table 1. Monoclonal gammopathy of undetermined significance (MGUS).
higher and all patients will eventually evolve into a
symptomatic MM.9-14
Serum M-protein < 30 g/L
Symptomatic multiple myeloma
Bone marrow clonal plasma cells < 10%
The diagnosis of symptomatic MM requires the
presence of an M-protein in serum and/or urine,
increased plasma cells in the bone marrow or plasmacytoma, and related organ or tissue impairment
(including bone lesions). The more common symptoms are weakness and fatigue from anemia and
bone pain due to the skeletal involvement. Some
patients may have no symptoms but have related
organ or tissue impairment. Clinical features may
include anemia, skeletal involvement (lytic lesions
and/or severe osteoporosis with or without compression fractures), renal failure, hypercalcemia, recurrent
bacterial infections, extramedullary plasmacytomas,
hiperviscosity (very rare) or associated amyloidosis
(Table 3). The criteria agreed by the International
Myeloma Working Group for the diagnosis of symptomatic MM are depicted in Table 4.1 Note that no
serum or urine M-protein levels were included.
About 40% of patients with symptomatic MM have
a serum M-protein lower than 30 g/L and 3% have
non-secretory MM (see Table 5 for the diagnostic criteria of non-secretory). Also, no minimal proportion
of bone marrow plasma cells was required, since
about 5% of patients with symptomatic MM have
less than 10% plasma cells in the bone marrow. In
fact, the most critical criterion for disease requiring
cytotoxic therapy is the evidence of organ or tissue
impairment (end organ damage) manifested by the
clinical features mentioned above.
The criteria for solitary plasmacytoma of bone and
extramedullary plasmacytoma are shown in Tables 6
and 7 respectively.1 Plasma cell leukemia is an aggressive variant of MM defined by a peripheral blood
absolute plasma cell number of at least 2×109/L and
more than 20% plasma cells in the peripheral blood
differential white cell count.1 Plasma cell leukemia is
classified as primary when it presents in the leukemic
phase (60% of cases) or as secondary when it results
from the transformation of a previously recognized
MM.
No evidence of other B-cell proliferative disorders
No related organ or tissue impairment
Table 2. Asymptomatic myeloma (smouldering myeloma).
Serum M-protein ≥30 g/L or urine light chain ≥1g/24h
or
Bone marrow clonal plasma cells ≥10%
No related organ or tissue impairment (no end organ damage including bone
lesions) or symptoms.
Table 3. Myeloma-related organ or tissue impairment (end
organ damage) (ROTI) due to the plasma cell proliferative
process.
Increased serum Calcium
Renal insufficiency
Anemia: hemoglobin 2 g/dL below the lowest normal limit
Bone lesions: lytic lesions or osteoporosis with compression fractures (possibly
confirmed by MRI or CT)
Other: symptomatic hyperviscosity (rare), amyloidosis, recurrent bacterial infections (> episodes in 12 months), extramedullary plasmacytomas.
CRAB (calcium, renal insufficiency, anemia or bone lesions).
Table 4. Symptomatic multiple myeloma*.
M-protein in serum and /or urine
Bone marrow (clonal) plasma cells or plasmacytoma**
Related organ or tissue impairment (end organ damage, including bone lesions)
* Some patients may have no symptoms but have related organ or tissue impairment; ** If flow cytometry is performed, most plasma cells (>90%) will show a
“neoplastic” phenotype.
Table 5. Non-secretory myeloma.
No M-protein in serum and/or urine with negative immunofixation*
Bone marrow clonal plasmacytosis ≥ 10% or plasmacytoma
Related organ or tissue impairment (end organ damage, including bone lesions)
*Oligosecretory: urine light chain excretion < 200 mg/24hrs (usually of kappa
type).
was defined as the presence of an M-protein equal or
higher than 30 g/L and/or ≥10% bone marrow plasma cells in the absence of symptoms or organ or tissue impairment due to the monoclonal gammopathy1 (Table 2). About 10% of patients diagnosed with
MM have smoldering disease.9,10 This situation is
clinically and biologically very close to that observed
in MGUS. However, the plasma cell mass is much
Prognostic factors and staging systems
The median survival of patients with MM is about
3 years. However, survival varies from one patient to
another. While some patients die within the first few
months from diagnosis others survive for more than
5 and even for more than 10 years. This variability in
survival relates mainly to prognostic factors associated with certain characteristics of both the host and
the tumor. Since the first report in 1967 by Carbone
et al.,15 many studies on prognostic factors have been
published resulting in the identification of a large
Table 6. Solitary plasmacytoma of bone.
No M-protein in serum and/or urine*
Single area of bone destruction due to plasma cell involvement
Bone marrow not consistent with multiple myeloma
Normal skeletal survey (and MRI of spine and pelvis if performed)
No related organ or tissue impairment (no end organ damage other than solitary
bone lesion)
*A small M-component may sometimes be present.
Table 7. Extramedullary plasmacytoma.
No M-protein in serum and/or urine*
Extramedullary tumour of clonal plasma cells
Normal skeletal survey
No related organ or tissue impairment (end organ damage including bone
lesions)
*A small M-component may sometimes be present.
Table 8. Cytogenetic prognostic subgroups in multiple
myeloma.
Good/average prognosis
Hyperdiploidy
t(11;14)(q32;q32): cyclin D1 upregulation
Bad prognosis
Hypodiploidy
t(4 ;14)(p16.3 ;q32) : FGFR3 & MMSET upregulation
t(14 ;16)(q32 ;q23) : c-MAF upregulation
Chromosome 13 deletion
Chromosome 1 abnormalities : 1q gains, 1p losses
number of features with prognostic impact. In this
review we will consider: 1) host factors, 2) factors
related to the malignant clone, and 3) factors associated with tumor mass and disease complications. We
will also review the different staging systems developed for MM, with particular emphasis on the
International Staging System (ISS) as well as the
impact of response to therapy on long-term outcome.
Host factors
Age is an important prognostic factor in MM.
Thus, the median survival of patients younger than
40 and 30 years was 54 and 87 months respectively.16,17 In contrast, the median survival of patients over
70 years was only 23 months.18 Another critical prognostic feature is the performance status (PS) at the
time of diagnosis. In fact, patients with a PS greater
than 2 have a significantly poorer survival compared
with those with a lower PS.19
Factors related to the malignant clone
In the Mayo Clinic experience, plasmablastic morphology is an important prognostic feature.20
However, plasma cell proliferative status, measured
either by plasma cell labelling index or by flow
cytometry, is one of the most reliable prognostic indicators in patients with MM.21
Cytogenetic status has emerged as the most important prognostic factor in MM. As shown in Table 8,
patients with hyperdiploidy have a good outcome in
contrast with those with hypodiploidy, and patients
with t(11;14) have an average survival. The cytogenetic poor prognostic features are: retinoblastoma
(RB) and P53 deletions and immunoglobulin heavyheavy chain (IgH) translocations, mainly the t(4;14)
and t(14;16).22 Although RB deletions have emerged
as an important prognostic indicator in many studies,
the coexistence of RB deletions with IgH translocations has raised the question of whether the prognostic impact of each abnormality may be influenced by
the other. In considearation of this, the Spanish
Myeloma Group has just reported that in a multivariate analysis that the only features independently
affecting survival were t(4;14), RB deletion associated
with other cytogenetic abnormalities, age > 60 years,
high proportion of S-phase cells and the advanced
disease stage according to the ISS.23Thus, RB deletion
as single cytogenetic abnormality would not have a
negative prognostic impact while t(4;14) is the worst
prognostic feature. On the other hand, gains in chromosome 1 is one of the more frequent cytogenetic
findings in patients with MM. The up-regulation of
1q genes as well as the down-regulation of 1p genes
is a poor prognostic feature.24 Thus, gene expression
profiling studies have shown that the high expression of the gene CKS1B (located in 1q21) is a marker
of very short survival. Thus, a high expression of
CKS1B, plus other cytogenetic abnormalities excluding t(11;14), define a poor prognosis population even
in patients undergoing a tandem transplant
approach.24
Prognostic factors associated with plasma cell mass and
disease complications
The most important prognostic marker in MM is
beta2-microglobulin.25 Its serum levels are related to
both the plasma cell mass and the renal filtering
capacity. Its value has been reproduced in many studies and has been successfully included in many staging systems (see below). It must be emphasized that
its measurement is only useful at the time of diagnosis and not for disease monitoring during follow-up.
The hemoglobin level, and in some series a low
platelet count, resulting from bone marrow involvement by plasma cells is an important prognostic factor. The presence of circulating plasma cells, identified either by morphology or immunophenotyping,
is associated with advanced disease and is an independent prognostic factor.26 Renal function impair-
Table 9. Main staging systems in multiple myeloma.
Author, year
Durie and Salmon, 1975
Merlini et al., 1980
MRC, 1980
Parameters
Other
Hb, Ca, M-protein, bone lesions
Renal function
%PC, Cr, and Ca (IgG)
Hb, Ca, M-protein (IgA)
Hb, urea, PS
than 11,000 patients included in cooperative studies
and from large individual institutions.37 The ISS is
based on the serum levels of beta2-microglobulin and
albumin. It defines three risk groups with median
survival of 62, 44 and 29 months respectively.
Importantly, this classification was reproduced in all
age groups, in patients with different geographic origins, and in patients treated with standard dose therapy as well as those undergoing autologous transplantation.
Cavo et al., 1989
D & S, platelet count
Greipp et al., 1988
β2-microglobulin, LI
Bladé et al., 1989
Albumin, urea
Response to therapy as prognostic factor
San Miguel et al., 1989
Hb, Cr, PS, PI
San Miguel et al.,1995
S-phase, β2-microglobulin, age, PS
Before the introduction of novel drugs, few
patients treated with conventional chemotherapy
achieved CR and the correlation between the
degree of tumor response and survival was the subject of debate.38 In fact, in many studies, the stabilization of tumor burden was a more powerful
prognostic indicator than the degree of tumor
reduction.38-42 In contrast with the low CR rate
attained with conventional chemotherapy, between
35 and 50 percent of patients enter CR after highdose therapy/stem cell support (HDT/SCT).43-45 A
correlation between the degree of response and survival has been shown after HDT/SCT.43,44-47 So
patients entering CR post-transplant have a significantly longer EFS and OS than those who enter in
PR or do not respond.46-48 This would suggest that
there is a difference in the quality of CR after conventional chemotherapy and after HDT/SCT.
However, in the MD Anderson experience, the EFS
and OS survival of patients achieving CR was similar irrespective of whether the CR was achieved
with conventional or with HDT/SCT.46,48 This thus
raises the question of whether patients already in
CR with primary therapy gain benefit from highdose intensification. With the incorporation of
novel therapies (thalidomide, bortezomib, lenalidomide) in the primary therapy, up the one-third of
patients with MM achieve CR.49-52 A longer followup is needed to establish the impact of these CR on
PFS and OS and to evaluate how far the increased
tumor reduction with initial chemotherapy can represent a real influence on the long-term outcome of
patients undergoing HDT/SCT intensification.
IMWG, 2005
β2-microglobulin, albumin
MRC, Medical Research Council; IMWG, International Myeloma Working
Group; Hb, haemoglobin; Ca, calcium; PC, plasma cells; Cr, creatinine; Ig,
Immunoglobulin; PS, performance status; LI, labeling index; PI, paraprotein
index.
ment due to tubular damage by nephrotoxic light
chains is one of the most adverse prognostic indicators.27,28 In the overall series, the median survival of
patients with renal failure is less than 1 year. The
reversibility rate of renal failure in patients with MM
ranges from 20% to 60%.27,28 The survival of patients
with reversible renal failure is similar to the survival
of patients with initial normal renal function. In the
author’s experience, the factors associated with renal
function recovery are a serum creatinine level lower
than 4 mg/dL, a 24-hour urine protein excretion
lower than 1 g, and a serum calcium level higher than
11.5 mg/dL.28
Staging systems for multiple myeloma
A number of prognostic classification systems for
MM have been developed during the last 30 years
(see Table 9). These staging systems are usually
based on prognostic factors derived from multivariate regression models. The most widely used classification was proposed by Durie and Salmon in
1975.29 Based on a mathematical model to assess
tumor cell mass, three stages based on the four
parameters showing a higher correlation with the
number of plasma cells (M-protein size, hemoglobin,
calcium, and extent of bone disease) were established. Each stage was subclassified as A or B according to renal function status. Despite the widespread
use of the Durie and Salmon staging system, there
has been no universal agreement on its prognostic
value. Unfortunately, neither have other proposed
systems been entirely satisfactory.30-36 In the search
for a more useful and reproducible prognostic classification, the International Myeloma Working Group
has recently developed the so-called International
Staging System (ISS) derived from a total of more
Criteria of response
First response criteria
Response criteria were first developed by the
Chronic Leukemia and Myeloma Task Force
(CLMTF) in 1968 and were reviewed by the same
group in 1973.53 The main response parameter was a
an minimum 50% a reduction in the M-protein. In
1972, the Southwest Cancer Chemotherapy Study
Group, now the Southwest Oncology Group
(SWOG), defined partial response as a reduction of at
Table 10. International uniform response criteria for multiple myeloma.
Response Category*
Criteria
CR
Negative IF (serum and urine)
5% BMPC
No soft tissue plasmacytomas
sCR
As above plus
Normal FLC ratio
Absence of clonal plasma cells**
VGPR
≥90% serum M-protein ⇓
Urine M-protein < 100 mg/24h
PR
≥50% serum M-protein ⇓
≥90% urine M-protein ⇓ or < 200 mg/24hrs
≥50% ⇓ soft tissue plasmacytomas
*All response categories require two consecutive measurements made at any time;
** Determined by immunohistochemistry or immunofluorescence; CR, complete
remission; sCR, stringent complete remission; VGPR, very good partial response;
PR, partial response; IF, immunofixation; BMPC, bone marrow plasma cells;
FLC, free-light chain.
least 75% in the calculated serum paraprotein synthetic rate and/or a decrease of at least 90% in urinary light chain urine protein excretion sustained for
at least two months.54 The CLMTF or SWOG criteria
were used in most clinical trials, although modifications to the original proposals were frequently made.
An exception to the above criteria was the Medical
Research Council Myelomatosis trials, in which
response was evaluated by the proportion of patients
achieving the so-called plateau phase.This phase consists of a period of stability after chemotherapy in
which tumor progression does not occur despite the
persistence of measurable disease. The minimum
period of stability required to define plateau was 3
months.55 Since complete remissions (CR) were
rarely observed with the old conventional dose
chemotherapy, neither the CLMTF nor the SWOG
response criteria included a definition of CR. In addition, there was no definition for disease progression
or relapse.
The EBMT, IBMTR and ABMTR criteria for response, relapse
and progression
With the introduction of high-dose therapy/stem
cell transplantation, the M-protein has disappeared in
a significant number of patients, ans is therefore
associated with a significant survival prolongation. In
this context, the EBMT developed new criteria defining CR (negative inmunofixation in serum and urine
in the absence of increased plasma cell in the bone
marrow), partial response (PR), minimal response
(MR), as well as criteria for relapse (reappearance of
the M-protein in patients who had achieved CR), and
progression from PR or MR.56 It is important that any
type of response should be maintained for a minimum of six weeks. These criteria have been used
over recent years in both transplant and in non-transplant series as well as in prospective phase II and III
clinical trials, and have been shown to be useful and
reproducible.
The international uniform response criteria for multiple
myeloma
The International Myeloma Working Group has
expanded the EBMT criteria and categories for stringent CR and very good partial response have been
added.57 Also, the serum free light chain measurements have been included for the definition of stringent CR and for the evaluation of response in
patients with non-secretory or oligosecretory disease
(Table 10). All response categories require two consecutive assessments at any time in contrast to the
EBMT which required an interval of at least six
weeks between the two measurements for response
confirmation. In addition, the time to event, duration
of response, clinical relapse and time to alternative
treatment are emphasized as critical end points.57
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Myeloma
Novel treatment approaches in multiple myeloma
A. Palumbo1
I. Avonto1
P. Falco1
F. Cavallo1
T. Caravita2
M.T. Petrucci3
M. Cavo4
M. Boccadoro1
1
Divisione di Ematologia
dell'Università di Torino, Azienda
Ospedaliera S. Giovanni Battista,
Torino;
2
Cattedra e Divisione di Ematologia,
Università Tor Vergata, Ospedale S.
Eugenio, Roma;
3
Dipartimento di Biotecnologie ed
Ematologia, Università La Sapienza,
Roma;
4
Istituto di Ematologia e Oncologia
Medica “Seragnoli”, Università di
Bologna, Italy.
2007;1:108-114
ultiple myeloma is the second
most common oncohematological disease. At diagnosis, the
majority of patients are older than 65
years. About 35% of myeloma patients
are aged under 65, 28% are between 6574 and 37% are over 75.1 The current
changes of the European demographic
curves will probably increase the incidence of elderly patients in the near
future. In newly diagnosed myeloma
patients under 65, high-dose melphalan
followed by autologous stem cell transplantation is considered the standard of
care. In elderly patients, usually over 65,
oral melphalan and prednisone (MP) have
been considered the standard.
The recent discovery of new drugs, such
as thalidomide, lenalidomide and the proteasome inhibitor bortezomib, targeting
both the myeloma cells and the bone marrow microenvironment, have significantly
increased the clinical efficacy of the old
chemotherapy regimens. The challenge is
now to define the optimal sequence and
combination of these drugs to allow a significant impact on the natural history of
the disease.
M
New induction regimens for younger patients
candidates for autologous transplantation
In patients younger than 65 years, melphalan 200 mg/m2 supported by autologous stem cell transplantation appears to
be the treatment of choice. Several studies
clearly demonstrated the superiority of
melphalan 200 mg/m2 in terms of
response rate and event-free survival
when compared with conventional treatments. Results were less consistent when
overall survival was examined.2 In a randomized study, tandem autologous transplantation improved complete response
(CR) rates, prolonged event-free and overall survival in comparison with the single
transplantation.3 These results were particularly evident among patients who did
not have a very good partial response
(VGPR) after one transplantation. The 7| 108 |
year overall survival was 11% in the single-transplant group and 43% in the double-transplant group (p<0.001).
Autologous transplantation is usually
preceded by de-bulking chemotherapy
with steroid-based regimens. High-dose
dexamethasone or vincristine-doxorubicin-dexamethasone (VAD) were the
most commonly used induction regimens.
Recently, new drugs such as thalidomide,
lenalidomide and bortezomib have been
used in association with dexamethasone
as induction treatment for newly diagnosed patients. In a randomized study, the
combination thalidomide-dexamethasone
increased partial response (PR) rate
(p=0.001) and 2-year event-free survival
(p<0.0001) when compared with dexamethasone alone.4 No marked survival differences were reported, although the trial
was not powered for a long-term followup (Table 1). A retrospective case-control
study showed superior PR rates for
thalidomide-dexamethasone in comparison with standard VAD, but higher rates
of deep-vein thrombosis (15%) were
reported.5 In a randomized study, the
combination thalidomide-dexamethasone
was compared with VAD. Before stem cell
collection, the VGPR rate was 24.7% vs
7.3% (p=0.0027), respectively. Six months
after transplantation the VGPR rate was
quite similar in both arms (44.4% vs
41.7%, p=0.87).6 Toxicity was higher in
the thalidomide-dexamethasone group.
Thalidomide-dexamethasone reduced the
mean duration of hospitalization before
stem cell collection from 20 to 8.3 days,
without any negative impact on the
amount of stem cell harvest.
When thalidomide was incorporated
into the high-dose therapy followed by
autologous transplantation, a higher CR
rate (62% vs 43%) and improved 5-year
event-free survival (56% vs 44%) was
observed compared with high-dose therapy without thalidomide.7 Unfortunately,
the 5-year overall survival was similar in
both groups (p=0.9). In the thalidomide
group, a higher rate of thromboembolism (30% vs
17%) and peripheral neuropathy (27% vs 17%) were
reported (Table 1).
The combination lenalidomide-dexamethasone
has been evaluated in 34 newly diagnosed patients.
The PR rate was 91%, including a CR plus VGPR rate
of 56%. In 13 patients 4 induction cycles of lenalidomide–dexamethasone were followed by autologous
transplantation. In 21 patients, the lenalidomide-dexamethasone induction regimen was delivered for a
prolonged period of time without autologous transplant consolidation, and the CR plus VGPR rate was
67%. The 2-year progression-free survival was 83%
in the group that received autologous transplantation
and 59% in the group that received lenalidomidedexamethasone only.8 These preliminary data support the proposal to incorporate high-dose melphalan after the induction treatment of myeloma
patients. Grade 3 or higher adverse events were very
limited, including neutropenia, pneumonia, and cutaneous rash. Deep-vein thrombosis was only 3%, but
prophylactic aspirin was introduced during treatment. A randomized phase III trial of lenalidomide
plus high-dose dexamethasone versus lenalidomide
plus low-dose dexamethasone (40 mg; days 1, 8, 15,
and 22) has been recently reported.9 Although efficacy data were not available, the incidence of adverse
events was significantly reduced in the low-dose
dexamethasone arm. Early deaths were reduced from
4.5% to 1.4%; infections from 19% to 9%, and deepvein thrombosis from 18% to 5% (Table 1).
Bortezomib, as single agent or in combination with
dexamethasone has been studied as first-line treatment. In this trial, dexamethasone was added to
bortezomib when suboptimal responses, such as less
than PR after 2 cycles or less than CR after 4 cycles,
were reported.10 This approach improved the suboptimal responses of bortezomib alone in 64% of
patients. After the addition of dexamethasone, the
PR rate was 90%, including 19% of CR and near CR.
Stem cell harvest and engraftment were successful in
all patients. The most common adverse events were
neuropathy (37%), fatigue (20%), constipation
(16%), nausea (12%) and neutropenia (12%). In a
recent randomized trial, the association bortezomibdexamethasone has been compared with VAD as
induction regimen before autologous transplantation.11 Bortezomib-dexamethasone significantly
increased both PR rate (82% vs 67%) and VGPR plus
CR rate (43% vs 26%). In the bortezomib-dexamethasone group, 78% of patients did not require a
second autologous transplantation. Serious adverse
events were similar in both arms. In 21 newly diagnosed patients, the combination of bortezomib,
pegylated-liposomal doxorubicin and dexamethasone induced a PR rate of 95%, including 29% of CR
plus nearCR.12 More frequent grade ≥3 adverse
events were infections (48%), neuropathy (5%), nausea and vomiting (5%) (Table 1).
New maintenance approaches
Maintenance therapy has been shown to prolong
response rate and event-free survival in patients who
have received induction treatment. However, the role
of maintenance remains controversial in myeloma.
After conventional or high-dose therapy, maintenance with interferon-alpha provided marginal benefits. In patients who responded to conventional
chemotherapy, maintenance therapy with 50 mg
alternate-day prednisone significantly improved progression-free and overall survival in comparison with
10 mg alternate-day prednisone.13
In a large randomized study conducted by the
French group, patients younger than 65 years were
randomly assigned to receive no maintenance,
pamidronate, or pamidronate plus thalidomide.14 The
3-year post-randomization probability of event-free
survival (p<0.009) and the 4-year overall survival
(p<0.04) were significantly prolonged in patients
who received thalidomide. The proportion of
patients who had skeletal events was not influenced
by the administration of pamidronate. Grade 3-4
Table 1. New induction regimens tested in myeloma patients < 65 years of age.
Diagnosis
Therapy
No.
of patients
Median
age (range)
≥PR
%
CR Progression-free
%
Survival
Overall
Survival
TD
TD
TD
ASCT-T
RD
VD
PAD
103
100
100
323
34
79
21
65 (38-83)
54 (49-59)
55 (ND)
ND
64 (32-78)
55 (ND)
55 (37-66)
63
70
25§
ND
91
82
95
4 50% at 22 months
10
ND
ND
ND
62 56% at 5 years*
18 74% at 2 years
9
ND
21
ND
72% at 2 years
ND
ND
65% at 5 years
91% at 2 years
ND
ND
PeripheralDVT/
Neutropenia Thrombocytopenia Infection References
Neuropathy
Embolism
grade 3-4 (%) grade 3-4 (%) grade 3-4 (%) grade 3-4 (%) grade 3-4 (%)
7
4
17°
27°°
ND
3
5
17
15
23°
30°°
3
3
0
9
0
ND
94°°
21
ND
5
ND
ND
ND
ND
0
ND
0
6
4
ND
ND
6
4
48
4
5
6
7
8+
11
12
ASCT-T: autologous transplant+thalidomide; CR: complete response; DVT: deep-vein thrombosis; ND: not determinate; PAD: bortezomib+doxorubicin+dexamethasone;
PR: partial response; RD: lenalidomide+dexamethasone; TD: thalidomide+dexamethasone; VD: bortezomib+dexamethasone; *event-free survival; §very good partial
response; °all grades; °°> grade 2; +updated information was presented at ASH 2006
Table 2. New induction regimens tested in myeloma patients > 65 years of age.
Therapy
Diagnosis MPT
MPT
MPR
VMP
No.
of pts.
124
129
54
60
Median >65 years ≥PR CR
age (range) %
%
ND (65-75)
72 (60-85)
71 (57-77)
75 (65-85)
100
97
96
100
81
76
85
89
16
16
24
32
Progression-free
survival
Overall
survival
PeripheralDVT/
neuropathy
embolism
50% at 28 months 78% at 2 years
6
54% at 2 years° 80% at 3 months 8
91% at 2 years
92% at 2 years
0
91% at 16 months 90% at 16 months 17
12
12
6
ND
48
16
66
43
14
3
34
51
13
10
8
16
18+
19
21+
22
CR: complete response; DVT: deep-vein thrombosis; MPR: melphalan+prednisone+lenalidomide; MPT: melphalan+prednisone+thalidomide; ND: not determinate; PR:
partial response; VMP: bortezomib+melphalan+prednisone; °event-free survival; +updated information was presented at ASH 2006.
neuropathy (7%), fatigue (6%) and constipation (1%)
were more prominent in the thalidomide group. The
incidence of thromboembolic events was not significantly different in the 3 arms. More recently, a randomized trial compared thalidomide-prednisone versus prednisone alone as maintenance therapy after
autologous stem cell transplantation. The 1-year progression-free survival was 91% vs 69%, and the 2year overall survival was 90% vs 81 respectively.
Neurological side effects were more common with
thalidomide, but no differences were observed in the
incidence of thromboembolic events.15
New induction regimens for elderly patients
The MP regimen has been considered the standard of care for elderly patients. In a randomized
trial, 4 treatment regimens have been evaluated: MP,
melphalan and dexamethasone, high-dose dexamethasone, or high-dose dexamethasone plus interferon-alpha.16 PR rates were significantly higher
among patients receiving melphalan-dexamethasone. Median progression-free survival was 21 and
23 months after MP or melphalan and dexamethasone, but only 12 and 15 months after high-dose
dexamethasone or high-dose dexamethasone plus
interferon-alpha respectively. No difference in overall survival was reported among the 4 different
groups. These data clearly show that melphalan
should be incorporated in the induction regimen for
elderly patients who are not candidates for autologous transplant.
In patients older than 65 years, melphalan 200
mg/m2 followed by autologous transplant is too
toxic, while intermediate-dose melphalan (100-140
mg/m2) seems more suitable. In the Italian study,
patients were aged 65-70 years and melphalan 100
mg/m2 was superior to MP.17 In the French study,
patients were aged 65-75 years and melphalan 100
mg/m2 was superior to MP in terms of response rate,
but not in terms of progression-free and overall survival.18 In the first study, 22% of patients did not
complete the assigned treatment and in the second
trial, 37% did not complete it. According to these
data, the age of 70 years might be considered the age
limit for intermediate-dose melphalan.
Recently, thalidomide has been added to the MP
regimen (MPT). In the Italian randomized trial, oral
MPT was compared with MP in patients aged 60-85
years.19 The PR rates were 76% in MPT patients and
47.6% in MP subjects. Near-CR or CR rates were
27.9% after MPT and 7.2% after MP. The 2-year
event-free survival rates were 54% for MPT and 27%
for MP (p=0.0006). The 3-year survival rates were
80% for MPT and 64% for MP (p=0.19). MPT was
associated with a higher risk of grade 3-4 neurological adverse events compared to MP regimen (10% vs
1%), infections (10% vs 2%, p=0.001), cardiac toxicity (7% vs 4%) and thromboembolism (12% vs 2%).
The introduction of enoxaparin prophylaxis significantly reduced the rate of thromboembolism from
20% to 3% (p=0.005) (Table 2).
In the French phase III trial, comparing MPT with
MP with intermediate-dose melphalan (100 mg/m2)
followed by autologous stem cell transplantation, a
higher PR rate in the MPT and in the melphalan 100
mg/m2 arms, compared with MP, was observed (81%
vs 73% vs 40%, respectively).18 Similarly, the CR
rates were significantly increased after MPT and
autologous transplant only. Progression-free survival
was superior in the MPT patients compared with
both MP (p<0.001) and autologous transplantation
(p=0.001). Furthermore, overall-survival was significantly improved in the MPT group in comparison
with both MP (p=0.001) and autologous transplantation (p=0.004). MPT was associated with a higher
risk of grade 3-4 neutropenia, infections, thrombocytopenia, thromboembolic complications, peripheral
neuropathy, constipation, and cardiac events (Table
2). These data strongly support the use of MPT as
standard of care in elderly patients with newly diagnosed myeloma. An increased risk of venous thromboembolism has been reported when thalidomide is
combined with chemotherapy, in particular at diagnosis. The risk of venous thromboembolism is particularly high in the first 4–6 months of therapy. At
diagnosis, antithrombotic prophylaxis is recom-
mended. At present there is no evidence of the best
prophylaxis. Low-molecular-weight heparin, therapeutic doses of warfarin, or daily aspirin are the preferred options.20
Lenalidomide appears to be an attractive alternative to thalidomide. In a phase I/II trial dosing, safety
and efficacy of melphalan plus prednisone and
lenalidomide (MPR) have been evaluated in newly
diagnosed elderly myeloma patients.21 Aspirin was
administered as antithrombotic prophylaxis. At the
maximum tolerated dose (lenalidomide 10 mg plus
melphalan 0.18 mg/kg), 85% of patients achieved at
least a PR and 23.8% immunofixation negative CR.
The 1-year event-free and overall survivals were 92%
and 100%. In the MPT historical controls, the corresponding 1-year event-free and overall survivals were
78% and 87.4%. Grade 3-4 adverse events were
mainly related to hematological toxicities (neutropenia 66%). Severe non-hematological side effects
were less frequent and included febrile neutropenia
(8%), cutaneous rash (10%) and thromboembolism
(6%) (Table 2). Preliminary results showed that the
event-free survival of patients with deletion of chromosome 13 or chromosomal translocation (4;14) was
not significantly different from those who did not
have show such abnormalities. By contrast, patients
with high-levels of serum β2-microglobulin experienced a shorter event-free survival in comparison
with those who showed low-levels of β2-microglobulin. Neutropenia and deep-vein thrombosis are the
major complications with lenalidomide. The addition of aspirin markedly reduced the risk of thromboembolic events in newly diagnosed patients treated with lenalidomide in association with dexamethasone or chemotherapy. Although the optimal prophylaxis strategy has not been established, aspirin
seems to be the preferred choice.
The proteasome inhibitor, bortezomib, enhanced
chemosensitivity and overcame chemoresistance in
both preclinical and clinical studies. Bortezomib
seems a rational approach to combinational regimens
incorporating corticosteroids or chemotherapy
agents. The Spanish cooperative group conducted a
large phase I/II trial of bortezomib, melphalan, and
prednisone (VMP).22 The association showed encouraging results. PR rate was 89%, including 32%
immunofixation-negative CR, half of them achieved
immunophenotypic remission (no detectable plasma
cells at 10-4 to 10-5 sensitivity). The progression-free
survival at 16 months of VMP patients was significantly prolonged in comparison with historical controls treated with MP only (91% versus 66%).
Similarly overall survival at 16 months was improved
(90% versus 62%). Interestingly, response rate, progression-free and overall survivals were similar
among patients with or without retinoblastoma gene
deletion or IgH translocations. These data showed
that VMP overcome the poor prognosis induced by
such chromosomal abnormalities. Grade 3-4 adverse
events were thrombocytopenia, neutropenia, peripheral neuropathy, infections and diarrhea. (Table 2)
The treatment appeared more toxic in patients older
than 75 years and during early cycles. Bortezomib
may induce transient thrombocytopenia and peripheral neuropathy. Pre-existing neuropathy or previous
neurotoxic therapy increases the risk of peripheral
neuropathy, which can be reduced or resolved by
timely dose-adjustment of the drug. Bortezomib may
enhance the incidence of infections, in particular
Herpes Zoster reactivation, and prophylactic antiviral
medication is highly recommended.
New salvage combinations
The majority of the combinational approaches
have been studied in patients with relapsed-refractory myeloma. The thalidomide-dexamethasone combination23,24 induced a PR rate of 41-55%, and the 1year progression-free survival was around 50%
(Table 3). More than 50% of patients showed constipation, somnolence and peripheral neuropathy.
Thromboembolism was 7% and no antithrombotic
prophylaxis was performed. The association of
thalidomide with chemotherapy further increased
response rates. In relapsed-refractory patients the
combination of thalidomide, dexamethasone and
pegylated-liposomal doxorubicin induced a CR rate
of 26%, and a 22-month progression-free survival of
50%.25 Grade 3-4 neutropenia was low, but a higher
rate of infections and thromboembolic events was
noticed. Similar response rates were reported with
the oral combination cyclophosphamide-thalidomide-dexamethasone:26 PR rate was 57% and the 2year progression-free survival was 57%. The major
adverse events included constipation (24%) infections (13%) and thromboembolic toxicity (7%).
In previously treated patients, lenalidomide has
been combined with both steroids and chemotherapy. In two independent phase III randomized trials,
the combination of lenalidomide-dexamethasone
significantly increased the PR rate (59% and 58%) in
comparison with dexamethasone alone (21% and
22%, p< 0.001). Similarly, the 1-year progression-free
(p<0.001) and overall survival (p<0.03) were significantly improved in the lenalidomide-dexamethasone
groups.27,28 These results were unchanged in the subgroup of patients older than 65 years and in those
who received previous thalidomide treatment. Grade
3-4 neutropenia (16.5%) as well as thromboembolism (8.5%) were more frequent in the lenalidomide-dexamethasone group. Other side effects were
reported in similar frequencies in both arms. In 62
refractory patients, lenalidomide, pegylated-liposo-
Table 3. New salvage combinations.
Therapy
Relapsed TD
Refractory TD
TAD
CTD
RD
RD
RAD
CRD
V
VDoxo
VCP
VMPT
No.
Median >65 years ≥PR CR
of patients age (range) %
%
77
44
50
71
177
176
62
18
333
323
27
30
65 (ND)
67 (38-87)
68 (41-82)
ND
64 (33-86)
63 (33-84)
62 (57-70)
60 (34-76)
62 (48-74)
61(ND)
59 (48-74)
66 (38-79)
50
21*
44*
65
ND
ND
ND
ND
ND
ND
ND
57
41
55
76
57
59
58
75
70
38
48
93
67
3**
0
26
2
13
13
15
6
6
14**
43**
17
Progression-free
Survival
Overall
Survival
PeripheralDVT/
Neuropathy
Embolism
50% at 1 year
ND
50% at 4 months
50% at 1 year
50% at 22 months 64% at 2 years
57% at 2 yeras
66% at 2 yeras
50% at 11 months 50% at 29 months
50% at 1 year
51% at 2 years
ND
ND
50% at 6 months
80% at 1 year
47% at 11 year
83% at 1 year
61% at 1 year
84% at 1 year
17^
23^
2
6##
ND
ND
5
0
8
4
7°°
7
0
7^
12
7
15
8
9
11
ND
1
0
0
5^
9^
16
10°
24
16
32
44
14
30
ND
43
4^
0
2
0
12
10
13
ND
30
22
4
33
5^
ND
16
7°
ND
ND
13
22
13^^
3
48
16
23
24
25
26
27+
28+
29
30+
31
33+
34
35
CR: complete response; CRD: cyclophosphamide+lenalidomide+dexamethasone; CDT: cyclophosphamide+dexamethasone+thalidomide; DVT: deep-vein thrombosis; ND:
not determinate; PR: partial response; RAD: lenalidomide+ pegylated lyposomal doxorubicin+dexamethasone; RD: lenalidomide+dexamethasone;
TAD: thalidomide+ pegylated lyposomal doxorubicin+dexamethasone; TD: thalidomide+dexamethasone; V: bortezomib; VCP: bortezomib+cyclophosphamide+prednisone; VDoxo: bortezomib+pegylated lyposomal doxorubicin; VMPT: bortezomib+melphalan+prednisone+thalidomide; * >70 years; **CR+nCR; ^WHO all grade; ^^
Herpes Zoster infections; #OMS score; ##OMS score>2; °°grade>2; +updated information was presented at ASH 2006.
mal doxorubicin and dexamethasone showed a PR
rate of 75%, including a CR rate of 15% and the 1year progression-free survival was 50%.29 The most
common grade 3-4 adverse events were neutropenia
(32%), thrombocytopenia (13%), infections (13%),
gastrointestinal events (12%) and thromboembolism
(9%) despite aspirin prophylaxis. High responses
were also observed with the association of oral
lenalidomide, cyclophosphamide and dexamethasone. The PR rate was 70%, including 6% CR, but
grade 4 neutropenia, neutropenic fever and deep-vein
thrombosis were observed (Table 3).30
Bortezomib has been effectively used alone or in
combination with steroids or chemotherapy to treat
relapsed-refractory myeloma patients. In the randomized phase III APEX study, 669 relapsed patients
received bortezomib or oral dexamethasone.31 The
response rate was significantly superior in the bortezomib arm compared with the dexamethasone arm
(PR 38% versus 18%; CR 6% vs 1% respectively).
Similarly, duration of response, 1-year progressionfree and 1-year survival of the bortezomib group
were significantly increased. Serious adverse events
included peripheral neuropathy, thrombocytopenia
and gastrointestinal disorder in the bortezomib arm;
psychotic disorder and hyperglycemia in the dexamethasone arm (Table 3). A recent update of this trial,
confirmed the higher response rate and longer survival of the bortezomib group, even if dexamethasone-resistant patients in the control arm were
allowed to crossover to receive bortezomib.
Jagannath et al. recently analyzed the impact of
chromosomal abnormalities on response rate and
survival after treatment with bortezomib.32
Matched-pairs analysis demonstrated that borte-
zomib overcomes the negative impact of chromosome 13 deletion as an independent prognostic factor, both responses and survival appeared comparable in bortezomib-treated patients with or without
chromosome 13 deletion. In advanced myeloma, the
association of bortezomib-doxorubicin has been
compared with bortezomib alone.33 Bortezomibdoxorubicin significantly improved time to progression even in those patients previously treated with
thalidomide or doxorubicin or in patients with
unfavourable cytogenetic markers. Another combination of bortezomib-cyclophosphamide-prednisone proved to be very effective with a high PR
rate of 93%, including 43% of CR or near CR.34 Most
frequent adverse events were infections. The synergistic activity showed by bortezomib with thalidomide or melphalan provided the rationale to combine bortezomib, melphalan, prednisone and
thalidomide (50 mg/day).35 In relapsed-refractory
patients, the PR rate was 67%, including 17% CR.
The 1-year progression-free survival was 61%, and
the 1-year survival from study entry was 84%.
Patients treated in the earlier phases of disease (first
relapse) achieved a higher PR rate of 78%, including
36% CR. The most common grade 3 adverse events
were hematologic toxicity (56%), infections (9%),
Herpes Zoster reactivation (7%) and peripheral neuropathy (7%) (Table 3).
Conclusions
High-dose melphalan followed by autologous stem
cell transplantation in the younger patients and oral
melphalan-prednisone in the elderly have been considered the standard of care for the initial therapy of
myeloma.
Figure 1. A proposed treatment algorithm using novel
agents, according to Level of Evidence Ib based on >1 randomized controlled trial. *Thalidomide as maintenance
was evaluated in patients who have not received any previous thalidomide therapy. Abbreviations: ASCT, autologous
stem cell transplantation; DEX, dexamethasone; DOXO,
doxorubicin; MEL200, Melphalan 200 mg/m2; MPT, melphalan plus prednisone plus thalidomide; REV, Lenalidomide; THAL, thalidomide; VEL, bortezomib
Survival after transplant appears to be related to
the achievement of CR or VGPR.3,7,36 An improved
response rate after induction treatment, prior to
transplant, could translate into better results after
high-dose therapy and into a prolonged survival. In
younger patients, combinations incorporating
thalidomide or lenalidomide or bortezomib with
dexamethasone or doxorubicin significantly increase
the pre-transplant CR rate before high-dose melphalan and autologous transplantation. These combinations might further improve the CR rate achieved
after transplant. Based on the data available, the combination of thalidomide and dexamethasone is recommended as induction treatment in younger
patients. Further randomized prospective trials are
needed to definitively assess the role of bortezomib
and lenalidomide combinations and their impact on
remission duration in newly diagnosed patients.
Based on the French randomized study, thalidomide
could also be suggested as optimal maintenance
treatment after autologous stem cell transplantation.
In elderly patients, the MPT combination has been
shown to achieve more rapid and higher response
rates and, most significantly, achieved improved
event-free survival compared with conventional MP
in two independent randomized trials.18,19 The MPT
regimen is now recommended as the new standard
of care for the elderly and for patients who are not
candidates for high-dose chemotherapy with stem
cell support. Other regimens such as MPR21 or VMP22
are currently under investigation and might be introduced in the clinical practice in the near future.
Whether a sequential single agent treatment would
yield similar survival benefits with less toxicity in
comparison with a more complex combinational regimen administered at diagnosis remains an unanswered question. If a combinational approach is
superior to single agent therapy, it should be considered at diagnosis, when there is the best chance to
induce a prolonged remission duration. A sequential
approach should then be considered at first and subsequent relapses, when less intense and more palliative regimens are needed. The combination of bortezomib-dexamethasone or bortezomib-doxorubicin
or lenalidomide-dexamethasone are currently recommended in the setting of relapsed myeloma patients.
The choice of combination relies on previous exposure to such drugs as well as concomitant co-morbidities which might contraindicate the delivery of a
specific compound (Figure 1).
Cytogenetic abnormalities, such as deletion of
chromosome 13 or chromosomal translocation (4;14)
are considered negative prognostic factors.37
Unfortunately, most of the studies reported to date
have not prospectively stratified patients based on
cytogenetic abnormalities, making conclusions difficult. In the VMP patients,22 as well as in a smaller
cohort of MPR patients,21 the event-free survival of
patients with deletion of chromosome 13 or chromosomal translocation (4;14) was not significantly different from those who did not show such abnormalities. If these data are confirmed, it seems likely that
a cytogenetically adapted strategy will represent the
most rational, molecularly targeted approach to
myeloma therapy.
Potential conflicts of interest
Two of the authors (AP, MB) have received scientific
advisory-board and lecture fees from Pharmion, Celgene,
and Janssen-Cilag. M.C. has received scientific advisoryboard, lecture fees and research funding from Janssen-Cilag
and Novartis. The other authors declare that they have no
conflict of interest.
Acknowledgments
Supported in part by the Università degli Studi di Torino,
Fondazione Neoplasie Sangue Onlus, Associazione Italiana Leucemie, Compagnia di S. Paolo, Fondazione Cassa
di Risparmio di Torino, Ministero dell’Università e della
Ricerca (MIUR), and Consiglio Nazionale delle Ricerche
(CNR), Italy.
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et al. PAD combination theraphy (PS-341/bortezomib, doxorubicin and dexamethasone) for previously untreated
patients with multiple myeloma. Br J Haematol 2005;129:75562.
Berenson JR, Crowley J, Grogan TM, Zangmeister J, Briggs
AD, Mills GM et al. Maintenance therapy with alternate-day
prednisone improves survival in multiple myeloma patients.
Blood 2002;99:3163-8.
Attal M, Harousseau JL, Leyvraz S, Doyen C, Huylin C,
Benboubker L et al. Maintenance therapy with thalidomide
improves survival in patients with multiple myeloma. Blood
2006;108:3289-94.
Spencer A, Prince M, Roberts AW, Bradstock KF, Prosse IW
First Analysis of the Australasian Leukaemia and Lymphoma
Group (ALLG) Trial of Thalidomide and Alternate Day
Prednisolone Following Autologous Stem Cell Transplantation
(ASCT) for Patients with Multiple Myeloma (ALLG MM6).
Blood 2006;108:58a[abstract].
Facon T, Mary JY, Pegourie B, Attal M, Renaud M, Sadoun A
et al. Dexamethasone-based regimens versus melphalan-prednisone for elderly multiple myeloma patients ineligible for
high-dose therapy. Blood 2006;107:1292-8.
Palumbo A, Bringhen S, Petrucci MT, Musto P, Rossini F,
Callea V et al. Intermediate-dose melphalan improves survival
of myeloma patients aged 50 to 70: results of a randomized
controlled trial. Blood 2004;104 :3052-7.
Facon T, Mary J, Harousseau J, Huguet F, Berthou C, Grosbois
B et al. Superiority of melphalan-prednisone (MP) + thalidomide (THAL) over MP and autologous stem cell transplantation in the treatment of newly diagnosed elderly patients with
multiple myeloma. J Clin Oncol 2006 24(18S):1a[abstract].
Palumbo A, Bringhen S, Caravita T, Merla E, Carapella V,
Callea V et al. Oral melphalan and prednisone chemotherapy
plus thalidomide compared with melphalan and prednisone
alone in elderly patients with multiple myeloma: randomised
controlled trial. Lancet 2006; 367:825-31.
20. Bennet CL, Angelotta C, Yarnold PR, Evens AM, Zonder A,
Raisch W et al. Thalidomide and lenalidomide-associated
thromboembolism among patients with cancer. JAMA
2006;296:2559-60.
21. Palumbo A, Falco P, Falcone A, Corradini P, Di Raimondo F,
Giuliani N et al. Oral Revlimid plus melphalan and prednisone
(R-MP) for newly diagnosed multiple myeloma: a phase I-II
study. Blood 2006;108(11):800a[abstract].
22. Mateos MV, Hernandez JM, Hernandez MT, Guiterrez N-C,
Palomera L, Fuertes M et al. Bortezomib plus melphalan and
prednisone in elderly untreated patients with multiple myeloma: results of a multicenter phase I/II study. Blood
2006;108:2165-72.
23. Palumbo A, Giaccone L, Bertola A, Pregno P, Bringhen S, Rus
C et al. Low-dose thalidomide plus dexamethasone is an effective therapy for advanced myeloma. Haematologica
2001;86:399-403.
24. Dimopoulos MA, Zervas K, Kouvatseas G, Galani E, Grigoraki
V, Kiamouris C et al. Thalidomide and dexamethasone combination for refractory multiple myeloma. Ann Oncol
2001;12:991-5.
25. Offidani M, Corvatta L, Marconi M, Visani G, Alesiani F,
Brunori M et al. Low-dose thalidomide with pegylated liposomal doxorubicin and high-dose dexamethasone for
relapsed/refractory multiple myeloma: a prospective, multicenter, phase II study. Haematologica 2006;91:133-6.
26. Garcia-Sanz R, Gonzales-Porras JR, Hernandez JM, Zarzuela
MP, Sureda A, Barrenetxea C et al. The oral combination of
thalidomide, cyclophosphamide and dexamethasone
(ThaCyDex) is effective in relapsed/refractory multiple myeloma. Leukemia 2004;18:856-63.
27. Weber DM, Chen C, Niesvizky R, Belch A, Stadtmauer E, Yu
Z et al. Lenalidomide plus high-dose dexamethasone
improved overall survival compared to high-dose dexamethasone alone for relapsed or refractory multiple myeloma (MM):
Results of a North American phase III study (MM-009). J Clin
Oncol 2006;24(18S): 7521a [abstract].
28. Dimopoulos MA, Spencer A, Attal M, Prince M, Harousseua
JL, Dmazynsk A et al. Study of lenalidomide plus dexamethasone alone in relapsed or refractory multiple myeloma (MM):
results of a phase 3 study (MM-010). Blood 2005;106:6a
[abstract].
29. Baz R, Walker E, Karam MA, Jawde RA, Bruening K, Reed J et
al. Lenalidomide and pegylated liposomal doxorubicin-based
chemotherapy for relapsed or refractory multiple myeloma:
safety and efficacy. Ann Oncol 2006;17:1766-71.
30. Morgan G, Schey S, Wu P, Srikanth M, Phekoo M, Jenner MW
et al. Lenalidomide (Revlimid), in combination with
cyclophosphamide and dexamethasone (CRD) is an effective
regimen for heavily pre-treated myeloma patients. Blood
2006;108:355[abstract].
31. Richardson PG, Sonneveld P, Schuster MW, Irwin D,
Stadtmauer EA, Facon T et al. Bortezomib or high-dose dexamethasone for relapsed multiple myeloma. N Engl J Med
2005;352:2487-98.
32. Jagannath S, Richarson PG, Sonneveld P, Schster MW, Irwin D,
Stadtmauer EA et al. Bortezomib appears to overcome the
poor prognosis conferred by chromosome 13 deletion in phase
2 and 3 trials. Leukemia 2007;21:151-7.
33. Orlowski RZ, Zhuang SH, Parekh T, Xiu L, Harouseau JL. The
combination of pegylated liposomal doxorubicin and bortezomib significantly improves time to progression of patients
with relapsed/refractory multiple myeloma compared with
bortezomib alone: results from a planned interim analysis of a
randomized phase III study. Blood 2006;108: 404a[abstract].
34. Reece DE, Piza G, Trudel S, Pantoja M, Chen , Mikhael JR et
al. A phase I–II trial of bortezomib plus oral cyclophosphamide and prednisone for relapsed/refractory multiple
myeloma. Blood 2006;108:3536a[abstract].
35. Palumbo A, Ambrosini MT, Benevolo G, Pregno P, Pescosa N,
Callea V et al. Bortezomib, melphalan, prednisone and
thalidomide for relapsed multiple myeloma. Blood 2006 Dec
5; [Epub ahead of print].
36. Child JA, Morgan GJ, Davies FE, et al. Medical Research
Council Adult Leukaemia Working Party. High-dose
chemotherapy with hematopoietic stem-cell rescue for multiple myeloma. N Engl J Med. 2003;348:1875-83.
37. Fonseca R, Blood R, Rue M, et al. Clinical and biologic implications of recurrent genomic aberrations in myeloma. Blood
2003;101:4569-70.
Chronic Lymphocytic Leukemia
Genetics in chronic lymphocytic leukemia:
impact for prognosis and treatment decisions
U. Jäger
M. Shehata
D. Heintel
R. Hubmann
B. Kainz
E. Porpaczy
A. Hauswirth
A. Gaiger
Medical University of Vienna
Department of Internal Medicine I,
Division of Hematology and
Hemostaseology
Währinger Gürtel
Vienna, Austria
2007;1:115-121
he prognosis of patients with B-cell
chronic lymphocytic leukemia (BCLL) has long been determined by
the clinical staging systems of Binet and
Rai.1-4 Unfortunately, these systems fail to
identify patients in early stages whose
disease will rapidly progress. In addition,
the response of individual patients to specific therapies can scarcely be predicted
by this approach. In recent years, numerous genetic approaches have provided
new markers for prognosis and response
prediction.5 The predictive potential of
genetic factors associated with CLL outcome is determined by (i) its accessibility
for routine use; (ii) its stability throughout
the course of disease; and (iii) its sensitivity and specificity. Prognostic factors are
largely dependent on the conventional
therapeutic regimens used (i.e. chemotherapy) as well as on the availability of
specific targeting drugs. This review will
discuss the advances in the genetics of
CLL with a special focus on molecular
markers. The first part will focus on prognostic markers which have already been
firmly established, while the second part
will review the current status of molecular predictive markers and their potential
influence on treatment decisions.
T
Prognostic markers
Conventional cytogenetics
Karyotypes from CLL cells collected
from peripheral blood or bone marrow
are difficult to assess. However, stimulation by CD40L or CpG oligonucleotides
has yielded positive results in the majority of cases.6 One of the most important
findings was that the number of chromosomal translocations has largely been
underestimated. Patients with translcations have a poor prognosis with short
treatment free and overall survival times.6
Recent advances were also made in identifying chromosomal regions predisposing for familial CLL. Regions on chromosomes 13q as well as 1, 3, 6, 12, and 17
have been linked to pathogenesis of
inherited disease.7-9 However, while relatives of CLL patients have increased numbers of circulating CD19+/CD5+ B-cells (a
phenomenon called B-cell monoclonal
lymphocytosis, MBL), the risk factors
leading to the final development of overt
CLL are still unclear.10 Moreover, familial
CLL does not seem to be associated with
shorter survival in affected individuals.11
MicroRNAs (MiRs) are also connected to
familial disease.12 However, their impact is
not yet clear.
Fluorescence in situ hybridization
The prognostic value of aberrations has
long been established following the hierarchical model described by Döhner et al.13,14
Half of the B-CLL cases have a del 13q
indicating good prognosis. On the other
hand, 11q deletions are associated with
bulky disease and short survival times.
This is even more pronounced for 17p
deletions which predict for very poor outcome.15,16 Interphase cytogentics have been
extended to a molecular level by microarray analysis in several instances.17–19 Thus, a
number of genes over- or underexpressed
in association with chromosomal aberrations have been identified. Some of these
are directly related to the location on
deleted or duplicated chromosomes (gene
dosage effect). However, a number of genes
from unaffected chromosomes change
their expression, suggesting trans-acting
effects. These genes have only partially
been evaluated.20
Molecular prognostic markers
Molecular markers can be classified
according to their presence on the DNA
level (mutations, polymorphisms) or on
the RNA level (gene expression) (Table
1). Most markers are predictive at diagnosis, but dynamic markers predicting
response to therapy (e.g.minimal residual disease) are also available (see also
Table 4).
hematology - the european hematology association education program | 2007; 3(1) | 115 |
Gene mutations
Immunoglobulin VH-mutational status is now well
established as a strong predictor of outcome.21-28 By
convention, a 98% sequence homology to germline
has been defined as a useful clinical cut-off between
good (mutated) or poor (unmutated) prognosis. More
detailed analysis has revealed that the 98% threshold
is indeed relevant, but that some variation exists.29
Further analysis has revealed the prognostic relevance of specific VH-families: VH 1-69 cases are
always unmutated CLL and mutated cases with a VH
3-21 have a poor outcome.30, 31
A number of non-immunoglobulin genes have
been searched for mutations in CLL. Germline or
somatic mutations were found in 5 of 42 sequenced
microRNAs (miR) in 11 out 75 patients with CLL.12
Gene expression
Perhaps the most important value of IgVH mutational status was its use as a discriminator of patient
subgroups for further analysis in microarray studies.
Gene expression profiling revealed the association
of a number of genes previously thought to be unrelated to CLL. Depending on the use of CD19-selected/T-cell depleted or unpurified B-CLL samples, a
variety of markers have been found since the initial
experiments of Klein and Rosenwald in 2001.19,32-34
(Table 2) When CD19+ cells were used, the receptor
kinase ZAP-70 was identified. The prognostic value
of this genetic marker has now been well established by FACS-analysis or PCR.35-41 ZAP-70 protein
expression predicts for treatment-free survival (TFS)
and overall survival independently of mutation status. While convincing results were obtained by several groups using different protein detection
methodology, harmonization efforts by the
European Initiative in CLL Research (ERIC) have
shown problems and were not successful to date.42
Moreover, contrasting results concerning association with IgVH mutational status have been
observed. Particularly, 17p- samples cluster in the
ZAP-70 negative group.43 Assessment of ZAP-70
mRNA expression by real time PCR requires positive (CD19) or negative selection of B-cells.39
Despite these drawbacks, ZAP-70 is a useful clinical
marker and may also serve as a future target for specific signal transduction inhibitors.
Among the markers with an even stronger correlation to IgVH mutational status,33,34 lipoprotein lipase
has been extensively studied44-48 (Table 3). Its association with other markers (cytogenetic risk groups,
molecular markers) as well as patient outcome (time
to treatment, overall survival) is also strong. LPL is a
stable marker which has been studied by real-time
PCR in several large series using purified or unpurified CLL cells or even whole blood. No difference
Table 1. Genetic markers in CLL with prognostic significance.
Cytogenetic markers
Conventional cytogenetics
translocations
FISH
17p-, 11q-, 13q-, +12
Molecular markers
Mutations
mRNA expression
Minimal residual disease
Ref.
6
13-19
IgVH-Status
Micro RNAs
ZAP70
LPL
PEG 10
sarcoglycan ε
septin 10
dystrophin
AID
telomerase
L-selectin
Integrin-β2
CLLU1
21-31
12
39
44-48
53
53
19
19
49-52
54
20
20
55
ASO-PCR
56-59
Table 2. Factors associated with IgVH mutational status by
gene expression profiling.
ZAP-70
LPL
Dystrophin
Gravin/AKAP12
BCL7A
FGL2
Rosenwald A,
J Exp Med
200132
CD19+ sorted
Klein U,
J Exp Med
200133
unsorted
Vasconcelos Y,
IWCLL
200334
CD19-selection
Bilban M,
Leukemia
200619
unsorted
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
between purified and unpurified samples was
observed in several studies, indicating its potential
for easy and general use.
Its specificity regarding IgVH mutational status is
89%, with a sensitivity of 68%, a positive predictive
value of 83% and a negative predictive value of
78%48 (Table 3). Contrasting results are also
observed.45 LPL can be combined with a downregulated marker (ADAM29) to increase specificity.44
While LPL protein can also be detected on normal Bcells, its cytoplasmatic expression correlates well
with RNA levels.45
We have used the level of LPL expression as a discriminator for microrray analysis.19 Several markers
emerging from this experiment have been validated
by real time PCR. Among those, septin 10 and dystrophin (DMD) were strongly associated with time
to treatment. The prognostic significance of some of
these factors has also been confirmed by other
groups (septin 10, DMD, AKAP12/gravin).32-34, 44-48
(Tables 2 and 3) In addition, LPL expression correlated with several functional modules including the
MTA3 and fatty acid degradation pathways.19 Using
special statistical methods exploiting the LPL-associated gene expression signature we have shown that
LPL-positive CLL cells show similarities to other tissues like fat, muscle and dendritic cells. This suggests that LPL mRNA and protein expression may be
of functional importance.
Expression of other markers was also linked to
prognosis.
The gene responsible for somatic mutations in
immunoglobulins (activation induced cytidine
deaminase – AID) is overexpressed in high-risk
(unmutated) CLL cases.49-52 We have investigated the
prognostic significance of PEG10. This maternally
expressed gene is overexpressed in high-risk CLL in
parallel with sarcoglycan ε which resides within the
same locus on chromosome 7q21. Knock-down of
PEG10 expression in primary CLL patient cells
results in increased apoptotic cell death.53
Another functionally important target is the
telomerase gene which is overexpressed in CLL cells
of patients with poor prognosis.54 Additional prognostic factors include L-selectin and integrin-β2.20 A
very CLL-specific gene is CLLU1 whose mRNA
expression level can predict time to initiation of
treatment and survival in CLL patients.55
One of the most significant findings in the field of
CLL research was the detection of certain microRNA
genes which are over- or underexpressed in conjunction with certain chromosomal aberrations.12,18
Specific miR expression signatures are correlated
with IgVH mutational status, ZAP-70 expression
and treatment-free survival indicating prognostic
importance.
Molecular monitoring of residual CLL cells during
therapy has been used as a dynamic marker during
therapy. Eradication of MRD below detection levels
of tailored PCR (ASO-PCR) or multicolour FACS is a
predictor of favourable outcome.56,57 This has been
shown for induction therapy with the antibody
alemtuzumab as well as for autologous or allogeneic stem cell transplantation.58,59
Table 3. Studies on lipoprotein lipase mRNA and protein expression as a prognostic factor.
Method
n
Microarray
purified CLL cells
Association with IgVH
mutation status
OS/EFS
TFS
Association
yes
-
-
Specific comments
with other markers
Publication
author/ref.
ZAP70, BCL7A and others
J Exp Med
2001
Rosenwald A,32
J Exp Med
2001
Klein U,33
signature related to fat, Leukemia
muscle, DC’s;
2006
functional modules:
MTA3, fatty acid degradation
Bilban M,19
Microarray
purified
+ unpurified CLL cells
34
+
NA
NA
BCL7A, ZAP70, gravin,
DMD, FGL2 and others
RQ-PCR
+Microarray
Purified CLL cells
42
NA
NA
NA
Septin10,
DMD, gravin
RQ-PCR +
+ competitive PCR
Unpurified cells
127
+
99%
NA/yes
?
ADAM29, ZAP70
significance in Binet A
Blood
2005
Oppezzo P, 44
RQ-PCR
Unpurified cells
104
+
Odds ratio 25.9
84% discrimination
no/NA
yes
cytogenetics
significance in Binet A,
LPL protein correlates
with mRNA
Leukemia
2005
Heintel D,45
RQ-PCR
Unpurified cells
130
+
yes
-ZAP70, ADAM29, Septin10,
AKAP12, DMD, NRIP1,
TPM2, CLECSF2
10 markers
selected
unpurified
more realiable
than ZAP70
Haematologica van’t Veer MB,46
2006
RQ-PCR
Unpurified cells
RQ-PCR
whole blood
CD19-selected
133
NA
yes?/NA
-
ADAM29, CD38, ZAP70
stable over time
Leuk Lymphoma
2006
Nückel H,47
50
+
83% pos. pred. v.
78% neg. pred. v.
yes/NA
yes
ZAP70
unpurified + CD19
selected equal
Clin Chem
2006
van Bockstaele F, 48
Prediction of response and influence on treatment
decisions
Cytogentics/FISH
Chromosome 17p deletions or p53 mutations result
in poor response to fludarabine and rituximab which
can be overcome by therapy with alemtuzumab60,61
(Table 4). Alemtuzumab is particularly effective when
combined with high-dose steroids.62 The predictive
value of trisomy 12 is annulled when fludarabine-containing regimens are used (Stilgenbauer, personal communication). Patients with deletions in 17p or 11q have
been shown to respond to flavopiridol.63 While combination chemoimmunotherapy with rituximab, pentostatin and cyclophosphamide was not effective in 17ppatients, it was very effective in patients with 11q
deletions.64 These novel data will obviously have consequences for treatment selection and expected
response and will lead the way to tailored therapy.
MRD detection may not only be used as a measure
of outcome, but could potentially serve to guide therapy in individual patients. In particular, response
assessment after induction therapy could be used to
tailor maintenance treatment with alemtuzumab or
rituximab.58,65 Since the clinical outcome of patients
who become MRD-negative is substantially improved these molecular data may also impact on
transplant decisions. Thus, MRD data will contribute
to challange the paradigm that CLL should not be
treated aggressively.57,66-68
In vitro evidence for response to specific targeting drugs
Molecular markers have led to the detection of
potentally active novel agents or off-label use of
known drugs against CLL (Table 4). While these
data are still based on in vitro or ex vivo observations, some of the agents used are close to clinical
testing. Inhibitors of activated heat shock protein
90 (HSP90) have been shown to influence survival
of B-cells expressing high levels of ZAP70.69 Specific
ZAP70 inhibitors are currently being developed.
Since there is strong evidence for the functional
importance of LPL in high-risk CLL, it is noteworthy that lipoprotein lipase inhibitors are already on
the market for metabolic disorders.
There is preliminary evidence that one of these
agents (orlistat) leads to apoptosis in B-CLL cells.70
Since orlistat has low toxicity, its off-label use in BCLL seems called for. We have shown that PI3kinase/AKT inhibitors (Wortmannin, Ly294002)
also effectively induce apoptosis of primary B-CLL
cells.71 A number of genes important for CLL survival have been knocked down by siRNA technology. Thus, even miR genes or others may eventually serve as targets for therapeutic interventions.12,18
Molecules detected by molecular methods may
also serve as targets for T-cell immunotherapy. One
such example is the CLL specific antigen fibromodulin which allows expansion of specific CD8+
autologous T lymphocytes in vitro.72
Table 4. Impact of selected genetic markers on treatment decisions.
Prognostic
Cytogenetics /FISH
17p-
Influence of treatment decision, response to specific drugs
Reference
(yes)
effective: alemtuzumab, flavopirdol, high-dose steroids;
ineffective: fludarabine, rituximab
60-62
11p-
yes
effective: flavopiridol, pentostatin (PCR) not very effective: alemtuzumab
63, 64
+12
yes
prognostic power lost after treatment with FC
yes
yes
no
unclear
unclear
effect of rituximab dependent on genotype
73
yes
HSP90 inhibitors effective in vitro, direct inhibitors in development
69
yes
no
no
yes
LPL inhibitor orlistat effective in vitro
Antiapoptotic effect of Wortmannin, Ly294002 in vitro
Elicits autologous CD8+ T-cell response in vitro
Multidrug resistance, target for specific agents
70
71
72
75
no
yes
fludarabine response influenced
maintenance treatment with alemtuzumab
Molecular markers
Mutation
IgVH
MiR genes
FCγReceptor IIIA
Expression
ZAP70
LPL
PI3-Kinase
Fibromodulin
MDR1
Dynamic markers
microarray p53 signature
74
58, 65
Pharmacogenomics
In addition to general static and dynamic prognostic markers related to the disease, several genetic
markers associated with response to specific therapies have been investigated (Table 4). Polymorphisms of the FC receptorγIIIA (FCGR3A) generally predict response to rituximab.73 Rosenwald et al. have
studied the gene expression pattern of CLL cells after
therapy with fludarabine in vivo and in vitro and have
shown that fludarabine induced changes which
resulted in a p53-related expression signature.74 The
results predict that fludarabine treatment will lead to
selection of p53-mutated CLL clones. The phenomenon of multi-drug resistance has recently been investigated.75
Conclusions
Genetic markers have already contributed many
important insights into the biology of B-CLL. Some
genetic markers have been established as routinely
used prognostic factors in addition to the traditional
staging systems.76 This is particularly true for cytogenetic aberrations detected by FISH. Other markers
like ZAP70 have shown their prognostic power in
large patient series, but need to be more thoroughly
harmonized for standard use; markers like IgVH
mutational status and minimal residual disease detection by PCR are harmonized, but cannot easily used
in daily routine. The least developed group including
lipoprotein lipase, AID, or micro RNAs have not
reached either one of these stages, but are potentially interesting prognostic markers.
There is strong evidence that several of the predictive markers will influence treatment decisions for
tailored therapy.77,78 While generalized routine use of
these data cannot be recommended to date, results
from randomized trials are imminent. These trials
have been powered to answer the questions related
to the rational use of specific therapies on the basis of
identification of molecular genetic markers.
Acknowledgements
This work has been supported by the Austrian Human
Genome Project (GEN-AU c.h.i.l.d.).
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State-of-the-art treatment of chronic lymphocytic
leukemia
M. Hallek
On behalf of the German CLL Study
Group
Klinik I für Innere Medizin
Universität zu Köln,
Germany
2007;1:122-128
he last decade has seen rapid
progress in the management of
chronic lymphocytic leukemia
(CLL). Fludarabine and a monoclonal antibody, alemtuzumab, have been approved
by the European and American regulatory
agencies. Additional monoclonal antibodies (anti-CD20; anti-CD23; anti-MHC II;
anti-CD40) as well as other drugs (flavopiridol, bendamustine) are currently being
tested in clinical trials. In addition, the
increased experience with allogeneic progenitor cell transplantation has offered this
intensified treatment option to physically
fit patients at very high risk of relapse or at
the time of relapse. Similarly, rapid
progress has been achieved with regard to
new diagnostic tests to identify prognostic
subgroups in CLL and to assess their
response to therapy. However, the optimal
use of these different therapeutic and diagnostic modalities remains to be determined. This review attempts to summarize the best developments today in the
initial management of CLL.
T
Clinical staging and prognostic markers
The survival period from the time of
diagnosis of CLL varies between 2 and
more than 10 years, depending on stage.
The staging systems of Rai et al.1 and Binet
et al.2 are used to predict the prognosis.
Both are based on the extent of lymphadenopathy, splenomegaly, and hepatomegaly on physical examination and on
the degree of anemia and thrombocytopenia in peripheral cell counts. These simple
studies are inexpensive and can be applied
to every patient without sophisticated
technical equipment. Both staging systems
describe three major prognostic subgroups.
However, it has long been recognized that
the above clinical staging systems are not
sufficient to predict the individual prognosis, especially in the early stages of disease
(Binet stage A, Rai stages 0–II) and in
younger patients. Therefore, additional
parameters have been identified allowing a
more accurate prediction of the prognosis
| 122 |
of CLL patients (Table 1). While these
parameters are effective in predicting the
prognosis (survival, time to progression) of
individual patients independent of the
Binet or Rai stage, only molecular cytogenetics hasve proven useful in predicting the
response to chemotherapy in CLL.
Therefore, the assessment of most parameters remains a task of clinical trials, but is
not recommended in general practice. The
only exception is the use of molecular
cytogenetics (FISH), since the occurrence
of del(17p13.1) or del(11q22.3) is associated with a shorter PFS or overall survival.3,4
Therefore, molecular cytogenetics should
be performed prior to first treatment.
Treatment decision
Any decision to treat should be guided
by clinical staging, the presence of symptoms, and disease activity. Evidence that
current treatment can improve outcome is
only available for patients with Rai III and
IV or Binet B and C stages. Patients in earlier stages (Rai 0-II, Binet A and B) are generally not treated but monitored with a
watch and wait strategy. In early stages,
treatment is necessary only if symptoms
associated with the disease occur (e.g., B
symptoms, decreased performance status,
or symptoms or complications from
hepatomegaly, splenomegaly, and lymphadenopathy). High disease activity,
often defined by a lymphocyte doubling
time of less than 6 months or by rapidly
growing lymph nodes, is also an indication
to treat. In contrast, even in advanced disease (Binet C), the absence of disease progression (e.g., with a stable platelet count
around 80,000/µL) may sometimes justify
a watch and wait strategy.
Response assessment
As with other malignancies, eradication
of the disease is a desired endpoint of CLL
treatment, especially in younger patients.
New detection technologies have found
that most patients who achieve a complete
response as defined by the National
Table 1. Parameters predicting the prognosis of CLL independent of disease stage.
treatment (chlorambucil plus prednisone vs. cladribine, 82% vs. 78% at 2 years).
Parameter
Reference
Combination chemotherapy with purine analogs
Aberrations in chromosomes 13 (13q-), 11
(11q-) and 17 (17p-)
45
Cytoplasmic ZAP70 in CLL cells
46,47
Expression of CD38 on CLL cells
48,49
Lymphocyte doubling time
50
Serum β2-microglobulin concentration
51
Serum levels of soluble CD23
52,53
Serum thymidine kinase activity
54
Somatic hyper-mutations of the immunoglobulin VH-gene region
48,55
Cancer Institute-sponsored working group (NCI-WG)
guidelines5 typically have minimal residual disease
(MRD). Critical in this assessment is a standardization
of the techniques used to define MRD. The most sensitive techniques are 4-color flow cytometry and realtime quantitative polymerase chain reaction (PCR).
Fortunately, the techniques for assessing MRD have
now become standardized.6 Nevertheless, the clinical
relevance of MRD testing needs to be verified by
prospective clinical trials and is not recommended for
general practice. Clinical trials that aim to achieve
long-lasting complete remissions should include a test
for MRD, because eradication of leukemia may have
a strong prognostic impact..7-9
First-line treatment
Monotherapy with purine analogs
Three purine analogues are currently used in CLL:
fludarabine, pentostatin, and cladribine (2-CdA).
Fludarabine remains by far the best studied compound of the three in CLL. Fludarabine monotherapy
produces superior overall response (OR) rates compared with other treatment regimens containing alkylating agents or corticosteroids.10-12 In three Phase III
studies in treatment-naïve CLL patients (Table 2), fludarabine induced more remissions and more complete remissions (CR) (7-40%) than other conventional chemotherapies, like CHOP (cyclophosphamide,
doxorubicin, vincristine, prednisone), CAP (cyclophosphamide, doxorubicin, prednisone), or chlorambucil.12-14 Despite the superior efficacy of fludarabine,
overall survival is not improved by this drug when
used as single agent.12-15
Similarly, the comparison of cladribine monotherapy to chlorambucil plus prednisone in one Phase III
trial has yielded a higher CR rate of 47% versus 12%
respectively (Table 2). However, this difference did
not result in a longer survival after cladribine first-line
Fludarabine has been evaluated in a variety of combination regimens. The combination of fludarabine
and another purine analog, cytarabine, appears to be
less effective than fludarabine alone, while the combination of fludarabine with chlorambucil or prednisone increases hematological toxicity without
improving the response rate compared with fludarabine alone (response rates 27–79%).12,16
The most thoroughly studied combination chemotherapy for CLL is fludarabine plus cyclophosphamide (FC) (Table 2).16-19 In preliminary, non-comparative trials, the overall response rates did not
appear to be better than with fludarabine alone, but
the addition of cyclophosphamide appeared to
improve the quality of response. This combination,
with or without mitoxantrone, has achieved response
rates of 64% to 100%, with CR rates of up to 50%.16
Variations on this regimen have shown that a slightly decreased cyclophosphamide dose improves the
safety profile of the regimen without compromising
efficacy, and that results were similar with concurrent
or sequential administration of the two therapies.16
The addition of mitoxantrone to FC in 37 patients
with relapsed/refractory CLL produced a high CR rate
(50%), including 10 cases of MRD negativity, with a
median duration of response of 19 months.9 All MRDnegative patients were alive at analysis. The median
duration of response had not been reached in the CR
patients compared to 25 months in non-CR patients.
A Phase II study of cladribine in combination with
cyclophosphamide has also been seen to be active in
advanced CLL, but the results seemed inferior to
those of FC.20
In a prospective trial of the German CLL study
group (GCLLSG) comparing fludarabine versus FC,
results for 375 patients showed superior response
rates for the combination.3 The FC combination
chemotherapy resulted in a significantly higher complete remission rate (16%) and overall response rate
(94%) compared to fludarabine alone (5% and 83%;
p=0.004 and 0.001 respectively). The FC treatment
also resulted in a longer median duration of response
(48 vs. 20 months; p=0.001), and a longer event-free
survival (49 vs. 33 months; p=0.001). So far, no difference in the median overall survival could be observed
within a median observation period of 22 months. FC
caused significantly more thrombocytopenia and
neutropenia, but less anemia than fludarabine. FC did
not increase the number of severe infections3 (Table
2). Two additional phase III trials have confirmed
these findings. The American study21 has included 278
previously untreated patients with CLL randomly
Table 2. Summary of recent major randomized trials for advanced chronic lymphocytic leukemia in treatment-naïve
patients.
Study
n
CR (%)
PR (%)
Response Duration Survival
(median, months) (median, months)
Rai12
Fludarabine
Chlorambucil
170
181
20
4
43
33
25
14
66
56
Johnson13
Fludarabine
CAP
52
48
23
17
48
43
NR
6.9
60% at 4 years
60% at 4 years
Leporrier14
Fludarabine
CAP
CHOP
341
240
305
40.1
15.2
29.6
31
43
41.9
31.7
27.7
29.5
69
70
67
Robak56
Cladribine + prednisone
Chlorambucil + prednisone
126
103
47
12
40
45
21
18
78% at 2 years
82% at 2 years
Eichhorst3
Fludarabine
Fludarabine, cyclophosphamide
182
180
4.9
16.5
78
78
20
48
Median not reached
Median not reached
Flinn21
Fludarabine
137
141
4.6
23.4
59.5
50.9
19.2
31.6
79% at 2 years
80% at 2 years
Robak23
Cladribine
Cladribine, cyclophosphamide
Cladribine, cyclophosphamide, mitoxantrone
166
162
151
21
29
36
56
54
44
23.5
22.4
23.6
51.2
Median not reached
Median not reached
O’Brien24
Fludarabine, cyclophosphamide, oblimersen
121
120
7*
17*
ND
ND
ND
ND
32.9
33.8
CAP,cyclophosphamide, doxorubicin, prednisone; CHOP, cyclophosphamide, doxorubicin, vincristine, prednisone; CR, complete response; PR, partial response; NR, not
reached. Please note that the response definitions varied considerably among the 4 trials. In the study by Leporrier et al.,14 a systematic evaluation of the bone marrow
was not performed routinely to confirm a CR. Thus, the CR rates are somewhat higher than in the other trials. *included nPRs. ND = not determined.
assigned to receive either fludarabine 25 mg/m2 intravenously (IV) days 1 through 5 or cyclophosphamide
600 mg/m2 IV day 1 and fludarabine 20 mg/m2 IV
days 1 through 5. These cycles were repeated every
28 days for a maximum of six cycles. Treatment with
fludarabine and cyclophosphamide was associated
with a significantly higher complete response (CR)
rate (23.4% v 4.6%; p<0.001) and a higher overall
response (OR) rate (74.3% v 59.5%, p=0.013) than
treatment with fludarabine as a single agent.
Progression-free survival (PFS) was also superior in
patients treated with fludarabine and cyclophosphamide than those treated with fludarabine (31.6 v
19.2 months, p<0.0001). Fludarabine and cyclophosphamide caused additional hematologic toxicity,
including more severe thrombocytopenia (p=0.046),
but it did not increase the number of severe infections
(p=0.812).21 The UK study was presented at the
American Society of Hematology Meeting in 2005
and has shown very similar results.22
Finally, the Polish study group compared 2-CdA
alone to 2-CdA combined with cyclophosphamide
(CC) or to cyclophosphamide and mitoxantrone
(CMC) in 479 cases with untreated progressive CLL.23
Surprisingly, the CC combination therapy did not
produce any benefit in terms of progression free sur-
vival or response rates when compared to 2-CdA
alone. Compared with 2-CdA, CMC induced a higher CR rate (36% vs 21%, p=0.004), and a trend for a
higher CR rate with CC was observed (29% vs 21%,
p=0.08). Furthermore, the percentage of patients who
were in CR and were MRD negative was higher in
the CMC arm compared with 2-CdA (23% vs 14%,
p=0.042). There were no differences in overall
response, progression-free survival, and overall survival among treatment groups. Grade 3/4 neutropenia
occurred more frequently in CC (32%) and CMC
(38%) than in 2-CdA (20%) (p=0.01 and p=0.004,
respectively). Infections were more frequent in CMC
compared with 2-CdA (40% vs 27%, p=0.02). Based
on these results, cladribine combination therapies do
not seem to offer any advantage when used as first
line treatment for CLL (Table 2).
Finally, Susan O’Brien and her colleagues examined
the effect of oblimersen, an anti-Bcl2 antagonist,
when added to the FC regimen in a study on 241
patients. Fludarabine 25 mg/m2/d plus cyclophosphamide 250 mg/m2/d were administered intravenously for 3 days with or without oblimersen 3
mg/kg/d as a 7-day continuous intravenous infusion
(beginning 4 days before chemotherapy) for up to six
cycles. CR/nPR was achieved in 20 (17%) out of 120
Table 3. Efficacy of combination regimens using fludarabine plus concurrent monoclonal antibodies in chronic lymphocytic leukemia in Phase II trials.
Reference
Fludarabine + rituximab
Schulz28
Byrd29
(Phase III)
randomized
No. of
evaluable
patients
31
104
Stage
Prior
therapy
no.
Treatment
regimen
100%
Binet B or C
Yes 20; No 11
FLU 25 mg/m2 d1-5 q 4 wk
rituximab 375 mg/m2
d1 q4wk
Rai I/II 59%
Rai III/IV 41%
No
FLU 25 mg/m2 d1-5 q4wk
rituximab 375 mg q 4 wk
concurrent vs. sequential
Yes
CYC 250 mg/m2 d1-3 q4wk
rituximab* 375/500 mg/m2
d1 q4wk
CYC 250 mg/m2 d1-3 q4wk
rituximab 375 mg/m2 d1 q4wk
Fludarabine + cyclophosphamide + rituximab
177
Rai IV 44%
Wierda57
Keating31
224
Fludarabine + alemtuzumab
Kennedy39
6
Elter40
36
Rai III + IV 33% No
Binet B 50%
Binet C 50%
Binet B 24%
Binet C 76%
Yes
Yes
CR
Clinical response
CR + PR
32%
87%
Survival/duration
of response
Median survival 33 mths;
median duration of
response 75 weeks
47% concurrent 90% concurrent After 23 months median
28% sequential 77% sequential duration of response
not yet reached
in both arms
25%
73%
Median survival 42 months;
median time to progression
28 months
70%
95%
69% failure free at 4 years
17%
alemtuzumab 30 mg tiw 8-16 wk
30%
alemtuzumab 30 mg d1-3 q4wk
83%
83% survival after
12 months
n.a.
83%
CR: complete response; CYC: cyclophosphamide; FLU: fludarabine; PR: partial response. *rituximab 375 mg/m2 course 1, 500 mg/m2 all subsequent courses.
patients in the oblimersen group and eight (7%) of
121 patients in the chemotherapy-only group (p=
0.025). Achievement of CR/nPR was correlated with
both an extended time to progression and survival (p
<0.0001). However, the overall survival and the progression-free survival did not show any difference
indicating that oblimersen did not strongly improve
the efficacy of the FC regimen.24
Rituximab-based chemo-immunotherapy
Rituximab, an anti-CD20 monoclonal antibody, has
only recently provoked interest for the treatment of
CLL. As a single agent rituximab is less active than in
follicular lymphoma, unless very high doses are
used.25,26 Somewhat surprisingly, combinations of rituximab with chemotherapy have proven to be very
effective in CLL. There is preclinical evidence for synergy between rituximab and fludarabine.27 The majority of rituximab combination studies in CLL have
focused on combinations with fludarabine or fludarabine-based regimens (Table 3). A multicenter Phase II
study of the German CLL study group has evaluated
the efficacy and safety of rituximab plus fludarabine
in patients with previously treated or untreated CLL.28
Of 31 patients treated, 27 (87%) responded, with 10
patients (32%) achieving a complete response. Byrd
and colleagues combined rituximab with fludarabine
in either a sequential or concurrent regimen in a randomized study (CALGB 9712 protocol).29 Patients
(n=104) with previously untreated CLL received six
cycles of fludarabine, with or without rituximab, followed by four once-weekly doses of rituximab.
Overall and complete response rates were higher in
the concurrent regimen group (90% and 47% vs. 77%
and 28%). More recently, in a retrospective analysis,
all patients under the CALGB 9712 protocol treated
with fludarabine and rituximab were compared with
178 patients from the previous CALGB 9011 trial,
who received only fludarabine.30 The basic characteristics of patients were comparable, except for an 8year longer observation time in the CALGB 9011 protocol. The patients receiving fludarabine and rituximab had a better progression-free survival (PFS) and
overall survival (OS) than patients receiving fludarabine alone. Two-year PFS probabilities were 67% versus 45% and 2-year OS probabilities were 93% versus 81%. Similarly, in a large Phase II trial conducted
at the MD Anderson Cancer Center on 224 patients
with previously untreated CLL, rituximab plus fludarabine/cyclophosphamide (FC) achieved a response
rate of 95% with 71% complete responses.31 Median
overall survival was not reached in patients treated
with rituximab plus FC, and was significantly longer
than in patients treated with FC alone in an historical
comparison (Table 3).
Taken together, these results suggest that rituximab
plus fludarabine-based therapies represent a significant advance in therapy for CLL. However, the analysis by Byrd and colleagues is retrospective and could
be confounded by differences in supportive care or
different prognostic subsets included in the two trials.
Therefore, these findings need to be confirmed by
prospective trials. The results from the GCLLSG
CLL8 trial comparing FC to FCR are expected next
year.
Alemtuzumab-based chemo-immunotherapy
Alemtuzumab is a recombinant, fully humanized,
monoclonal antibody against the CD52 antigen.
Monotherapy with alemtuzumab has produced
response rates of 33% to 53%, with a median duration of response ranging from 8.7 to 15.4 months in
patients with advanced CLL who were previously
treated with alkylating agents and had failed or
relapsed after second-line fludarabine therapy.32-34 In
addition, alemtuzumab has proven effective even in
patients with poor prognostic factors, including highrisk genetic markers such as deletions of chromosome
11 or 17 and p53 mutations.35,36 If these results are confirmed in larger, prospective trials, alemtuzumab
might be a rational choice for first-line treatment of
patients with these poor prognostic factors.
Alemtuzumab consolidation therapy after fludarabine-based chemotherapy also improved the quality
of response, achieved molecular remissions in a substantial proportion of patients, and increased PFS
compared with patients who had no further treatment.8,37,38 Results of a Phase III trial by the GCLLSG
showed improved PFS with alemtuzumab consolidation therapy compared to the observation arm (no
progression vs. 24.7 months, p=0.036) when calculated from the start of fludarabine-based treatment0.8
When PFS was calculated from the date of alemtuzumab administration, the same benefit was apparent, with no progression compared to 17.8 months
(p=0.036). O’Brien and colleagues reported an overall
response rate of 53%, in 9 out of 23 patients (39%) at
a 10 mg dose and 17 out of 26 (65%) at a 30 mg dose
(p=0.066)0.38 Residual disease was cleared from the
bone marrow in most patients, and 11 (38%) of the 29
patients with available data achieved a molecular
remission. Median time to disease progression had
not yet been reached for patients who achieved MRD
negativity, compared to 15 months for patients who
still had residual disease after alemtuzumab consolidation treatment.38 While the GCLLSG trial was
stopped early because of infectious adverse events,
this was not the case in the study by O’Brien et al.
This was perhaps due to a longer time interval
between induction therapy and consolidation with
alemtuzumab (6 months versus 3 months in the
GCLLSG study).
Perhaps the most potent regimen for CLL is the
combination of the most effective single chemotherapeutic agent with the most effective monoclonal antibody fludarabine plus alemtuzumab (Table 3). The
synergistic activity of these two agents was initially
suggested by the induction of responses, including
one CR, in 5 out of 6 patients who were refractory to
each agent alone.39 The combination of fludarabine
and alemtuzumab was investigated in a Phase II trial
enrolling patients with relapsed CLL (Table 3).40 Using
a four-weekly dosing protocol, this combination has
proven feasible, safe, and very effective. Among the
36 patients, the ORR was 83% (30/36 patients),
which included 11 CRs (30%) and 19 PRs (53%). In
addition, one patient achieved stable disease. Sixteen
out of 31 (53%) evaluated patients achieved MRD
negativity in the peripheral blood by 3 months’ follow-up, and resolution of disease was observed in all
disease sites, particularly in the blood, bone marrow
and spleen. The fludarabine/alemtuzumab combination therapy was well tolerated. Infusion reactions
(fever, chills, and skin reactions) occurred primarily
during the first infusions of alemtuzumab, and were
mild in the majority of patients. While 80% of
patients were cytomegalovirus immunoglobulin G
(CMV IgG)-positive before treatment, there were
only two subclinical CMV reactivations. The primary
grade 3/4 hematological events were transient,
including leukocytopenia (44%) and thrombocytopenia (30%). Stable CD4+ T-cell counts (> 200/µL) were
seen after 1 year. A Phase III prospective randomized
study evaluating the effectiveness of fludarabine and
alemtuzumab combination in comparison with fludarabine alone is currently underway.
The combination of both monoclonal antibodies
(alemtuzumab and rituximab) has been studied in
patients with lymphoid malignancies, including those
with refractory/relapsed CLL, producing an ORR of
52% (8% CR; 4% nodular PR, nPR; 40% PR).41 A larger trial with a longer follow-up is needed to confirm
these preliminary results and determine the long-term
efficacy of this combination.
Conclusions
Treatment of CLL in first line
Given the potential of the chemoimmunotherapy
regimens described above, choosing the right treatment for a patient with CLL has become a task that
requires skill and experience. Table 4 proposes an
algorithm for the selection of the best treatment
option, which is based on three potentially relevant
considerations. a) Patient physical condition (fitness
Table 4. Summary of the current options for first second
line treatment in CLL.
Binet stage
Fitness
First line treatment
GCLLSG trialk
A, asymptomatic B
Irrelevant None
CLL7
(FCR at high risk?)
C, symptomatic B
Go Go
Slow Go
FCR, FC
CLB, F (reduced dose)
CLL8
CLL9
Relapse
Fitness
Second line
GCLLSG trial
Early (<1 year) =
refractory disease
GoGo
Slow Go
Allo Tx
Alemtuzumab (17p-)
Bendamustin, R-CHOP
CLL3X
CLL2G,
CLL2M
Late(>1 year)
Go Go &
Slow Go
Repeat first line
and comorbidity) is independent of calendar age; b)
the prognostic risk of the leukemia as determined by
the factors mentioned above; c) Rai or Binet disease
stage
Patients at early stage (Binet A and B without symptoms) should not be treated outside clinical trials.
Treatment may be indicated in clinical trials in
patients at high risk of disease progression.
Patients with advanced disease (Binet C, or symptomatic stage A or B) should start treatment. In this situation, patients need to be evaluated for their physical condition (or comorbidity). For patients in good
physical condition (go go) as defined by a normal creatinine clearance and a low score at the cumulative illness rating scale (CIRS),42 patients should be offered
more combination therapies such as FC or FR or FCR.
Patients with an impaired physical condition (slow go)
may be offered either chlorambucil or a dose-reduced
fludarabine monotherapy for symptom control.
Patients with symptomatic disease and with
del(17p) or p53 mutations should receive an alemtuzumab-containing regimen as first-line treatment,
because these patients respond poorly to fludarabine
or fludarabine-cyclophosphamide.
Treatment for relapsed or refractory disease
While an extensive review of all treatment options
for relapsed or refractory CLL is beyond the scope of
this paper, Table 4 summarizes some principles of the
management of patients at relapse according to the
duration of remission and the physical fitness.
In general, first-line treatment may be repeated, if
the duration of the first remission exceeds 12 months
(or with the modern chemoimmunotherapies, 24
months).
The choice becomes more difficult and limited in
treatment-refractory CLL (as defined by an early
relapse within 12 months after the last treatment) or in
cases with the chromosomal aberration del(17p). In
principle, the initial regimen should be changed. There
options are: Alemtuzumab alone or in combination.33,40
Flavopiridol (if available).43 Allogeneic stem cell transplantation with curative intent.44
The choice of one of these options depends on the
fitness of the patient, the availability of drugs and the
molecular cytogenetics. Physically fit patients with a
del(17p) should be offered an allogeneic transplantation, since their prognosis remains poor with conventional therapies. The EMBT consensus has defined
criteria for including patients in allogeneic transplantation protocols.44
Finally, it is important to emphasize that all patients
with refractory disease should be treated within clinical trials whenever possible.
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New drugs for chronic lymphocytic leukemia
A
J. Gribben
Institute of Cancer,
Barts and
The London School of Medicine,
University of London,
United Kingdom
2007;1:129-133
B
S
T
R
A
C
T
Recently there has been an improvement in response rates in patients with chronic
lymphocytic leukemia (CLL) using combination chemotherapy and chemoimmunotherapy, most commonly with rituximab or alemtuzumab. After relapse, retreatment is more difficult in patients who have received combination chemoimmunotherapy and is particularly difficult once patients become refractory to fludarabine, and new agents are needed. Ongoing clinical trials are investigating the role of
new agents, including alternative nucleoside analogs, monoclonal antibodies, novel
agents including lenalidomide and flavopirodol, signal transduction inhibitors/small
molecules targeting novel pathways capable of overcoming the failure of apoptosis in
CLL cells.
he outcome of patients with CLL is
improving with the use of combination chemotherapy1,2 and chemoimmunotherapy3and the use of purine
analog combinations have resulted in the
highest response rates observed to date in
CLL. Most studies have used fludarabine
while, similar responses have been seen
with pentostatin.4 Although an early
report noted responses to cladribine in
patients with fludarabine-refractory CLL5
this has not been confirmed in later studies which also demonstrated considerable
toxicity from cladribine due to myelosuppression and infections.6 Trials with the
novel purine analog clofarabine, administered daily for five days, demonstrated
minimal activity in CLL.7 Therefore, there
is no evidence to support the use of an
alternative nucleoside analog in fludarabine-refractory CLL.
Once patients relapse after combination
chemoimmunotherapy, and particularly
as they become resistant to fludarabine,
there are few therapeutic options available. Alemtuzumab is the only drug currently licensed for use in fludarabine
refractory CLL following an important
demonstrating improved survival in this
setting.8 There is clearly a need for additional agents with activity in this disease
New drug development in CLL has
been seriously compromised both by the
lack of suitable cell lines derived from
T
patients with CLL as well as appropriate
animal models of the disease. The EµTCL1 mouse develops a B cell lymphoproliferative disorder resembling CLL9
and although there are concerns regarding
how accurately the model reflects the
human disease, CLL cells in the Eµ-TCL1
mouse model express relevant therapeutic
targets including anti-apoptotic proteins,
survival kinases and DNA methlytrasferases. Specifically, the TCL-1
leukemia cells express BCL-2, MCL-1 and
DNMT1, with corresponding phosphorylation of AKT and PDK1 and also demonstrate similar in vitro and in vivo sensitivity
to agents including fludarabine, flavopiridol and OSU03012 and resistance to
paclitaxel, as occurs in the human disease.10 Therefore, this model has sufficient clinical and therapeutic similarities
to human CLL to suggest exciting new
opportunities to screen new drugs and
novel combinations in vivo and speed therapeutic development in this still incurable
disease.
Targeting anti-apoptotic pathways in CLL
Over-expression of BCL-2 in CLL is
associated with chemotherapy resistance
and decreased survival and inhibition of
this antiapoptotic proteins is an attractive
strategy for either restoring normal apoptotic process in CLL cells or making them
more susceptible to conventional chemo-
therapy. A number of agents are under investigation
which target not only BCL-2, but also other antiapoptotic proteins in CLL cells. Oblimersen is an
antisense oligonucleotide compound designed to
bind to the first six codons of human BCL-2 mRNA,
resulting in degradation of subsequent decrease in
Bcl-2 protein translation. Studies have investigated
the role of this agent in improving responses to
chemotherapy in patients with relapsed or refractory CLL. Twohundredandforty-one patients were
randomly assigned to 28-day cycles of fludarabine
plus cyclophosphamide (FC) with or without
oblimersen 3 mg/kg/d as a 7-day continuous intravenous infusion (beginning 4 days before chemotherapy) for up to six cycles. Complete remission
(CR) or nodular partial remissions (nPR) were
achieved in 17% of the oblimersen group but only
7% of the chemotherapy-only group (p=0.025) and
those patients who achieved CR/nPR had increased
time to progression and survival (p<0.0001).11 This
was particularly the case in those patients who
remained fludarabine sensitive. The major toxicity
was thrombocytopenia and, rarely, tumor lysis syndrome and cytokine release reactions. The incidence
of opportunistic infections and second malignancies
was similar in both groups.
Novel agents specifically targeting anti-apoptotic
proteins include the pan-BCL-2 inhibitor GX15-070
which mimics BH3-only proteins by binding to multiple antiapoptotic BCL-2 members. A number of
agents targeting these pathways are in pre-clinical
development. It is also clear that a number of agents
that are thought to target other pathways, also
mediate at least some of their effect by alteration of
the balance of pro-and anti-apoptotic proteins in
malignant cells, including the proteosome inhibitor
bortezomib. Although this agent has impressive
activity in a number of B cell malignancies, disappointing response rates were observed in a clinical
trial in CLL.12
Lenalidomide
There is increasing interest in agents that target the
tumor-cell microenvironment. Immunomodulating
agents alter cytokine expression, co-stimulate
immune effector cells, and have direct and indirect
effects upon CLL cells. Lenalidomide is a synthetic
analog of thalidomide and while its mechanism of
action is uncertain, lenalidomide downregulates
tumor necrosis factor-α and enhances antibodydependent cytotoxicity. Significant activity of
lenalidomide has been observed in both 5qmyelodysplasia and multiple myeloma and now also
in CLL.13 In a study of CLL patients, lenalidomide
was administered orally at 25 mg on days 1 through
to 21 of a 28-day cycle and patients continued treat-
ment until disease progression, unacceptable toxicity, or complete remission. Rituximab could be added
to lenalidomide on disease progression. Forty-five
patients were enrolled, 51% refractory to fludarabine. Overall response was 47%, with 9% of
patients attaining CR. The most common adverse
effects were fatigue, thrombocytopenia, and neutropenia. Lenalidomide is clinically active in patients
with relapsed or refractory B-CLL and is under
investigation in ongoing clinical trials.
Flavopiridol
Flavopiridol is a synthetic flavone that inhibits
CDK1, CDK2, CDK4 and other kinases, including
cyclin-dependent kinase 9, leading to inactivation of
RNA polymerase II and global inhibition of gene
transcription. Flavopiridol induces apoptosis in CLL
cells in vitro at concentrations attainable in the clinic
and induces apoptosis in CLL cells by activating caspase 3. Importantly, induction of apoptosis by
flavopiridol occurred in a p53 independent manner.
It is likely that alternative mechanisms of action are
also involved in producing flavopiridol’s rapid cytotoxic effect on CLL cells in vivo. A variety of different
schedules of administration have been explored
with flavopiridol, including 72-hour continuous
infusion, 24-hour continuous infusion, and 1-hour
bolus. The 24-hour infusion schedule was examined
at two doses in 28 patients and no clinical responses
were noted.14 Pharmakinetic modeling studies at
Ohio State University suggested that a 30-minute
bolus followed by a 4-hour CI schedule could provide a sufficient concentration of flavopiridol to
induces apoptosis in primary CLL cells. In a subsequent clinical trial 42 patients were enrolled in 3
cohorts. Cohort 1 was treated at 30 mg/m2 loading
dose followed by 30 mg/m2 4-hour infusion, cohort
2 at 40 mg/m2 loading dose followed by 40 mg/m2 4hour infusion, and cohort 3 at cohort 1 dose for
treatments 1 to 4, then a 30 mg/m2 loading dose followed by a 50 mg/m2 4-hour infusion. The dose-limiting toxicity was hyperacute tumor lysis syndrome,
which may be prevented by aggressive prophylaxis
and exclusion of patients with high white blood
counts. Of the 42 patients treated, 45% achieved a
PR and responses were noted in patients with del
17p13 and del 11q22, justifying a further study of
flavopiridol in CLL and other diseases. In addition,
other studies with flavopiridol, both to eliminate
minimal residual disease and in combination with
other agents, are being developed.
Monoclonal antibodies
Alemtuzumab is the only monoclonal antibody
currently licensed for use in CLL and is licensed for
fludarabine refractory CLL following a pivotal trial
demonstrating improved survival in this setting.8
Alemtuzumab is being investigated as consolidation
therapy to eradicate minimal residual disease.15,16
There have been concerns regarding the safety of
this approach17 and ongoing studies are examining
the optimal timing, duration and route of administration of this agent as consolidation. Alemtuzumab
has been investigated in combination with fludarabine,18 and with rituximab.19
Despite its impressive activity in B cell nonHodgkin’s lymphoma, the activity of rituximab as a
single agent in CLL is modest when rituximab is used
at the standard 375 mg/m2 weekly for 4 to 8 weeks as
for lymphoma, but does have increased activity at
higher doses or with increased frequency of scheduling.20,21 Patients with del 17p did not respond to rituximab monotherapy.22 Most interest in rituximab is in
its potential synergistic activity with chemotherapy,
including fludarabine alone,23 FC,3 or pentostatin and
cyclophosphamide.4 Ongoing randomized trials will
demonstrate whether the addition of rituximab to
chemotherapy is associated with improved response
rates, durations of response and overall survival.
Rituximab is also being investigated as monotherapy
in early stage disease in the elderly and in combination
with chlorambucil, lenalidomide and bortezomib.
Since CD23 expression is a characteristic feature
of CLL cells, the anti-CD23 antibody lumiliximab is
of interest and is being investigated in combination
with chemotherapy and rituximab. HuMax-CD20 is
the first fully human monoclonal antibody and targets a different epitope of the CD20 molecule
expressed by B cells and is currently in clinical trials.
Other monoclonal antibodies of interest in CLL
include anti-CD40. CHIR-12.12 is a fully humanized
anti-CD40 monoclonal antibody that blocks interaction with CD40 ligand, thereby preventing an antiapoptotic stimulus to CLL cells. Based on a promising clinical study, successful xenograft lymphoma
studies, and acceptable toxicity in vivo, a phase I
study of CHIR-12.12 has recently been initiated in
relapsed and refractory CLL. SGN40 is another fully
humanized CD40 directed antibody that does not
block interaction with CD40. Preclinical studies
with SGN40 are underway both as a single agent
and in combination with other biologic therapies.
Tru 16.4 is a single-chain protein with a modified
CD37 binding Fv domain linked to a modified
human IgG1 hinge, CH2 and CH3 domains, and is a
member of a novel composition class called small
modular immunopharmaceuticals (SMIPs). Preclinical studies with Tru 16.4 demonstrated that it
binds to CD37 on primary CLL cell surface and
induces caspase-independent apoptosis. Further
modifications of Tru16.4 are underway to enhance
ADCC mechanisms. Hu1D10 is a humanized mon-
oclonal antibody that targets a β-chain epitope of
HLA-DR and induces apoptosis via a novel pathway.
Up to 70% of patients express this antigen on their
CLL cells. A phase I trial administering this agent
three times a week in CLL patents has been completed with clinical responses noted, including patients
with del 17p13 and a phase II study is underway in
fludarabine-refractory CLL. Combination studies
with Hu1D10 and granulocyte colony-stimulating
factor and Hu1D10 and rituximab are also ongoing.
Additionally, there are other broad HLA-DR–directed antibodies that are currently being considered for
clinical trials.
Small molecules in CLL
Increased understanding of CLL pathophysiology
has led to the investigation of a variety of targeted
therapies directed at specific signal transduction
pathways. Many of these either have preclinical
rationale or have been demonstrated to have preclinical activity in CLL and merit further investigation. Notable among these agents are 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) and
17-dimethylaminoethylamino-17-demethoxygeldanamycin (DMAG) that inhibit function of activated heat shock protein 90 (hsp90), thus preventing chaperoning of client proteins including AKT
and ZAP70 in CLL cells..25 The nutlins are smallmolecule activators of p53 that have been developed and who inhibit binding of p53 to MDM2.
Nutlin-3 stabilized p53, induced p53 target genes
and synergized with chemotherapy, suggesting
that MDM2 antagonists alone or in combination
with chemotherapy may have potential in CLL.26
Of note, nutlin-3 activates the p53 pathway and
effectively induces apoptosis in cells with dysfunctional ATM, but not mutant p53.
Other agents
Protein kinase C inhibitors including UCN-01 (27)
and PKC412,28 and alternative cyclin-dependent
kinase inhibitors including Roscovitine,29 may also be
promising in CLL. Chromatin remodeling agents
which have been studied include the histone
deacetylase inhibitors depsipeptide30 and MS275,31 as
well as decitabine, which inhibits DNA methyltransferase which has shown promise in preclinical testing. Other agents including epigallocatechin-3 gallate
(EGCG)32 have shown promising preclinical activity
and are entering clinical trials. R-etodolac (SDX-101)
is an isoform of the non-steroidal anti-inflammatory
drug etodolac which shows activity against CLL
cells33 and is currently being tested in phase II clinical
trials for the treatment of refractory B-cell.
Deletion and dysfunction of p53 in CLL are associated with poor response to chemotherapy and
poor outcome. Corticosteroids have long been recognized to have activity in CLL, and recent studies
have focused on the use of high dose methylprednisolone to induce response in patients with p53
dysfunction.24 When high-dose corticosteroids are
used for the treatment of CLL, it is necessary to
administer antibiotic and antiviral prophylaxis and
to monitor patients closely. High dose methylprednisolone is being investigated in combination with
monoclonal antibodies including rituximab and
alemtuzumab and, in particular, the combination of
alemtuzumab and high dose corticosteroids appears
attractive to target CLL cells with dysfunctional p53.
Future directions
After many years in which few agents showed
documented activity in CLL, there is increased interest in the development of agents that target specific
pathways. These agents have often been chosen on
the basis of a fuller understanding of the underlying
pathophysiology of CLL and selected for further
study according to their preclinical activity. The
potential role of novel agents in CLL will be dependent upon the results of ongoing clinical trials and the
subsequent use of these agents will probably be
dependent upon particular agents showing activity
in subsets of patients with specific genetic risk factors.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
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Sickle Cell Disease
Hemolysis-associated pulmonary hypertension
in sickle cell disease and thalassemia
G.J. Kato1,2
M.T. Gladwin1,2
1
Vascular Medicine Branch, National
Heart Lung and Blood Institute;
2
Critical Care Medicine Department,
Clinical Center National
Institutes of Health, Bethesda,
Maryland, USA
2007;1:134-139
| 134 |
edical advances in the management of patients with sickle cell
disease, thalassemia, and other
hemolytic anemias have led to significant
increases in life expectancy. Improved
public health, neonatal screening, parental
and patient education, advances in red cell
transfusion medicine, iron chelation therapy, penicillin prophylaxis for children,
pneumococcal immunization, and hydroxyurea therapy have all probably contributed to this effect on longevity.
Importantly, as a generation of patients
with sickle cell disease and thalassemia
ages, new chronic complications of these
hemoglobinopathies emerge. In this context, pulmonary hypertension is emerging
as one of the leading causes of morbidity
and mortality in adult sickle cell and thalassemia patients, and in patients with
other hemolytic anemias.
A common feature of both sickle cell
disease and thalassemia is intravascular
hemolysis and chronic anemia. Recent
data suggests that chronic intravascular
hemolysis is associated with a state of
endothelial dysfunction. This is characterized by reduced nitric oxide (NO)
bioavailability, pro-oxidant and proinflammatory stress and coagulopathy,
leading to vasomotor instability and ultimately producing a proliferative vasculopathy, with the development of pulmonary hypertension in adulthood.
This article will briefly review the role
of NO in homeostatic endothelial and
vasomotor function and the specific
mechanisms of endothelial dysfunction in
sickle cell disease. The mechanisms held
responsible for the development of pulmonary hypertension in hemolytic diseases and the role of pulmonary hypertension as a risk factor for death in patients
with sickle cell anemia will then be discussed. Finally, a case will be made that
hemolytic anemia produces a clinical subphenotype in patients with sickle cell disease that is shared by other hemolytic disorders, a phenotype characterized by pul-
M
monary hypertension, priapism, cutaneous leg ulceration, sudden death, and
possibly stroke.
Hemolysis, a forgotten complication of sickle
cell disease
Sickle cell disease is basically a form of
hemolytic anemia. The average patient
with SCD has a hemoglobin level that is
approximately one-half that of normal.
The red cell survival is as low as 10-20
days, requiring massively increased red
cell production to maintain even this very
low hemoglobin level, indicated by a reticulocyte count elevation often five to twenty times normal. The red cell mass
hemolyzed in a single day has been estimated to release 30 gm of hemoglobin.
Although approximately two-thirds of the
hemolysis takes place extravascularly in
the reticuloendothelial system, the
remaining one-third of the hemolysis
occurs intravascularly, decompartmentalizing up to 10 gm of hemoglobin and
other red cell contents into blood plasma
daily.1 Almost 40 years ago, Naumann and
Neely and their colleagues documented
the high levels of hemoglobin and lactate
dehydrogenase (LDH) in the plasma of
patients with SCD.2,3 They also demonstrated crisis-associated hyperhemolysis,
with sharply higher levels of plasma
hemoglobin and LDH during vaso-occlusive pain crisis. Using more modern methods, these pioneering findings have
recently been confirmed by our group and
by Ballas and colleagues.4,5 These levels far
exceed the hemoglobin-scavenging capacity of the haptoglobin-hemopexin system,
resulting in prolonged exposure of the
blood vessel wall and blood plasma to
high levels of hemoglobin.
NO and vascular function
Nitric oxide (NO) is a soluble diatomic
gas molecule, produced by the endothelial
cells that line the blood vessel. It serves as
a master regulator of vascular function and
enhances blood flow.6 NO diffuses from
the endothelial cell, binding to its primary receptor in
vascular smooth muscle cells, guanylyl cyclase, activating the conversion of GTP to cGMP. This cyclic
nucleotide induces a signal transduction cascade that
effects smooth muscle relaxation and vasodilation.
NO also represses a whole program of pathways that
contribute to vascular obstruction. NO represses activation of platelets and endothelial cells, release of
coagulation factors, and expression of endothelial
adhesion molecules. This response seems to have
evolved so that loss of NO signaling results in hemostasis, including vasoconstriction, endothelial adhesiveness and thrombosis.
Intravascular hemolysis and impaired NO bioavailability
There are multiple lines of evidence indicating
impaired nitric oxide bioavailability in patients and
mice with SCD, linked to intravascular hemolysis.
This decompartmentalization of red cell contents
into blood plasma releases hemoglobin, which inactivates NO in a diffusion-limited stoichiometric reaction, generating plasma methemoglobin and inert
nitrate.5 The large amount of hemoglobin released
into blood plasma in SCD saturates and overwhelms
the hemoglobin scavenging system, resulting in prolonged exposure of endothelial cells to levels of plasma hemoglobin in the 10-20 µM range, at times
reaching levels of 50-100 µM. This degree of hemoglobinemia results in readily identifiable blunted
vasodilatory responses to exogenous nitric oxide
donors in several human and animal blood flow
physiology studies, constituting a form of nitric
oxide resistance.6-12 These data imply that in patients
with SCD, a portion of NO released from endothelial
cells is intercepted by plasma hemoglobin, diminishing its bioavailability to effector cells.
Intravascular hemolysis also releases red cell
arginase into blood plasma, which worsens the
state of impaired NO bioavailability. This ectopic
arginase activity depletes blood plasma of L-arginine, limiting the expected compensatory increase
in NO production due to accelerated NO turnover.
Plasma arginase or its proxy (decreased arginine:
ornithine ratio) is associated with pulmonary hypertension and risk of early mortality.13 Parallel findings
are seen in thalassemia.14 Increased plasma levels of
methylated arginine species are also detectable in
patients with SCD, which may potentially further
impair L-arginine transport or NO synthase activity
in endothelial cells.15
Reactive oxygen species can also impair NO
bioavailability in SCD.16-18 These oxygen radicals can
react stoichiometrically with NO, contributing to NO
depletion and giving rise to highly oxidative peroxynitrite.19 Besides inhibiting NO production, depletion of L-arginine can uncouple NO synthase activity,
Table 1. Contributory factors to hemolysis-associated vasculopathy.
Scavenging of nitric oxide by cell-free plasma hemoglobin
Diversion of plasma L-arginine by cell-free plasma arginase from nitric oxide synthase
Generation of oxygen radicals:
Xanthine oxidase
NADPH oxidase
Uncoupled nitric oxide synthase activity
causing it to generate reactive oxygen species.20
Xanthine oxidase and NADPH oxidase, present in
large amounts in sickle cell blood vessels, also produce superoxides and hydrogen peroxides.17,21
Oxygen radical production in SCD is associated with
endothelial dysfunction (Table 1).
Sickle vasculopathy: pulmonary hypertension
Pulmonary hypertension was the first sickle cell
complication to be linked to NO scavenging due to
chronic intravascular hemolysis. Once considered a
rare occurrence in SCD, more recent reports indicate
the frequency of pulmonary hypertension, and its association with early death.22,23 Prospective echocardiography screening studies indicate that approximately
one-third of adults with SCD have a tricuspid regurgitant jet velocity (TRV) of 2.5 m/sec or higher.24-26
Furthermore, such patients have a 9-10-fold risk for
early mortality compared to those with a normal
TRV.24,27 Further characterization of a subset of these
patients with right heart catheterization studies indicate that the vast majority of patients with a high
TRV have pulmonary arterial hypertension (PAH), a
state of chronic pulmonary vasoconstriction leading
to chronic proliferative vascular changes.24 Independent correlates of high TRV include older age,
systolic hypertension, and markers of high hemolytic rate, renal insufficiency, iron overload and a subtle
cholestatic hepatopathy. Frequency of vaso-occlusive
pain or acute chest syndrome episodes do not appear
to contribute. Interestingly, SCD males with a high
TRV are more likely to have a history of priapism.24
Concurrent with hyperhemolysis during vaso-occlusive crisis, the TRV rises acutely, then later falls back
to baseline levels.28
There has been some controversery as to
whether pulmonary hypertension in SCD might be
due to left ventricular diastolic dysfunction
(LVDD). In a recent study by our group, tissue
Doppler measurements and other detailed echocardiographic analysis indicate that LVDD and PAH
are independent and additive risk factors for
death.29 In this cohort, roughly three-quarters of
SCD adults with TRV ≥2.5 m/sec have isolated pulmonary arterial hypertension. The remaining quar-
ter have evidence of combined PAH and LVDD,
with a far worse prognosis than either PAH or
LVDD alone (Figure 1). Finally, only 7% of the
entire cohort had isolated LVDD without PAH. The
primary correlates of LVDD were systemic hypertension and renal insufficiency. Thus LVDD and
PAH are independent and overlapping risk factors
for early mortality in SCD, but isolated PAH
appears to be three times as common.
Pulmonary hypertension is now also known to
occur frequently in patients with thalassemia.30-36 Its
prevalence may exceed 65% in untransfused thalassemia intermedia patients, who have particularly
severe chronic intravascular hemolysis. Originally,
such cases were believed to be caused by recurrent
pulmonary emboli, but the mechanism of hemolysisassociated impairment of NO bioavailability, may
better define the clinical circumstances,14 especially
since recurrent pulmonary thrombosis in situ is a recognized common complication of all forms of pulmonary hypertension.37 Many reports indicate that
pulmonary hypertension is more frequently recognized in thalassemia patients who have undergone
splenectomy.38-40 Although it is possible that surgical
splenectomy serves only to identify a subset of
patients with more severe and high risk thalassemia,
there is circumstantial evidence that post-splenectomy thalassemia patients have higher levels of cellfree plasma hemoglobin.41 Even though splenectomy
may reduce the overall hemolytic rate, it might shunt
a portion of hemolysis from an extravascular to
intravascular pattern, resulting in the observed high
level of plasma hemoglobin, and high levels of plasma arginase which are probably associated. Since
NO is well known to suppress hemostasis at many
levels, including platelet activation and adhesion, and
release of tissue factor and other procoagulant proteins,42 decreased bioavailability of NO may account
in part for the thromboembolic events that are gaining increased recognition in thalassemia patients.43-49
Sickle vasculopathy: cutaneous leg ulceration
Leg ulceration occurs in about 20% of patients
with SCD, often a troublesome and recurrent problem. These ulcers develop most frequently in those
patients with clinical markers of more severe chronic
hemolysis: lower hemoglobin, higher serum bilirubin
and LDH.50 SCD patients with concurrent α-thalassemia, which serves to reduce hemolysis, have a
lower prevalence of leg ulcers.51,52 SCD males with leg
ulcers are more likely to have a history of priapism.
Polymorphisms are more common in SCD patients
with leg ulcers in genes implicated in pathways
affecting angiogenesis and vascular function: Klotho,
TEK, TGF-β and BMP.50 All of these data point to a
pattern of hemolysis-associated vascular dysfunction
PAH Only
29%
RR 3.4
Normal
51%
LVDD
Only
7%
RR 4.8
Both
11%
RR 12.0
Figure 1. Relative contribution of diastolic dysfunction in
sickle cell pulmonary hypertension. In a subset of 138
sickle cell patients studied in detail with echocardiography
and tissue Doppler measurements, 29% had isolated pulmonary arterial hypertension (PAH), as indicated by TRV
≥2.5 m/sec; 7% met criteria for isolated left ventricular
diastolic dysfunction (LVDD); and 11% had evidence of
simultaneous PAH and LVDD. Either PAH or LVDD alone
were associated with an increased relative risk (RR) of
early mortality, but simultaneous PAH and LVDD was associated with a particularly poor prognosis. Diagram adapted
from reference 29.
in the development of leg ulceration in patients with
SCD. So sickling does not appear to be necessary for
leg ulceration. Leg ulcers are also described in thalassemia, particularly thalassemia intermedia, and
hereditary spherocytosis.53–60 They have been reported in other hemolytic anemias, including pyruvate
kinase deficiency.61,62
Sickle vasculopathy: priapism
A very similar picture has emerged for the relationship of priapism and hemolysis associated vascular
dysfunction. Males with SCD and a history of priapism are more likely to have clinical markers of
accelerated hemolysis: lower hemoglobin, higher
reticulocyte counts, and higher serum levels of bilirubin, and LDH.63 Like leg ulcers, priapism has been
linked to polymorphisms of the Klotho gene.64 A link
to hemolysis, is further supported by reports of priapism in several other hemolytic disorders besides
SCD, particularly thalassemia.65-71 Since penile erection requires NO bioactivity, it would be expected
that the hemolysis-related impairment in NO
bioavailability would tend to inhibit, not promote,
priapism. However, consistent with the association
of priapism and NO consumption in SCD, the eNOS
Table 2. Subphenotypes of sickle cell disease.
Hemolysis-associated vasculopathy
Pulmonary arterial hypertension
Cutaneous leg ulceration
Priapism
Ischemic stroke?
Viscosity-vaso-occlusion syndrome
Vaso-occlusive pain crisis
Acute chest syndrome
Osteonecrosis
deficient mouse also develops priapism.72 The proposed cause of this paradoxical association of priapism with impaired NO bioavailability is loss of
NO-stimulated phosphodiesterase-5 (PDE5) expression. Intermittent treatment with PDE5 inhibitors is
thought to induce a compensatory increase in PDE5
protein expression, which ultimately serves to reduce
priapic activity in pilot studies in patients with
SCD.73,74
Is stroke part of sickle vasculopathy?
Cerebrovascular disease with ischemic stroke is
another example of large-vessel arteriopathy, histopathologically very similar to pulmonary hypertension.75,76 Like pulmonary hypertension, ischemic
stroke in SCD has been epidemiologically linked to a
low hemoglobin level and higher systemic systolic
blood pressure.77 Although the contribution of a
hemolysis-associated low NO state to cerebrovascular disease in SCD is an attractive hypothesis, it has
not been specifically investigated. However, our
group has published a case series of six patients with
SCD and PAH who developed cerebrovascular disease or stroke.78 The characteristics of these patients
support the hypothesis that cerebrovascular disease
may be part of the spectrum of sickle vasculopathy,
but further research in this area is needed.
Sickle vasculopathy and platelet activation
For many years, high levels of platelet activation
have been recognized in patients with sickle cell
disease.79-81 The level of platelet activation appears
to be even higher during vaso-occlusive crisis.80 Our
group has found that platelet activation correlates
significantly with the TRV in patients with SCD,
and to some extent also with markers of hemolytic
severity.82 This suggests that platelet activation may
join vasoconstriction as a downstream consequence
of hemolysis-associated impaired NO bioavailability. This interpretation is consistent with the known
strong anti-platelet effects of NO.83 Once again,
some parallel findings have been reported in thalassemia.84
Vasculopathy versus viscosity-vaso-occlusion
The concepts presented above describe a subset of
sickle cell complications that were previously
ascribed to sickling and vaso-occlusion. Although
sickling may play a role in these disorders, there are
now stronger mechanistic and epidemiologic data to
implicate hemolysis-associated impairment of nitric
oxide bioavailability. The steady state serum level of
lactate dehydrogenase is in large part an indicator of
intravascular hemolysis in SCD, and is associated
with impaired NO bioavailability, PAH, leg ulcers,
priapism and early mortality.85 These complications
are associated with particularly severe hemolytic
anemia. In contrast, vaso-occlusive crisis, acute chest
syndrome, and osteonecrosis of the femoral head
have been associated with less severe hemolysis and
higher hemoglobin, presumably leading to high
blood viscosity and poor microvascular blood flow
and tissue infarction (Table 2). We have described in
detail the body of data supporting this model.86 In
support of this concept, as described above, these
features of the hemolytic vasculopathy syndrome
have all been reported in thalassemia and other
hemolytic disorders, demonstrating that sickling is
not required for these complications to occur.
However, it is likely that sickling and cell adhesion
mechanisms increase the vasculopathy defect.
Pulmonary hypertension: diagnosis and treatment
The most useful screening modality for pulmonary
hypertension has been echocardiography, with careful measurement of TRV in two or three views. Our
screening data and confirmation by others indicate
that 2.5 m/sec is a threshold for abnormal values.24
There may be a role for measurement of serum Nterminal pro-brain-type natriuretic peptide (NTproBNP) to identify the highest risk patients for
echocardiography, but this requires further study.87 A
small minority of patients may have mitral valvular
insufficiency rather than pulmonary hypertension,
but this should be readily detected on echo. Patients
with TRV 2.5-2.9 m/sec should be followed clinically for any other cardiopulmonary concerns, with follow up studies every 6-24 months. In patients with
repeated TRV ≥ 3 m/sec, consideration should be
given to right heart catheterization studies by a pulmonologist or cardiologist experienced with pulmonary hypertension. More detailed studies may be
clinically indicated.88 Right heart catheterization data
will confirm whether pulmonary hypertension is
present, its severity and whether this is due to PAH,
or to LVDD.29 Encouraging pilot data indicate a
potential role for sildenafil, and there are other
approved treatments for PAH for which specific studies are underway in SCD and thalassemia.89,90
Conclusions
Pulmonary hypertension is a complication of sickle
cell disease and thalassemia that is frequently underdiagnosed. Progressive dyspnea on exertion may be
misattributed to chronic anemia. Even mild pulmonary hypertension is poorly tolerated in SCD, is
associated with early mortality. Pulmonary hypertension, leg ulceration and priapism are epidemiologically associated, reflecting a new subphenotype of SCD
with a common pathophysiological mechanism.
Although the etiology is undoubtedly multifactorial,
one large component, marked by elevated steady
state serum LDH levels, involves impaired NO
bioavailability, mainly due to scavenging of NO by
cell-free plasma hemoglobin. Important new directions in SCD and thalassemia will include improved
screening for PAH and larger scale clinical trials of
therapeutic agents for PAH and associated vasculopathy.
Acknowledgements
The authors are supported by intramural funds from the
National Institutes of Health. M.T.G. also holds a cooperative research and development agreement with INO
Therapeutics.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
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Sickle Cell Disease
The contribution of asthma to sickle cell diseaserelated morbidity and mortality
M.R. DeBaun1
J.E. Jennings1
J.H. Boyd1
J.J. Field2
C. Hillery3
R.C. Strunk1
1
Department of Pediatrics,
Washington University School of
Medicine, St. Louis;
2
Department of Internal Medicine,
Washington University School of
Medicine, St. Louis;
3
Department of Pediatrics, Medical
College of Wisconsin, Milwaukee
Wisconsin, USA
2007;1:140-147
A
B
S
T
R
A
C
T
Pulmonary complications are the leading cause of death for individuals with sickle cell
disease (SCD). Recently, asthma has been identified as a significant contributor to SCDrelated morbidity and mortality. Among children with SCD, asthma has been associated
with an increased incidence of pain and acute chest syndrome (ACS) episodes when
compared to children without asthma. Additionally, children and adults with SCD and
asthma were noted to have a higher rate of death when compared to those without
asthma. The proposed biological mechanism for the disease-modifying role of asthma in
SCD is based on a combined inflammotory effect of the vasculature in SCD and the airways in asthma. Further, asthma results in ventilation-perfusion mismatch with regional hypoxia leading to an up-regulation of inflammatory proteins and increased adhesion
of sickled red blood cells. This abnormal lung process accelerates the sickling process in
the vascular beds which leads to vaso-occlusive episodes and other complications associated with SCD. This article will focus on the epidemiology, pathophysiology, and potential mechanisms for the association between asthma and SCD-related morbidity and
mortality.
omozygous sickle cell disease
(SCD) results from a single
nucleotide substitution at the 6th
codon of the β-globin gene, yet SCD is
rather heterogenic in terms of disease
expression. The most common causes of
disease-related morbidity are pain and
ACS episodes. Pulmonary complications
also contribute significantly to premature
death. Although the relationship is not
completely understood, asthma, bronchial
hyperreponsiveness, and atopy are three
overlapping clinical phenotypes that occur
among individuals with SCD. When asthma is diagnosed among children with
SCD there is an associated increase in the
incidence of painful episodes, acute chest
syndrome and death. We will review the
epidemiology of how these separate clinical entities (Figure 1) influence the clinical
course of individuals with SCD.
H
lence of approximately 20% in the USA.
We are unaware of data relating to the
prevalence of asthma in ethnic groups outside of the USA. The co-existence of asthma and SCD was first published in a 1983.
This case report describes a six year-old
girl with SCD and severe asthma who,
several days following hospital admission
for a slight worsening of her astmatic condition, developed abdominal pain that
was identified as a vaso-occlusive
episode.2 The authors believed the pain
episode to be a result of asthma-induced
hypoxia and acidosis due to ventilationperfusion mismatch. Although the exact
mechanism has yet to be clarified, a complex interaction between SCD-related
complications. Asthma and asthma risk
factors such as atopy and bronchial hyperresponsiveness exists.
Prevalence of asthma in patients with SCD
Asthma in SCD
Asthma is a chronic lung disease that
affects approximately 9 million children in
the USA.1 Asthma disproportionately
affects African Americans with a preva| 140 |
Diagnosis of asthma among children
with SCD increases SCD-related complications. Although SCD and asthma are
both common diseases among the African
American population, conflicting evidence
exists as to whether asthma is more common among
children with SCD. In a case control study, Madden et
al.3 demonstrated that children with SCD have a two
times greater risk of developing asthma compared
children without SCD (48% vs. 22%, p=0.002). In
contrast, in cohort study of children with SCD from
birth to approximately 12 years of age, Boyd et al.7
demonstrated that in the absence of a diagnosis of
ACS, the prevalence of asthma does not differ from
that of the general African-American population
(17.1%). Each study has limitations. Madden et al.3
reported a high rate of asthma when compared to
other epidemiological surveys which suggests a selection bias. The study by Boyd et al.7 lacked prevalence
data obtained from concurrent ethnic-matched controls.
Atopy
Asthma
Phenotype
Bronchial
HyperResponsiveness
SCD
Morbidity
Asthma increases SCD morbidity and mortality
Pain and ACS episodes are common manifestations of SCD among children.
Many studies support the contributory effect asthma has on SCD-related complications, especially pulmonary complications (Tables 1 and 2).
Painful episodes in SCD can be precipitated by
known triggers, such as dehydration and cold exposure, but many precipitating factors remain
unknown. In a prospective infant cohort study of 291
infants followed for a total of 4,062 patient-years,4
Boyd et al. demonstrated a two-fold increase in the
incidence of pain episodes in individuals with SCD
and asthma compared to SCD alone (1.39 vs. 0.47
events per patient years p=0.001) (Figure 2). Boyd et
al., adjusted their risk analysis for established factors
associated with pain (age, gender, lifetime average
hemoglobin and percent fetal hemoglobin) thus
Figure 1. Proposed relationship between atopy, bronchial
hyperreponsiveness, asthma phenotype, and SCD morbidity. The intersection of the asthma phenotype, bronchial
hyper-responsiveness and atopy are thought to increase
the rate of sickle cell disease morbidity. For the moment,
no studies have demonstrated whether children with SCD
and asthma risk factors have an increased rate of pain or
ACS episodes.
strengthening the evidence of their findings.5
Furthermore, among children with SCD and asthma, mild respiratory symptoms either immediately
precede or occur simultaneously with painful
episodes more frequently when compared to with
children with SCD without asthma. Glassberg et al.6
showed that cough, wheeze, tachypnea, retractions,
or grunting occurred within 96 hours prior to the
Table 1. Influence of co-morbid conditions of SCD and asthma on the incidence or risk of painful episodes.
Study
Retrospective Study
Nordness (2005)
Case-control
Glassberg (2006)
Cohort
Number of Participants
(age range)
Pain
96 (3-18 yrs of age)
No significant difference in the rate of pain episodes in SCD and asthma vs. SCD alone
74 with SCD
(2-21 yrs of age)
Respiratory symptoms preceded pain episodes in patients with SCD and asthma vs. SCD alone
35% vs. 12%; p = 0.016
Respiratory symptoms were more likely to occur in association with a pain episode
in children with SCD and asthma
OR = 4.9 (95%CI:2.2-10.7)
Increased incidence of pain in children with SCD and asthma 1.7 vs. 1.2
episodes per patient–years; p=0.005
Prospective Study
Boyd (2006)
Cohort
291 with SCD
(<6 months-5 yrs of age)
Increased incidence of pain in children with SCD and asthma
1.39 vs. 0.47 episodes per patient-years; p<0.001
Table 2. Influence of co-morbid conditions of SCD and asthma on the incidence or risk of acute chest syndrome (ACS).
Number of participants
(age range)
Study
Retrospective
Boyd (2004)
ACS and asthma
139 (2-21 yrs of age)
Increased likelihood of developing ACS in children with SCD
and asthma hospitalized for painful episode;
OR = 4.0 (95%CI:1.7-9.5)
Case-control
Cases: ACS and pain
Controls: no ACS and pain
Knight-Madden (2005)
Case-control
160 (5-10 yrs of age)
Cases: SCD
Controls: no SCD
Recurrent ACS is associated with SCD and asthma;
OR = 6.0 (95%CI:1.5-23.4)
Nordness (2005)
Case-control
96 (3-18 yrs of age)
Cases: Asthma
Controls: no Asthma
ACS episodes are increased in children with SCD and asthma
7.5 vs.4.8 episodes per 100 patient-years; p=0.03
Bryant (2005)
Descriptive
60 (1.5-17 yrs of age)
SCD and ACS
ACS develops in children with a prior history of asthma and/or abnormal
pulmonary function tests 53%
Sylvester (2007)
Case-control
316 (0-18 yrs of age)
Cases: SCD
Controls: no SCD
Increased anti-asthmatic medication use in children with a
ACS history vs. no ACS history 18% vs. 5%; p=0.02
Asthma was diagnosed at a median of 3.5 (0.5-7)
years prior to children developing the first ACS episode
291 (< 6 months – 5 yrs)
Increased incidence of ACS in children with SCD and asthma vs. SCD alone;
0.39 vs. 0.20 episodes per patient-years; p<0.001
Prospective Study
Boyd (2006)
Cohort
400
Asthmatic
Not Asthmatic
Pain rate (/100 pt-yrs)
300
200
100
0
0-2
2-4
4-6
6-8
8-10 10-12
12-20
Age (yrs)
Figure 2. Age-specific incidence of pain in the infant sickle cell anemia (SCA) cohort. An infant cohort of 291
African-American children with SCA enrolled in the
Cooperative Study for Sickle Cell Disease (CSSCD) before
six months of age and followed beyond five years of age
for a total of 4,062 patient-years. A clinical diagnosis of
asthma was made in 17% of the cohort. Overall incidence
rate of painful events is higher in children with SCA and
asthma (1.39 events/patient-year) when compared to
children with SCA and without asthma (0.47
events/patient-year, p<0.001).*4
painful episode in children with SCD and asthma
more frequently than in children with SCD alone
(35% vs. 12%, p=0.016). Children with SCD and
asthma were approximately five times more likely to
have preceding or simultaneous respiratory symptoms associated with pain versus children with SCD
only (95% confidence intervals, odds ratio 2.2-10).
The incidence rate of painful episodes among children with SCD and asthma was higher compared to
children with SCD but without asthma 1.7 vs 1.2
episodes per patient year respectively.
ACS, defined as a new infiltrate on chest radiograph in combination with fever or respiratory
symptoms, affects primarily the pediatric population
with an incidence rate of 0.20 episodes per patient
years.7 Similarly, asthma predominantly affects children. Sufficient clinical overlap exists between the
presentation of an asthma exacerbation and ACS to
obscure a clinical diagnosis. Specifically, a clinical
diagnosis of ACS is associated with fever, tachypnea,
wheezing, cough, new radiodenisty on chest X-ray
and decreased oxygen saturation. As anticipated and
based on the strong clinical and epidemiological
overlap between the two co-morbidities ACS and an
asthma exacerbation, diagnosis of asthma has been
associated with an increased incidence of ACS. In a
single center retrospective study, approximately 53%
Table 3. Prevalence of bronchial hyper-responsiveness (BHR) in children with sickle cell disease (SCD).
Study
Number of Participants
(age range)
BHR Assessment
BHR prevalence
(SCD vs. controls)
Leong (1997)
50 (6-19 years of age)
Bronchodilator
Cold - air challenge
SCD overall - 73%
SCD and asthma - 83%
SCD and no asthma - 64%
Controls 0%
Bronchodilator
SCD - 54%
Bronchodilator
Exercise Test
SCD - 44%
Controls - 20%
Cases:
SCD and asthma
SCD and no asthma
Controls:
No SCD and no asthma
Koumbourlis (2001)
SCD
Knight-Madden (2005)
Cases: SCD
Controls: No SCD
of children with SCD were noted to have a diagnosis
of asthma or suggestive of obstructive airway disease
prior to their first ACS episode.8
Boyd et al.9 demonstrated in a retrospective study
that children with SCD and asthma had a four times
greater risk of developing ACS after being hospitalized for pain compared to children with SCD alone
(OR=4.0; 95%CI, 1.7-9.5). Longer hospitalizations
for ACS were observed in children with SCD and
asthma versus individuals with SCD alone (5.6 vs.
2.6 days, p=0.01). Due to the limitations of a retrospective study design, none of the before mentioned studies were able to establish a temporal
relationship between asthma and ACS. Based on
these studies, no data exist to determine whether
asthma increases the incidence of ACS or if SCD
increases the incidence of asthma exacerbations.
Despite this, collectively these studies firmly establish a strong association between asthma and ACS.
Additional evidence that there is an association
between asthma and ACS episodes comes from
Nordness et al.10 who conducted a retrospective
chart review and found that patients with SCD and
asthma had more episodes of ACS (7.5 vs. 4.8
episodes per 100 patient-years, p=0.03) than
patients with SCD without asthma. Furthermore,
this study illustrated that patients with both SCD
and asthma had more diseaserelated complications
with a resultant need for additional SCD treatment
including, total blood transfusion, and chronic
transfusion compared to controls.
In the previously mentioned prospective infant
cohort study, children with SCD and asthma were
also younger at the time of their first ACS episode,
median 2.4 years compared to 4.6 years of age for
children with SCD without astma (hazard ratio 1.64,
95% CI 1.13 to 2.39, p=0.0096).4 Children with SCD
and asthma also required more transfusions (1.00 per
patient-year v. 0.60 per patient-year, p=0.02) than
children with SCD without asthma.4 Sylvester et al.11
in a retrospective control study also found there was
a higher prevalence of asthma among children who
had a history of ACS. Specifically, 18% of the children that had an ACS episode were taking anti-asthmatic medication compared to 5% of the children
with ACS that were not.
Limited data exist regarding the affect of asthma
on mortality among individuals with SCD. To date
only one study addresses the association between
asthma and SCD in terms of mortality. In a prospective cohort study (n=1963), Boyd et al.9 demonstrated that asthma was associated with a shortened life
expectancy among individuals with SCD and asthma compared to SCD alone (52.2 years vs. 64.3
years). The existence of both SCD and asthma
increased the risk of mortality by two-fold (hazard
ratio 2.36, 95% CI 1.21-4.62, p=0.01).
Bronchial hyperreponsiveness in SCD
Bronchial hyper-responsiveness (BHR), a non-specific finding associated with asthma,10 also occurs in children without a clinical diagnosis of asthma. The presence of a positive BHR test is not a diagnosis of asthma. To diagnose asthma, the National Asthma
Education and Prevention Program recommends
obtaining a detailed medical history, physical examination, and pulmonary function testing to confirm airflow obstruction reversibility.12 In the absence of asthma, approximately 20% of children will have evidence
of BHR.13
Bronchial hyper-responsiveness in children with SCD and
without asthma
The relationship between the presence of BHR and
SCD-related pain or ACS is not known.
Bronchial provocation tests such as bronchodilator challenges,3,14,15 exercise tests,3 methacholine
tests,16 and cold air bronchial challenges14 have been
used to assess BHR among children with SCD
(Table 3). An increased rate of BHR has been
demonstrated in children with SCD without a previous diagnosis of asthma when compared to controls. Leong et al.14 conducted a case-control study in
children with SCD and no asthma compared to normal controls to determine the prevalence of BHR.
Pulmonary function testing was performed in 22
cases and 10 controls. BHR was assessed in each
participant using either the cold air challenge or
bronchodilator challenge based on the forced expiratory volume in 1 second (FEV-1). Patients with
SCD, but no history of wheezing, had a BHR prevalence of 64%. These results suggest an increased
propensity to airway hyper-responsiveness even in
the absence of a diagnosis of asthma. Cold air challenge is not specific for diagnosing asthma, thus
results should be interpreted cautiously. Exposure to
cold stimuli is known to precipitate sickling and a
vaso-occlusive process that may have contributed
significantly to bronchospasm. This is substantiated
by the association of pain episodes with a decrease
in ambient temperature,17 as well as an increase in
forearm vascular resistance (an indicator of vasoconstriction) in patients with homozygous SCD
compared to controls following repetitive exposure
to cool immersion stimuli.18
Atopy in SCD
A 10- to 20-fold increased risk of developing asthma
occurs in the presence of atopy, as determined by skin
testing.3 Savoy et al.19 reviewed medical records, questionnaires, and performed physical examinations to
evaluate the prevalence of atopy (i.e. asthma, atopic
dermatitis, and allergic rhinitis) in the SCD population.
Overall the atopy prevalence was 13.7% in the SCD
population with a similar rate in controls. Specifically,
the prevalence of bronchial asthma, allergic rhinitis,
and atopic dermatitis was not significantly different
from that found in similar population studies. This
observation was subsequently supported by KnightMadden et al.3 who demonstrated that the incidence of
positive skin prick test performed in children with
SCD was not significantly different compared to controls (36% vs. 34%, p=0.75). While these findings are
suggestive of two independent events, this conclusion
cannot be drawn because asthmatics are not always
atopic.
Pro-inflammatory status of SCD and its link to asthma
Hypoxia-induced polymerization of HbS is the initial step in occlusion of the vasculature by red blood
cells, ultimately leading to tissue injury. While the
exact mechanism of vaso-occlusion is poorly defined,
it involves a complex interaction of sickle and nonsickle erythrocytes, leukocytes, platelets, and vascular endothelial cells.20,21 Inflammatory mediators are
the driving force behind these cellular interactions
through the up-regulation of adhesion molecules (see
also Kato and Gladwin, this book).
At baseline, SCD is a pro-inflammatory state,
based on systemic increases in inflammatory markers
such as leukocyte count,22,23 platelet count,24 C-reactive protein,25-27 and cytokines.28-31 Inflammatory
mediators stimulate an elevation in up-regulation of
intracellular adhesion molecules (I-CAM), vascular
adhesion molecules (V-CAM), and selectins.20
Expression of these adhesion molecules promotes
the interaction of less deformable sickle erythrocytes
with endothelial cells. More specifically, significant
evidence suggests that sickled red blood cells result in
continued injury to the endothelium resulting in tissue specific inflammation. Circulating endothelial
cells and endothelial cell molecules (I-CAM, V-CAM,
and E-selectin) in the plasma provide evidence of this
vascular injury.32 Non-sickle erythrocytes, leukocytes,
and platelets also bind to the activated endothelium
of the microvasculature with resultant occlusion.
Upon reperfusion, activated leukocytes and radical
oxygen species released during tissue hypoxia further
increase cellular damage and inflammation. This
added elevation of inflammatory mediators further
promotes endothelial cell activation, and thus propagates a cycle of inflammation, tissue injury, and vasoocclusion.
Analogous to chronic endothelial inflammation in
SCD is chronic airway inflammation in asthma. The
stimulus for inflammation associated with asthma is
an interaction between an airway allergen and a specific IgE on the surface of airway mast cells, which
triggers the release of IL-4, IL-5, GM-CSF, histamine,
and leukotrienes.33 IL-5 stimulates the bone marrow
to increase eosinophil production, and circulating
eosinophils bind to vascular endothelium through
cell surface adhesion molecules.34 Inflammatory
mediators associated with asthma up-regulate the
expression of adhesion molecules to increase the
eosinophil-endothelial cell interaction.35 Following
attachment to the endothelium, eosinophils migrate
into airway tissues, inflicting injury and perpetuating
the inflammation that is characteristic of asthma.
Despite the observation that the primary basis for
inflammation differs between SCD and asthma,
inflammation is fundamental to the pathophysiology
of both disease processes. Furthermore, elevations of
Table 4. Elevated inflammatory markers common to sickle cell disease and asthma.
Inflammatory Markers
Sickle Cell Disease
Asthma
IL-5
Walter (2006) Br J Haematol39
Hoekstra (1997) Clin Exp Allergy40
sICAM-1
Shiu (2000) Blood
Oymar (1998) Ped Allergy Immunol42
El Sawy (1999) Int. Arch Allergy43
Marquet (2000) Am J Respir Crit Care Med44
sVCAM-1
Duits (1996) Clin Immunol and Immunopathol45
Sakhalkar (2004) Am J Hematol46
Tang (2002) Pediatr Pulmonol47
Hamzaoui (2001) Mediators Inflamm48
Koizumi (1995) Clin Exp Immunol49
TNF-α
Malave (1993) Acta Haematol29
Halasz (2003) Allergy Asthma Proc50
Koizumi (1995) Clin Exp Immunol49
Baysigit (2004) Mediators Inflamm51
LTB4
Setty (2002) J Lab Clin Med52
Baysigit (2004) Mediators Inflamm51
CRP
Hedo (1993) J Intern Med25
Hibbert (2004) Exp Bio Med53
Sinn (2004) Am J Respir Crit Care Med54
eNO
Pawar (2006) Pediatr Blood Cancer55
Girgis (2003) Am J Hematol56
Sullivan (2001) Am J Respir Crit Care Med57
Beck-Ripp (2002) Eur Respir J58
Kelly (2006) J Allergy Clin Immunol59
41
similar systemic inflammatory markers have been
observed in both diseases (Table 4). Thus, we think
that the combination of inflammatory processes in
SCD and asthma together contribute to the increase
in the rate of SCD-related complications, pain and
ACS.
Anti-inflammatory medication in the treatment of SCDrelated complications
Additional evidence of a pro-inflammatory state in
SCD is based on the quick return to baseline in individuals with either painful episodes or ACS after
treatment with steroids for their systemic antiinflammatory effects. Two separate trials have been
conducted using steroids to treat pain or ACS in
SCD. A double-blind placebo-controlled study by
Griffin et al.36 was conducted to determine the effect
of intravenous methylprednisolone on children and
adolescents with SCD (n=36) hospitalized for acute
pain episodes. Duration of hospital stay was significantly shorter in patients receiving IV methylprednisolone compared to those receiving placebo (mean,
41.3 vs. 71.3 hours; p=0.030). Recurrence of pain
leading to hospital readmission within two weeks of
discharge was observed in approximately 15% of
patients in the methylprednisolone study group (n=4)
compared to 3% in the placebo group (n=1).
Bernini et al.37 evaluated the efficacy and toxicity of
IV dexamethasone in children with SCD hospitalized
for ACS episodes (n=43) in a randomized, doubleblind placebo-controlled study. Hospital stay among
patients in the dexamethasone group was shorter
compared to the placebo group (mean hospital dura-
tion: 47 hours vs. 80 hours; p=0.005). The dexamethasone group was also observed to have less clinical deterioration and a decreased need for blood
transfusion (p<.001 and =0.013, respectively).
Additionally, the mean duration of oxygen therapy,
analgesic therapy, number of opioid doses, and duration of fever was significantly reduced in the dexamethasone group compared to the placebo group.
Although not statistically significant, seven patients
were readmitted within 72 hours, 6 patients from the
dexamethasone treated group (27%) compared to
one patient in the placebo group (4.7%), after hospital discharge. Only one patient was readmitted for an
ACS episode within this group. Similar to the treatment of asthma, both studies provide evidence that
anti-inflammatory therapy may be beneficial in the
treatment of acute complications of SCD. However,
the use of corticosteroids during the acute illness,
particularly for patients with ACS and without a previous diagnosis of asthma, must be measured against
the more recent evidence that the use of corticosteroids is temporally associated with cerebral hemorrhage in children with ACS38 and the high rate of readmission shortly after discharge.37
Summary
This review describes the epidemiology of asthma
and its associated increased rate of co-morbidity
among children with SCD. Among children with
SCD and asthma, the clinical management of SCD
should include adherence to evidence-based practice
of clinical care for asthma. Future research should be
directed at clarifying the mechanism for this associa-
tion. With a better understanding of the association
between SCD and asthma, targeted therapy can be
developed to decrease the morbidity and mortality
linked to SCD.
Acknowledgments
Funded by: National Heart, Lung and Blood Institute,
contract N01-HB-47110, RO1 HL079937 (MD, RS),
Doris Duke Foundation (JJ), T32 HL07873 (JHB).
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2002;33:249-54.
Hamzaoui A, Ammar J, El Mekki F, Borgi O, Ghrairi H, Ben
Brahim M, et al. Elevation of serum soluble E-selectin and
VCAM-1 in severe asthma. Mediators Inflamm 2001;10:33942.
Koizumi A, Hashimoto S, Kobayashi T, Imai K, Yachi A, Horie
T. Elevation of serum soluble vascular cell adhesion molecule1 (sVCAM-1) levels in bronchial asthma. Clin Exp Immunol.
1995;101:468-73.
Halasz A, Cserhati E, Kosa L, Cseh K. Relationship between
the tumor necrosis factor system and the serum interleukin-4,
interleukin-5, interleukin-8, eosinophil cationic protein, and
immunoglobulin E levels in the bronchial hyperreactivity of
adults and their children. Allergy Asthma Proc 2003;24:111-8.
Basyigit I, Yildiz F, Ozkara SK, Boyaci H, Ilgazli A. Inhaled corticosteroid effects both eosinophilic and non-eosinophilic
inflammation in asthmatic patients. Mediators Inflamm
2004;13:285-91.
Setty BN, Stuart MJ. Eicosanoids in sickle cell disease: potential relevance of neutrophil leukotriene B4 to disease pathophysiology. J Lab Clin Med 2002;139:80-9.
53. Hibbert JM, Hsu LL, Bhathena SJ, Irune I, Sarfo B, Creary MS,
et al. Proinflammatory cytokines and the hypermetabolism of
children with sickle cell disease. Exp Biol Med (Maywood)
2005;230:68-74.
54. Sin DD, Lacy P, York E, Man SF. Effects of fluticasone on systemic markers of inflammation in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2004;170:760-5.
55. Pawar SS, Panepinto JA, Brousseau DC. The effect of acute
pain crisis on exhaled nitric oxide levels in children with sickle cell disease. Pediatr Blood Cancer 2006 Apr 17.
56. Girgis RE, Qureshi MA, Abrams J, Swerdlow P. Decreased
exhaled nitric oxide in sickle cell disease: relationship with
chronic lung involvement. Am J Hematol 2003;72:177-84.
57. Sullivan KJ, Kissoon N, Duckworth LJ, Sandler E, Freeman B,
Bayune E, et al. Low exhaled nitric oxide and a polymorphism
in the NOS I gene is associated with acute chest syndrome.
Am J Respir Crit Care Med 2001;164:2186-90.
58. Beck-Ripp J, Griese M, Arenz S, Koring C, Pasqualoni B, Bufler
P. Changes of exhaled nitric oxide during steroid treatment of
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59. Kelly MM, Leigh R, Jayaram L, Goldsmith CH, Parameswaran
K, Hargreave FE. Eosinophilic bronchitis in asthma: a model
for establishing dose-response and relative potency of inhaled
corticosteroids. J Allergy Clin Immunol 2006;117:989-94.
Sickle Cell Disease
Hydroxyurea: benefits and risks in patients affected
with sickle cell anemia
M. de Montalembert
Service de Pédiatrie Générale,
Hôpital Necker, Paris, France
2007;1:148-153
ydroxyurea is increasingly being
used to treat patients with sickle
cell anemia (SCA). A recent survey
of 1,673 North-American patients enrolled
in the NIH-NHLBI funded Comprehensive
Sickle Cell Centers Program, found that
18% of children and 41% of adults were
treated with hydroxyurea.1 Approximately
10 years after the major publication by
Charache et al.2 reporting the benefits
related to hydroxyurea treatment, more
than 1,000 SCA patients are currently
being treated in the world.
H
Mechanisms of action of hydroxyurea
Hydroxyurea is a cytostatic agent (Sphase inhibitor) that was first used to
treat myeloproliferative diseases. It is
hypothesized that hydroxyurea increases
fetal hemoglobin (HbF) (α2γ2) production
through recruitment of new stem cells,
since the gamma globin expression of
such progenitors is still present. Higher
HbF levels are known to be associated
with an alleviation of the severity of
SCA. Saudi Arabian patients have high
HbF levels with a milder disease, SCA
newborns do not experience complications, and in the Cooperative Study of
Sickle Cell Disease, the mortality rate
and frequency of painful crises were
inversely correlated with HbF concentration.3,4 The clinical and pathological features of SCA are related to the occlusion
of the vessels of the microcirculation by
red cells sickled and stiffened after polymerization of the abnormal hemoglobin
S upon deoxygenation. There is a time
delay before polymerization occurs. This
time delay increases when the proportion
of HbF is raised, because in a mixture of
hemoglobin S (HbS) (α2β2S) and HbF, there
is a third tetrameric species, the hybrid
(α2βSγ), which co-polymerizes very poorly with the others. As a result, red cells
are able to exit from the capillary network before polymerization occurs.5
| 148 |
These biophysical and clinical findings
have led to a the search for ways of stimulating HbF synthesis to treat SCA. In a
study exploring the effects of hydroxyurea in 3 SCA patients treated over 24
weeks, sickle hemoglobin polymerization delay time increased by at least four
times after 12 weeks of treatment, and by
approximately another two times after
the second 12 week treatment period.6
Furthermore, as noted by others,7
increased K+ content and decreased K-Cl
cotransport after 12 weeks of therapy
indicated an improvement in red cell
hydration status. The earliest effect was
the striking decrease in erythrocyte adhesiveness to endothelial cells within 2
weeks of therapy. This is consistent with
the observation that clinical improvement precedes HbF increase, and that
HbF change is not correlated with clinical
benefit,8 suggesting additional mechanisms are involved. Investigators have
shown that hydroxyurea reduces expression of very late activation antigen-4 and
CD36 on sickle reticulocytes,9 and that
hydroxyurea therapy decreases the in
vitro adhesion of sickle erythrocytes to
thrombospondin and laminin.10 It is highly likely that Hydroxyurea also acts via a
decrease of polymorphonuclear neutrophil (PMN) adhesion to endothelial
cells which contributes to vascular
inflammation and vaso-occlusion. Hydroxyurea decreases the PMN count
wich is the measurement most strongly
related to the beneficial effect of the
drug,11 and as evidenced by a correction
of dysregulated L-selectin expression and
increased H2O2 production in SCA
patients.12 It has also been shown that
hydroxyurea enhances NO and cGMP
production in endothelial cells in a
cAMP-dependent protein kinase manner,
which could participate in the induction
of HbF as well as in the modification of
vascular tone.13
Clinical efficacy of hydroxyurea
Short-term and median-term (1 to 5 years of treatment)
controlled trials
The Multicenter Study of Hydroxyurea (MSH) in sickle
cell anemia2
This double-blind randomized study, enrolled SCA
adult patients with severe disease (at least 3 painful
crises per year). It assigned hydroxyurea to 152
patients and placebo to 147 controls. Patients given
hydroxyurea received an initial dose of 15
mg/kg/day. This was increased by 5 mg /kg/d every
12 weeks unless marrow depression occurred (indicated by neutrophil count <2.0×109/L, reticulocyte or
platelet count <80×109/L, or Hb level <4.5 g/dL). In
the case of marrow depression, treatment was
stopped until the blood count recovered and was
then resumed at a dose 2.5 mg/kg lower than that
associated with marrow depression. This defined the
maximum tolerated dose (MTD). The hydroxyureatreated patients had a lower annual rate of crises than
the controls (2.5 vs 4.5 crises per year, p<0.001),
fewer patients had chest syndrome (25 vs 51, p
<0.001), and fewer underwent transfusions (48 vs 73,
p=0.001). Treatment was stopped after a mean follow-up of 21 months because of the benefits
observed in the treated patients.
The Belgian pediatric trial14
Twenty-five children (median age 9 years) were
randomized to receive either hydroxyurea or placebo
for 6 months, and then switched to the other arm for
the next 6 months. The initial dosage was 20
mg/kg/d, increased to 25 mg/kg/d after 2 months if
no increase of HbF level >2% had occurred. No
attempt was made to reach the MTD. Among the 22
evaluable patients (median age 8 years) hydroxyureatreated children had significantly less hospitalizations (p=0.0016) and fewer hospitalized days (p=
0.0027).
Short-term and median-term (1 to 5 years of treatment) non
controlled trials
A Cochrane review15 focused on the use of hydroxyurea in SCA patients. It included studies involving
homozygous SS and Sbeta thalassemic adults16-18 and
children,19-23 and even very young children.24 All of the
studies but the last one enrolled patients with severe
forms of disease. North-American studies increased
the hydroxyurea dosage up to the MTD (the median
dosage in Kinney’s study was 26±6.2 mg/kg/d),
whereas European studies usually kept the dosage
around 20 mg/kg/d. The drug was provided in 500
mg capsules for adults and older children, and in a
liquid formula prepared by dissolution of the capsules for young children.
All the studies reported increases in HbF, MCV
and steady-state Hb, decrease of PMN, along with
improvement in the number of painful crises, hospitalizations, and acute chest syndrome rates.
However, clinical responses varied widely among
patients and the best results were observed in children.
Long-term trials
There are no controlled long-term trials. One of the
most important long-term studies is the extension of
the MSH study conducted between 1992-1995. After
completion of this initial study, patients were free to
continue, start or stop treatment with hydroxyurea
(25). Data for 233 patients were collected until 2001.
Ninety-six patients (32%) never received hydroxyurea, 48 (16%) received hydroxyurea for less than 1
year, and 156 (52%) received hydroxyurea for 1 or
more years. Twenty-five percent (n=75) of the 299
who originally volunteered for the MSH died during
follow-up, 28% from pulmonary disease. Cumulative mortality at 9 years was inversely related to
HbF level (p=0.03) and positively related to the occurrence of 1 or more episodes of acute chest syndrome
during the trial (p=0.02). Hydroxyurea treatment was
associated with a 40% reduction in mortality
(p=0.04).
Long-term studies on hydroxyurea use in children
confirm a sustained efficacy in young patients. In the
Belgian trial, there was a significant difference in the
number of hospitalizations (p=0.0002) and hospitalized days (p<0.01) during 5-year treatment, compared to previous hydroxyurea therapy.26 The NorthAmerican Hydroxyurea Safety and Organ Toxicity
(HUSOFT)24 which had included 28 infants was
extended for up to 4 years for 17 patients and up to 6
years for 11 patients.27 Patients experienced 7.5 acute
chest syndrome events/100 person-years, compared
with 24.5 events/100 person-years among historical
controls (p=0.001). Hydroxyurea-treated infants had
a comparatively preserved splenic function compared
to historical controls. The proportion of asplenic
patients assessed by Tc-99m sulphur colloid uptake
showed an absent uptake (i.e. functional asplenia) in
43% patients after study completion, versus the 94%
percent standard for that age. Remarkably, two
babies with markedly decreased or absent splenic
uptake prior to hydroxyurea treatment recovered
normal splenic uptake after 4 years of hydroxyurea.
Biologically, all of the studies reported a long-term
increase in Hb, MCV, and HbF levels, and a significant
decrease in reticulocyte, PMN, and platelet counts.26-29
Increases in Hb and HbF levels were greater in the
Duke cohort,29 where children received higher
dosages (average dose: 25.4 mg±5.4 mg/kg/d, with
17% receiving more than 30 mg/kg/d) than in the
Belgian cohort (where children received 20-25
mg/kg/d).26 The minimal HbF level increase needed
to observe clinical benefit has not yet been determined, therefore no recommendation can be made
for optimal dosage.
Predictors of the response to hydroxyurea
Clinical response to HU in SCA patients varies
widely, with children generally experiencing greater
benefits than adults. All of the patients in the Duke
pediatric cohort responded,29 whereas half the adults
in the Multicenter Study of Hydroxyurea showed an
almost stable fetal hemoglobin level after 2 years,30
despite identical treatment protocols. Most probably, mothers’ compliance is greater than that of adult
patients, but it cannot be excluded that children’s
erythroid progenitors also have a greater capacity to
increase HbF production than adult progenitors.
It is impossible to predict individual response to
treatment, regardless of patient age. To identify
determinants of response, 150 HU-treated adult
patients were grouped by quartiles of change in HbF
from baseline to 2 years of treatment.30 In the top
two quartiles, HbF increased to 18.1% and 8.8%.
These patients had the highest baseline neutrophil
and reticulocyte count and the largest treatmentassociated decrements in these counts. In the lower
two quartiles, 2-year HbF levels (4.2% and 3.9%)
and blood counts changed little from baseline. This
finding suggests that the capacity of the bone marrow to withstand HU might be an important factor
influencing response to treatment. In the quartile
with the highest HbF response, myelosuppression
developed in less than 6 months, compliance was
greatest, and final doses were 15 to 22.5 mg/kg/d.
All quartiles had substantial increases of F-cells (the
HbF containing cells). These were maintained for 2
years only in the top three quartiles. HbF response
was not associated with initial HbF in this study, but
was highly related to initial HbF in others.21,29,32
Pharmacokinetic differences among individuals
may account for the wide range of HbF response
among SCA patients. We studied pharmacokinetic
parameters in 15 adults and 11 children. Pharmacokinetics was not significantly different between
adults and children but considerable individual variation was noted.33 Genetic factors modulating HbF
response are most probably involved. In a study
exploring the influence of 226 single nucleotide
polymorphisms with possible roles in HbF regulation and hydroxyurea metabolism, aquaporin 9, a
membrane channel that stimulates urea transport
and allows the passage of uncharged solutes, and
CYP2CP, a member of the cytochrome P450 family,
appeared as possible modulators of HbF response to
hydroxyurea.34
Safety of hydroxyurea treatment
Short-term and mid-term safety
The most frequently reported side effect is myelosuppression, which is usually transient and resolved
by decreased dosage. Persistent pancytopenia has
occasionally been reported,35 and there is one publication of an opportunistic infection occurring during
hydroxyurea treatment.36 A complete blood count
must be performed before starting treatment, 2
weeks after starting treatment, at 2-4 week intervals
during the initial phase, and then every 6 weeks.
These results should be monitored by a medical professional. Rash, dizziness, headache and asthenia
have been reported, though these rarely led to cessation of treatment.37 Nail hyperpigmentation is common, and moderate alopecia can occur.37,38 The relationship with the development of leg ulcers is controversial, since leg ulcers are classic complications of
SCA.39
One case of azoospermia has been reported in a
SCA patient treated with hydroxyurea who before
treatment had a normal spermatic fluid analysis.
Azoospermia was reversible after stopping hydroxyurea.40 Many patients treated with hydroxyurea
have had children during hydroxyurea treatment, but
uncertainty remains over the long-term fertility of
boys treated since childhood.
Studies in rats showed an increase in fetal losses,
and reduced fetal and placental weights.41 Although
many healthy babies are now born from hydroxyurea-treated women, female patients should be
informed of the theoretical risks of teratogenicity and
advised to cease hydroxyurea treatment when planning a pregnancy.
A study of infant mice exposed to high doses of
hydroxyurea showed impaired brain and spleen
growth in mice aged less than 8 days (equivalent to 2
years old in humans) when receiving the dose.42
These findings led most pediatricians to wait until
the age of 2 to prescribe hydroxyurea. However, no
toxicities, and in particular no growth abnormalities,
have been reported in the trial in very young children.24 Hankins et al. have in fact reported improved
growth in infants receiving hydroxyurea than in controls.27 In older children, hydroxyurea treatment had
no adverse effect on height or weight gain or pubertal development.43
The possibility that hydroxyurea increases the risk
of splenic sequestration in children is still under discussion. In fact, hydroxyurea has been shown to
delay splenic infarction or even to restore splenic
function in older patients.27,44,45 We reported 6 cases of
hypersplenism leading to withdrawal of the drug in
our cohort of 225 hydroxyurea-treated children, with
recurrent splenic sequestration episodes in 2
homozygous for HbS children aged 6 and 7 years.37 In
children, we therefore recommend careful monitoring of spleen size and blood tests at each evaluation
particularly for children with prior splenomegaly or
past history of splenic sequestration before starting
hydroxyurea treatment.
Compliance remains the most frequent problem
encountered. Using analyses of laboratory parameters and review of peripheral blood smears (MCV
increase in particular), and pill counts when possible,
Zimmerman estimated that non-compliance led to
hydroxyurea withdrawal in 12% of her pediatric
patients.29 This percentage was 7.5% in our series.37
Table 1. Malignancies in SCD patients treated with hydroxyurea.
ALL Ph +
AML/MDS
AML
Hodgkin
AML
ALL
Testis cancer
Breast cancer
Age (years)
Duration of treatment
Reference
10
27
42
8
21
14
39
58
7 weeks
8 years
6 years
6 months
8 years
unknown
unknown
unknown
22
47
48
49
50
51
51
51
Long-term safety
As discussed above, uncertainties remain as to the
long-term consequences on fertility of boys treated
with hydroxyurea for several years. Storage of frozen
sperm must be proposed systematically to mature
boys and adults, though this is rarely accepted.
The risk of malignancies is also cause for concern
Hemoglobinopathies are not thought to increase the
risk of developing of secondary malignancies. A
study of 64 patients treated for 2-15 years with
hydroxyurea for cyanotic congenital heart disease
showed no increased malignancy.46 So far, 8 malignancies have been reported in SCA patients receiving
hydroxyurea (Table 1):22,47-51 Five cases have been published as single reports, 3 come from a NorthAmerican register which enrolled 16,613 SCA
patients followed in 52 centers.51 Two (1 ALL, 1
Hodgkin disease) occurred in the first 6 months after
starting treatment, reducing the probability that
hydroxyurea is responsible for the malignancies.
Three other hematological cases (3 AML) occurred
after 6, 8, and 8 years of treatment. The register mentions one ALL occurring in a 14 year old child, testicular cancer in a 39 year old man, breast cancer in a 47
year old woman. However, time intervals between
the start of hydroxyurea treatment and malignancy
are not noted. While it is not possible to identify the
role of hydroxyurea in the pathogenesis of these
malignancies, great caution must be observed. In
vitro, quantitative analyses of acquired DNA mutations suggest that the mutagenic potential of hydroxyurea is low.52
Indications for hydroxyurea treatment
Randomized studies assessed the efficacy of
hydroxyurea in preventing recurrences of painful
crises in adults and children severely affected with
SCA, and in adults with repeated episodes of acute
chest syndrome. It seems reasonable to also expert
such efficacy for prevention of acute chest syndrome
recurrences, in children, although this has not been
demonstrated by a pediatric controlled study. The
Food and Drug Administration has approved its use
in adult SCA patients with recurrent moderate to
severe painful crises (at least 3 over 12 months), and
European regulatory authorities are currently approving a coated breakable 1,000 mg tablet for patients
with repeated vaso-occlusive events.
However, hydroxyurea is currently used in many
other indications. In addition to prevention of painful
crises and acute chest syndromes, hydroxyurea is
mainly used in adults with early chronic organ damage (respiratory, renal, hepatic, myocardial insufficiencies), or in association with an auto-immune disease requiring steroids, conditions for which chronic
transfusion therapy was usually employed prior to
the introduction of hydroxyurea. However, there has
been no randomized study comparing transfusion to
hydroxurea in these indications. The decision to use
hydroxyurea or chronic transfusions is usually based
on better tolerability to hydroxyurea treatment compared with chronic transfusions which carry the risk
of allo-immunization, iron overload, venous access
problems, and transfusion-related infections, particularly in developing countries. There are also some
general reports of hydroxyurea use in children with
hepatic53 or myocardial54 failure. Hydroxyurea is
sometimes also prescribed in patients with severe
anemia, after elimination of an aggravating factor
such as iron or vitamin deficiency, inflammation, or
renal insufficiency. It may be prescribed either systematically when Hb level is below 6 g/dL, or in
patients whose tolerance of anemia is decreased.
Erythropoietin is sometimes associated with hydroxyurea therapy in patients with pulmonary hypertension or mild renal insufficiency, since erythropoietin
may allow more aggressive hydroxyurea dosing.55
The most controversial use of hydroxyurea is in
the prevention of cerebro-vascular events. The
pathophysiology of these events associates sickling
and adhesion of sickle red cells to the vascular
endothelium, progressive narrowing of the lumen of
medium and large intracranial vessels leading to
stenosis and occlusion, collateral circulation with
moya-moya disease, microcirculation perfusion
defects, a procoagulant state, chronic hemolysis and
abnormal vasomotor tone.56 Given that hydroxyurea
improves deformability, decreases sickling and adhesion of red cells, and is probably a NO donor, a protective effect on the brain could be expected. In fact,
two studies argue for this protective effect. Thirtyfour Belgian SCA children at high-risk of primary
stroke on the basis of transcranial Doppler velocities
(TCD) > 200 cm/s were given hydroxyurea rather
than transfusion because of a high rate of allo-immunization. Only one of them experienced a cerebrovascular event (seizures) after a follow-up of 96
patient-years.28 Another study reports a stroke recurrence rate of 10% in 20 children who had had a
stroke, had been transfused during a median period
of 27±23 months, and had then discontinued transfusion and been treated with hydroxyurea after an
overlap period of 6±3 months.57 However, there are
many reports of cerebrovascular accidents, sometimes fatal, in patients receiving hydroxyurea.20,24,35,37,58
The incidence of stroke was identical in the treated
and control groups in the MSH study.2 We therefore
believe that chronic transfusion which keeps the HbS
level permanently below <30%, whenever feasible
and safe, remains the best option for children who
have had an overt stroke, or who have abnormal
TCD studies. One study reports stopping transfusion
in 10 patients initially transfused for abnormal TCD,
but without stroke history. After checking normalization of TCD velocities during transfusion and normality of MRA, hydroxyurea was prescribed after an
overlap period with transfusions, and TCD measured
every 3 months.59 Four of these ten patients redeveloped high velocities off transfusion and transfusions
were therefore resumed. The other six remain transfusion-free after a mean follow-up of 4.4 years. This
could suggest that i) hydroxyurea is better than no
treatment at all after a stroke in countries without
adequate blood supplies, ii) hydroxyurea may prevent a first overt stroke in patients with moderate
narrowing of vessels lumen, but that iii) hydroxyurea
is insufficient to prevent a stroke in patients with
advanced cerebral vessel disease.
It is not possible to say today whether hydroxyurea
is an appropriate treatment for the 20% of SCD children and 40% of SCD adults who currently receive it.
Efficacy and safety data are encouraging but we lack
long-term follow-up on cohorts of patients with longterm exposure to the drug. Randomized studies are
needed. We would like to conclude by quoting
Charache: «We’d best be cautious in what we tell our
patients and their parents. We can hold out our hope of
improvement, but we should not promise it».60
The author is indebted to Sam Charache for his helpful
comments.
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Kagambega F, et al. Hydroxyurea for sickle cell disease in children and for prevention of cerebrovascular events. The Belgian
experience. Blood 2005;105:2685-90.
29. Zimmerman SA, Schultz WH, Davis JS, Pickens CV, Mortier
NA, Howard TA, et al. Sustained long-term efficacy of
hydroxyurea at maximum tolerated dose in children with
sickle cell disease. Blood 2004;103:2039-45.
30. Steinberg MH, Lu ZH, Barton FB, Terrin ML, Charache S,
Dover GJ. Fetal hemoglobin in sickle cell anemia: determinants of response to hydroxyurea. Blood 1997;89:1078-88.
31. Maier-Redelsperger M, de Montalembert M, Flahaut A,
Neonato MG, Ducrocq R, Masson MP, et al. Fetal hemoglobin
and F-cell response to long-term hydroxyurea treatment in
young sickle cell patients. Blood 1998;91:4472-9.
32. Ware RE, Eggleston B, Redding-Lallinger R, Wang WC, SmithWhitley K, Daescher C, et al. Predictors of fetal haemoglobin
response in children with sickle cell anemia receiving hydroxyurea therapy. Blood 2002;99:10-4.
33. De Montalembert M, Bachir D, Hulin A, Gimeno L, Mogenet
A, Bresson JL, et al. Pharmacokinetics of the 1,000 mg coated
breakable tablets and 500 mg capsules in pediatric and adults
with sickle cell disease Haematologica 2006;91:1685-8.
34. Wyszynski DF, Baldwin CT, Cleves MA, Farrell JJ, Bisbee A,
Kutlar A, et al. Genetic polymorphisms associated with fetal
haemoglobin response to hydroxyurea in patients with sickle
cell anemia. Blood 2004;104: 34a.
35. Vichinsky EP, Lubin BH. A cautionary note regarding hydroxyura in sickle cell disease. Blood 1994;83: 1124-8.
36. Venigalla P, Motwani B, Nallari A, Allen S, Agarwal M, Alva
M, et al. A patient on hydroxyurea for sickle cell disease who
developed an opportunistic infection. Blood 2002;100: 363-4.
37. De Montalembert M, Brousse V, Elie C, Bernaudin F, Shi J,
Landais P. Long-term hydroxyurea treatment in children with
sickle cell disease:tolerance and clinical outcomes.
38. O’Branski EE, Ware RE, Prose NS, Kinney TR. Skin and nail
changes in children with sickle cell disease receiving hydroxyurea therapy. J Am Acad Dermatol 2001;44: 859-61.
39. Chaine B, Neonato MG, Girot R, Aractingi S. Cutaneous
adverse reaction to hydroxyurea in patients with sickle cell
disease. Archives of Dermatology 2001;137: 467-70.
40. Carozzo G, Disca S, Fidone C, Bonomo P. Azoospermia in a
patient with sickle cell disease treated with hydroxyurea.
41. Spencer F, Chi L, Zhu MX. Hydroxyurea inhibition of cellular
and developmental activities in the decidualized and pregnant
uteri of rats. Journal of Applied Toxicology 2000;20:407-12.
42. Bennett T, Bal H, Chen Q, Abdulmalik O, Yang J, Iyamu E, et
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Wang WC, Helms RW, Lynn HS, Redding-Lallinger R, Gee B,
Ohene-Frempong K, et al. Effect of hydroxyurea on growth in
children with sickle cell anemia: results of the HUG-KIDS
study. J Pediatr 2002;140:225-9.
Claster S, Vichinsky E. First report of reversal of organ dysfunction in sickle cell anemia by the use of hydroxyurea:
splenic regeneration. Blood 1996;88:1951-3.
Santos A, Pinheiro V, Anjos C, Brandelise S, Fahel F, Lima M,
et al. Scintigraphic follow-up of the effects of therapy with
hydroxyurea on splenic function in patients with sickle cell
disease. Eur J Nucl Med Mol Imaging 2002;29:536-41.
Triadou P, Maier-Redelsperger M, Krishnamoorty R,
Deschamps A, Casadevall N, Dunda O, et al. Fetal haemoglobin variations following hydroxyurea treatment in patients
with cyanotic congenital heart disease. Nouv Rev Fr Hematol
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Rauch A, Borromeo M, Ghafoor A, Khoyratty B, Maheshwari
J. Leukemogenesis of hydroxyurea in the treatment of sickle
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treated with hydroxyurea. Ann Intern Med 2000;133:925-6.
Moschovi M, Psychou F, Memegas D, Tsangari GT,
Tzortzatou-Stathopoulou F, Nikolaidou P. Hodgkin’s disease
in a child with sickle cell disease treated with hydroxyurea.
Pediatr Hematol Oncol 2001;18:371-6.
Ferster A, Sariban E, Meulemann N. Malignancies in sickle cell
disease patients treated with hydroxyurea. Br J Haematol
2003;123:368-9.
Schultz WH, Ware RE. Malignancy in patients with sickle cell
disease. Am J Hematol 2003;74:249-53.
Hanft VN, Fruchtman SR, Pickens CV, Rosse WF, Howard TA,
Ware RE. Acquired DNA mutations associated with in vitro
and in vivo hydroxyurea exposure. Blood 2000;95:3589-93.
Jeng MR, Rieman MD, Naidu PE, Kaste SC, Jenkins III JJ,
Serjeant G, et al. Resolution of chronic hepatic sequestration in
a patient with homozygous sickle cell disease receiving
hydroxyurea. J Pediatr Hematol Oncol 2003;25:257-60.
De Montalembert M, Maunoury C, Acar P, Brousse V, Sidi D,
Lenoir G. Myocardial ischemia in children with sickle cell disease. Arch Dis Child 2004;89:359-62.
Little JA, McGowan VR, Kato GJ, Partovi KS, Feld JJ, Maric I,
et al. Combination erythropoietin-hydroxyurea therapy in
sickle cell disease: experience from the National Institutes of
Health and a literature review. Haematologica 2006;91:107683.
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and treatment of stroke in sickle-cell disease: present and
future. Lancet Neurol 2006 5:501-12.
Ware RE, Zimmerman SA, Sylvestre PB, Mortier NA, Davis JS,
Treem WR, et al. Prevention of secondary stroke and resolution of transfusional iron overload in children with sickle cell
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2004;145:346-52.
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Acute Lymphoblastic Leukemia
Genetics of T-cell acute lymphoblastic leukemia
C.J. Harrison
Leukaemia Research Cytogenetics
Group, Cancer Sciences Division,
University of Southampton, UK
2007;1:154-160
-cell acute lymphoblastic leukemia
(T-ALL) is a high risk malignancy of
thymocytes, which accounts for
approximately 15% of childhood and
25% of adult ALL.1 It is a heterogeneous
disease, classified according to the expression of specific cytoplasmic or surface
markers.2 The development of normal
thymocytes and their regulation mechanisms have been studied extensively and
it has been shown that the significant
genes in T-cell development are also
involved in T-ALL.3 This is supported by
the gene expression signatures of T-ALL,
which mirror the specific stages of thymocyte development.4 These observations
indicate a multistep process of pathogenesis in T-ALL.3 De Keersmaecker et al.5
defined four pathways based on different
classes of mutations that: 1) provide a proliferative advantage; 2) impair differentiation and survival; 3) affect the cell cycle; 4)
provide self renewal capacity. The recurrent chromosomal abnormalities and
molecular changes as defined within these
pathways are described below and summarised in Table 1.
T
The role of cytogenetics and molecular
analysis in the detection of mutations
Cytogenetic analysis, and more recently
fluorescence in situ hybridisation (FISH),
have been instrumental in revealing chromosomal rearrangements, which have
identified a number of important oncogenes in T-ALL. Visible chromosomal
changes are seen in approximately 50% of
T-ALL, while the remainder show a normal karyotype. Cryptic translocations, for
example t(5;14)(q35;q32) involving TLX3,
and deletions, such as TAL1, may be
detected by FISH using appropriate
probes6 (Figure 1). In T-ALL, translocations
involving the T-cell receptor (TCR) loci, α
(TRA@) and δ (TRD@), located to chromosomal band 14q11; β (TRB@) and γ
(TRG@) located to 7q34 and 7p15, respectively, are found in approximately 35% of
| 154 |
T-ALL by FISH, many of which are cryptic
at the cytogenetic level.7 Breakpoints
within the TCR loci give rise to illegitimate recombination. This may result in
oncogenes becoming juxtaposed to the
promoter and enhancer elements of the
TCR genes leading to their aberrant
expression and the development of TALL. Alternatively, aberrant expression of
oncogenic transcription factors may result
from loss of the upstream transcriptional
mechanisms that normally downregulate
the expression of these oncogenes during
T-cell development.8
The Krüppel-like zinc-finger gene,
BCL11B, encodes a transcription factor
essential in T-cell development.9 In the
translocation, t(5;14)(q35;q32), BCL11B
specifically associates with TLX3, leading
to upregulation of TLX3.10 There are an
increasing number of reports of the
involvement of BCL11B in other
rearrangements in T-ALL. For example,
t(5;14)(q35.1;q32.2), in which NKX2-5,
another homeobox gene, is upregulated,11
and inv(14)(q11.2q32.31), which results in
expression of the BCL11B-TRD@ fusion
in association with the absence of the
wild type BCL11B transcript.12 A number
of other novel BCL11B partners have
been identified by FISH, which are in the
process of being characterized.13 The
interrelationships between the TCR
genes, BCL11B and partners are shown in
Figure 2.
Alternatively, chromosomal rearrangements may produce fusion genes, formed
by in frame fusion of part of the two partner genes located at the chromosomal
breakpoints. The fusion gene encodes a
new chimeric protein with oncogenic
potential. Translocations of this type
involving MLL fusions and PICALMMLLT10 (CALM-AF10) are classified
among the mutations which impair differentiation, while those involving the tyrosine kinase, ABL1, play a role in the proliferation and survival pathway.
Four classes of mutations in the pathogenesis of T-ALL
Mutations which impair differentiation
Aberrant expression of one or more transcription
factors is a critical component of the molecular
pathogenesis of T-ALL. These include the class B
basic helix-loop-helix (bHLH) genes: TAL1, TAL2,
LYL1, bHLHB1 and MYC, as well as genes involved
in transcription regulation, for example, the cysteinerich LIM-domain-only genes, LMO1 and LMO2.
Abnormalities also affect the homeodomain genes,
TLX1 and TLX3, and members of the HOXA cluster.
The TAL1 gene maps to 1p32. TAL1 deregulated
expression is common in childhood T-ALL, and is
found in ~17% childhood cases.14 It results from the
translocation, t(1;14)(p32;q11), in which TAL1 is
translocated into TRA/D@,15 or more frequently a
submicroscopic interstitial deletion of part of SIL and
the 5’ untranslated region (UTR) of TAL1 generating
the SIL-TAL1 fusion gene16,17 It was shown that both
the t(1;14) translocation and TAL1 deletions disrupt
the 5' part of the TAL1 gene, placing its entire coding
sequence under the control of the regulatory elements of TRD@ or SIL, both of which are normally
expressed in T-cell development.17,18 High expression
levels of TAL1 in the absence of detectable rearrangements have been described in about 40% of T-ALL.8,19
TAL2 is upregulated in T-ALL as a result of the
translocation, t(7;9)(q34;q32), which juxtaposes TAL2
and TRB@.20
LYL1 is the partner gene of TRB@ in the translocation, t(7;19)(q34;p13).21 It is also constitutively overexpressed in a subset of T-ALL in the absence of
chromosomal rearrangements.22 T-ALL with LYL1
overexpression shows an immature phenotype and is
associated with a poor prognosis.
bHLHB1 is upregulated in T-ALL by the translocation, t(14;21)(q11,2;q22), which juxtaposes bHLHB1
and TRA@.23
In T-ALL with the translocations, t(7;12)(q34;p13.3)
and t(12;14)(p13;q11), massive upregulation of
CCND2 is produced by translocation to TRB@ and
TRA@, respectively. This expression is associated
with overexpression of TAL1, TLX1, TLX3,
NOTCH1 mutations and CDKN2A deletions indicating a role for CCND2 in the multistep leukemogenesis of T-ALL.24
The MYC gene is well known for its upregulation
by the IGH promoter in Burkitt’s lymphoma and
mature B-cell ALL. In T-ALL, upregulation is brought
about by the juxtaposition of MYC to TRA@ or
TRB@ promoters.
The LIM-domain-only genes, LMO1 and LMO2,
are located at 11p15 and 11p13 respectively. They are
frequently rearranged in T-ALL.25 The most common
translocations are t(11;14)(p15;q11) and t(11;14)
(p13;q11), juxtaposing LMO1 and LMO2 to the
Figure 1. Break apart probe for TLX3 showing the split red
and green signals on chromosomes 5 and 14, respectively. A fused red/green signal is present on the normal chromosome 5.
LYL1
NOTCH1
TLX1
TAL2
TRB@
LMO1
LCK
CDK6
MYC
TLX3
CCND2
TRA/D@
HOXA@
LMO2
2p21
IGH@
bHLHB1
TAL1
Single Case
BCL11B
Recurrent (n=2-9)
Recurrent (n=>10)
Figure 2. Interrelationships between TCR loci, BCL11B and
relevant oncogenes.
TRA/D@ locus respectively.26,27 Translocations with
TRB@ have also been reported.7 Cryptic deletions of
the short arm of chromosome 11, del(11)(p12p13),
have been identified in ~4% paediatric T-ALL by
array based comparative genomic hybridization
(aCGH).28 The deletion activates the LMO2 oncogene
in the same way as the translocation which together
Table 1. The recurrent chromosomal abnormalities and
molecular changes as defined within four pathways.
HOXA@
TLX1*
TLX3
PICALM-MLLT10*
MLL
2. Proliferation and survival
BCR-ABL1
NUP214-ABL1
ETV6-ABL1
EML1-ABL1
ETV6-JAK2
LCK
FLT3
N-RAS
t(9;22)(234;q11)
t(9;9)(q34;q34)**
t(9;12)(p24;p13)
t(9;14)(q34;q32)
t(9;12)(p24;p13)
t(1;7)(p34;q34)
mutations
mutations
3. Cell cycle defects
CDKN2A/CDKN2B
del(9)(p21)
4. Self-renewal capacity
NOTCH1
mutations
t(7;9)(q34;q34.3)
T
T
T
B
T
T
B
T
CDK6
?
F
F
F
F
F
F
F
T
F
*Upregulated expression in the absence of visible abnormalities; v, various; **episomal or HSR; T, translocations involving TCR; B, translocation involving
BCL11B; F, translocations (arising from) gene fusions; CDK6, upregulated by
CDK6 promoter; ?, involved gene unknown
account for about 9% of pediatric T-ALL. Overall,
abnormal expression of LMO1 and LMO2 occurs in
45% of T-ALL including cases with no evidence of
chromosomal changes.22,29 Their deregulation often
occurs in association with deregulation of LYL1
(LMO2) or TAL1 (LMO1 and LMO2) confirming their
involvement within common oncogeneic pathways.
Homeobox (HOX) genes are key regulators in
embryonic development and normal hematopoiesis.
They are divided into two classes. Class 1 HOX genes
BCL11B
IGH@
3’ TRG@
HOXA@
3’ TRA/D@
3’ TRG@
HOXA@
3’ TRA/D@
3’ BCL11B
5’ BCL11B
3’ BCL11B
IGH@
der(14)
LMO2*
5’ TRA/D@
5’ TRG@
TRA/D@
Normal 14
LMO1*
T
F
T
T
T
T
T
T
T
T
T
T
T
B
IGH@
5’ BCL11B
der(7)
MYC
t(1;14)(p32;q11)
del(1)(p32)
t(7;9)(q34;q32)
t(7;19)(q34;p13)
t(14;21)(q11;q22)
t(7;12)(q34p13.3)
t(12;14)(p13;q11)
t(8;14)(q24;q11)
t(7;8)(q34;q24)
t(11;14)(p15;q11)
t(7;11)(q34;p15)
t(11;14)(p13;q32)
t(7;11)(q34;p13)
t(11;14)(p13;q32)
del(11)(p13p13)
inv(7)(p15q34)
t(7;7)(p15;q34)
t(7;14)(p15;q32)
t(7;14)(p15;q32)
t(10;14)(q24;q11)
t(7;10)(q34;q24)
t(5;14)(q35;q32)
t(5;14)(q35;q11)
t(5;7)(q35;q21)
t(2;5)(p21;q35)
t(10;11)(p13;q14)
t(11;v)(q23;v)
Normal 7
1. Differentiation impairment
TAL1*
SIL-TAL1
TAL2*
LYL1*
bHLHB1
CCND2
HOXA@
TRG@
Figure 3. Idiogram showing the position of probes specific
for TCR, BCL11B, IGH@ and HOXA@ loci in a complex
rearrangement involving TRA/D@, TRG@, BCL11B and
HOXA@.
comprise 39 genes distributed in four clusters (A-D)
located to 7p15, 17q21, 12q13, and 2q31 respectively.
In T-ALL, HOXA10 and HOXA11 are upregulated as
a consequence of an often cryptic inversion of chromosome 7, inv(7) (p15q34) or the translocation,
t(7;7)(p15;q34), which brings the TRB@ enhancer
within the HOXA@ locus.31,32 HOXA is also upregulated by TRD@33 and BCL11B, specifically HOXA13.34
HOXA@ rearrangements are found in up to 3% of TALL by FISH.35 Rare reports of HOXA@ involved in
complex rearrangements with TCR genes35 (Figure 3)
indicate an additional mechanism of transcriptional
activation of HOXA@ cluster genes, overexpression
with gene dosage. Expression studies have identified
a subgroup of HOXA@ expressing T-ALL32 which
include, as well as cases with TCR-HOXA@, MLL,36
PICALM-MLLT10,37 cases without these rearrangements. This suggests the presence of additional as yet
undisclosed mechanisms of HOX activation.
The genes TLX1 and TLX3 belong to the class II
homeobox genes. TLX1 is not normally expressed in
developing T-cells although its oncogeneic potential
is well known. It is located at 10q24 and is involved
in the translocations, t(10;14)(q24;q11) and t(7;10)
(q34;q24).38;39 As a result of juxtaposition of promoter
elements of TRA@ and TRB@ respectively, the full
length protein is expressed at a high level. TLX1 is
also frequently activated in T-ALL in the absence of
visible genetic rearrangement.8,22,40 TLX1 is expressed
in ~30% T-ALL, more often in adults than children.
These leukemias show an early cortical phenotype
and a more favourable outcome than other classes of
T-ALL.41
In the majority of cases, expression of TLX3 in TALL results, from the cryptic translocation t(5;14)
A
B
Figure 4. A. Extrachromosomal amplification of the ABL1
signals (red) on episomes. B. Co-localization of 3’ ABL1
(red) and NUP214 signals (green) on episomes.
(q35;q32), juxtaposing TLX3 to the distal region of
BCL11B.42 This translocation is found in ~20% childhood and 13% adult T-ALL.43 Rare variants have been
reported, as well as subtle genomic deletions and
insertions within 5q.43,44 The latter abnormalities
highlight that TLX3 expression in T-ALL rarely
occurs in the absence of 5q abnormalities. The occasional findings of a t(5;14)(q35;q11) juxtaposing TLX3
and TRA/D@,45 a t(5;7)(q35;q21), a t(2;5)(p21;q35)
involving TLX3 with CDK6 and an as yet uncharacterized locus on 2p21, respectively,43 indicate that the
regulatory elements of TCR, along with other genes,
may contribute to the upregulation of TLX3.
Although their expression is mutually exclusive, gene
expression studies have shown that T-ALL expressing either TLX3 or TLX1 cluster together, suggesting
a common mechanism of action.22 TLX3 expressing
T-ALL do not have the favourable outcome associated with TLX1 expression.14,22,46
The PICALM-MLLT10 fusion of the t(10;11)
(p13;q14) is found in approximately 10% childhood
and adult T-ALL and is associated with a poor prognosis.47-49 The fusion may occur in a cryptic rearrangement, as expression has been shown in the absence
of the translocation. Expression arrays showed associated upregulation of HOXA5, HOXA9, and
HOXA10 and their coregulator MEIS137 in these
patients.
The MLL gene at 11q23 is known to rearrange with
more than 50 partners in translocations encoding
chimeric proteins in which the N-terminal portion of
MLL is fused to the C-terminal portion of the new
partner.50 MLL fusions are rare in T-ALL, accounting
for about 5%.51,52 The most frequent partners in TALL are MLLT1 (ENL) in the t(11;19) (q23;p13.3) and
MLLT4 in the t(6;11) (q27;q23).51 Gene expression
profiling demonstrated increased expression of the
HOX@ gene: HOXA9, HOXA10, HOXC6, as well as
MEIS1, to be a central mechanism of leukemic transformation in MLL positive T-ALL.36 This strongly
indicates common oncogeneic pathways in PICALMMLLT10 and MLL T-ALL.
Mutations providing a proliferative and survival advantage
ABL1 is a ubiquitously expressed cytoplasmic tyrosine kinase, encoded by the ABL1 gene at 9q34.
Although the BCR-ABL1 fusion of chronic myeloid
leukemia and B-lineage ALL is exceptionally rare in
T-ALL1 an alternative ABL1 fusion with NUP214 has
recently been described as a secondary abnormality
in 6% of T-ALL53 and 4% of adult patients.54 This was
initially discovered by FISH screening for evidence of
the BCR-ABL1 fusion which revealed multiple extrachromosomal ABL1 signals on episomes53,55 and,
rarely, homogeneously staining regions (HSR).56
Cohybridisation of 3’ ABL1 and NUP214 signals confirmed the presence of both genes in the same episomes (Figure 4). An in-frame fusion between introns
23 to 34 of NUP214 and intron 1 of ABL1 was confirmed in patients with this abnormality. The results
were compatible with a model in which the genomic region from ABL1 to NUP214 was circularized to
provide the NUP214-ABL1 fusion. The copy number
of the episome is increased due to unequal segregation during cell division. The NUP214-ABL1 fusion is
associated with increased HOX@ expression22 and
deletion of CDKN2A,57 consistent with a multistep
pathogenesis of T-ALL. Like BCR-ABL1, NUP214ABL1 acts as a constitutively phosphorylated tyrosine kinase, which is also sensitive to imatinib, a
selective inhibitor of ABL1 kinase activity. However,
in a recently published case, the NUP214-ABL1 positive patient showed no response to imatinib.58
Other rare ABL1 fusions have been reported in TALL: ETV6-ABL1 of t(9;12)(q34;p13),59 the cryptic
t(9;14)(q34;q32) encoding the EML1-ABL1 fusion
protein.60 The latter case also had a deletion of
CDKN2A and expression of TLX1 consistent with a
multistep pathogenesis of T-ALL.
The JAK2 gene is essential for transmission of signals from cytokine receptor to downstream signaling. In one case of pediatric T-ALL, a t(9;12)
(p24;p13),61 encoding the ETV6-JAK2 fusion protein,
was shown to result in constitutive tyrosine kinase
activity. Its transforming role was demonstrated in
mice.62
LCK is a tyrosine kinase gene specifically expressed
in T cells. It is overexpressed in rare cases with
t(1;7)(p34;q34), joining LCK to the TRB@ locus.63
FLT3 is a receptor tyrosine kinase. Activating mutations, such as internal tandem duplication (ITD) in
the juxtamembrane domain or point mutations in the
activation loop of the kinase domain are common in
AML but rare in T-ALL. They are restricted to cases
with a very immature phenotype expressing LYL1,
LMO2 and the KIT receptor.64
Activating mutations of N-RAS have been detected
in 10% of pediatric T-ALL.65 It is highly activated in
50% T-ALL66 suggesting a key role in T-ALL pathogenesis.
Mutations affecting the cell cycle
The CDKN2A and CDKN2B loci at 9p21 contain
genes coding for p16INK4A, p14ARF and p15INK4B
respectively, all involved in cell cycle regulation.67,68
Deletion of CDKN2A, often with CDKN2B, is the
most frequent abnormality in T-ALL.57,69 Deletions
may be homozygous or heterozygous seen in 65%
and 15% of cases respectively. This abnormality usually occurs as a secondary change and is associated
with all other primary genetic abnormalities in TALL.13 Inactivation of these loci may also arise from
promoter hypermethylation or mutation at both the
transcriptional and post transcriptional levels.70,71 The
functional inactivation of CDKN2A in the majority of
T-ALL implies a direct involvement in T-cell leukemogenesis72 through the RB1 and TP53 pathways.5
Mutations providing self renewal capacity
NOTCH1 is a direct regulator of cell growth, playing a critical role in T-cell development.73 MYC is an
important target of NOTCH1 in T-ALL and also in
normal pre-T cell development.74 In T-ALL, NOTCH1
forms a fusion with TRB@ in the rare translocation
t(7;9)(q34;q34.3). This results in aberrant expression
of a truncated activated form of NOTCH1.75
Altogether, NOTCH1 activating mutations are found
in more than 50% T-ALL, occurring most frequently
in the heterodimerization (HD) and PEST domains,
and are associated with short survival in adults but
not children.76-78 NOTCH1 acts cooperatively with
oncogene transcription factors in T-ALL pathogenesis.79 As NOTCH1 needs a gamma secretase enzyme
for activation, there are ongoing trials on inhibitors of
gamma secretase for treatment of aberrant NOTCH1
T-ALL.
Molecular classification
Interlaced with the four major classes of mutations
involved in the molecular classification of T-ALL, as
described by De Keersmaecker et al.,5 is the molecular classification which has emerged from gene
expression profiling.4,22 That the relative expression
of genes is of fundamental importance in T-ALL
leukemogenesis comes from the observations that
many of the significant transcription factors are aberrantly expressed in a large proportion of T-ALL in the
absence of chromosomal abnormalities affecting the
locus. Expression profiling of T-ALL has identified
several gene expression signatures indicative of arrest
at specific stages of thymocyte development: a LYL1
positive signature represents immature thymocytes
(pro-T), TLX1 positive represented early cortical thymocytes and TAL1 correlated with late cortical thymocytes. Thus gene expression profiling has
improved our understanding of the biological heterogeneity of the disease while revealing clinically rele-
vant subtypes. Five different multistep molecular
pathways have been defined that involve the activation of different oncogenes that lead to T-ALL: 1)
TLX1; 2) TLX3; 3) TAL1 plus LMO1 and/or LMO2; 4)
LYL1 plus LMO2; 5) MLL-MLLT1. TLX1, TLX3 and
TAL1 positive patients show high levels of MYC
expression and share the loss of CDKN2A locus
whereas LYL1 positive cases show high level expression of MYC-N. The MLL-MLLT1 group has low levels of MYC expression but high levels of HOXA9,
HOXA10, HOXC6 and MEIS1.4
It is evident that the interrelationships between the
different molecular aberrations in T-ALL define multistep pathogenesis of the disease which may currently be oversimplified. New abnormalities may be
uncovered as intensive research in the genetics of TALL continues. However, to date, molecular analysis
has shown its capacity to clarify significant pathways
relevant to the future treatment of T-ALL.
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Treatment of Philadelphia chromosome positive acute
lymphoblastic leukemia
O.G. Ottmann
H. Pfeifer
B. Wassmann
Johann Wolfgang Goethe Universität,
Frankfurt/Main, Germany
2007;1:161-167
A
B
S
T
R
A
C
T
Introduction of imatinib-based therapy into front-line treatment of Ph+ALL has greatly improved the rates of complete remission and there is evidence of improved overall
outcome in adult patients with Ph+ALL. Accordingly, Imatinib combined with
chemotherapy is now becoming the gold standard for treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia. The optimal treatment combinations,
particularly with respect to consolidation and maintenance therapy remain to be determined for different populations, and are likely to differ substantially between elderly
patients and younger patients, in whom SCT in first CR is still widely considered an
essential element of curative therapy. Overcoming or preventing acquired imatinib
resistance remains the predominant therapeutic challenge; the introduction of several
novel kinase inhibitors that are more potent inhibitors of the Abl-kinase and are active
against a wide spectrum of BCR-ABL mutations conferring imatinib resistance is an
important addition to our therapeutic armament. The lessons learned during the clinical
development of imatinib will have to be applied to these compounds as well to facilitate further rapid advances in treatment of Ph+ALL.
he Philadelphia (Ph) chromosome is
the result of a reciprocal translocation
between chromosomes 9 and 22
[t(9;22)] and is characterized at the molecular level by expression of the BCR-ABL
fusion gene. It is the most frequent genetic
aberration in adult acute lymphoblastic
leukemia (ALL), being detectable in 30-40%
of patients with B-precursor ALL.1,2 In particular, its incidence increases with age, so
that leukemic cells from approximately
50% of ALL patients older than 60 years
have the Ph chromosome and/or express
BCR-ABL transcripts. The prognosis of
Ph+ALL is extremely poor even in younger
patients, even though the complete remission rates with dose-intensive induction
chemotherapy (approximately 60-80%) are
only moderately inferior to those achieved
in Ph negative.1-5 Conventional chemotherapy regimens that have been effective in
other precursor B-cell ALL cases are largely
unable to cure patients with this disease.
Median remission duration ranges from 916 months in patients treated only with
chemotherapy but there are almost no longterm survivors.
(SCT) is considered to be the treatment of
choice in adult Ph+ALL, with probabilities
of long-term survival ranging from 27% to
65% in patients undergoing SCT in first
complete remission (CR1). However, even
T
after SCT in CR1, the probability of
relapse is approximately 30%.6
The highly successful introduction of
the first ABL-kinase inhibitor imatinib into
the treatment of Philadelphia chromosome positive chronic myeloid leukemia
(CML) has led to clinical testing of imatinib in Ph+ALL. It has recently been
approved for Ph+ALL in Europe and Japan.
Given the much more aggressive nature of
Ph+ALL when compared with chronic or
even accelerated phase CML, a variety of
imatinib-based treatment strategies have
been explored in ALL. These studies will
be the focus of this review.
Induction therapy with single-agent imatinib
The prognosis of elderly patients with
acute lymphoblastic leukemia (ALL) is
considerably inferior to that of younger
patients, irrespective of cytogenetic aberrations. Chemotherapy induces complete
remissions (CR) in approximately 50% of
patients but responses are not sustained,
with remission duration ranging from 3 to
12 months and a probability of long-term
survival below 10%.7-10 Because of its high
frequency in elderly patients, the Ph chromosome contributes significantly to the
poor prognosis in this patient population,
in addition to the greater frequency of
comorbidity and reduced tolerability of
cytotoxic drugs. Allogeneic SCT is not
appropriate applicable in elderly patients, as treatment-related mortality (TRM) increases substantially
with age.6 Accordingly, monotherapy with an agent
that selectively targets the leukemogenic BCR-ABL
oncoprotein is an attractive option. The initial phase
II studies of imatinib monotherapy in Ph+ALL were
conducted in patients who had failed previous
chemotherapy and, in some cases, also SCT;11-13 the
overall response rate was 60-70%, with a CR rate of
17-30%. In addition, median time to progression was
only 2.2 months, and median survival about 4.9
months.12,13 Together with the favorable toxicity profile of imatinib, these date led to the start several
studies in which imatinib was used either alone or in
conjunction with chemotherapy of varying intensities in elderly patients with newly diagnosed rather
than advanced Ph+ALL.
The efficacy of induction therapy with imatinib
plus prednisone followed by maintenance treatment
with imatinib alone was examined by the GIMEMA,
as recently reported by Vignetti et al.14 Patient median age was 69 years, and all 29 patients evaluable for
response achieved a CR within the first 45 days of
treatment. However, only one patient had a complete molecular response as defined by undetectable
BCR-ABL transcripts. Treatment was well tolerated
with no deaths in CR. Relapse during imatinib maintenance was frequent, with a median remission duration of 8 months and an approximately 40% relapse
rate within the first 4-6 months of treatment.
The first prospective, randomized trial comparing
the efficacy and tolerability of imatinib monotherapy
with age-adapted multi-agent chemotherapy as
induction treatment in de novo Ph+ALL was conducted by the German Multicenter Study Group for
Adult ALL (GMALL). This study also examined the
tolerability and outcome of subsequent uniform consolidation therapy in which all patients received imatinib concurrently with successive cycles of consolidation chemotherapy. Fifty-five patients with a
median age of 68 years were enrolled.15 The overall
CR rate was 96% in patients randomly assigned to
imatinib induction and 50% in patients allocated to
induction chemotherapy (p=0.0001). No patient
failed imatinib induction whereas 35% of patients
were refractory and 8% of patients died during
induction chemotherapy. As expected, severe
adverse events were significantly more frequent during front-line chemotherapy (90% vs. 39%; p=0.005).
This initial benefit associated with imatinib induction was lost during the prolonged consolidation
phase because of a substantial rate of death in CR
that was attributable to cytotoxic chemotherapy. As
a result, estimated overall survival (OS) was not significantly different between the two cohorts (42±8%
at 24 months).
The treatment strategy adopted by the GRAALL
Study Group to assess the use of imatinib in previously untreated elderly patients was different in several respects.16 ALL patients aged 55 years or older
were given steroids for the one week needed to
establish the diagnosis of BCR-ABL positive ALL.
Ph+ patients were then offered a chemotherapybased induction followed by a consolidation phase
with imatinib and steroids lasting 2 months. Patients
in CR after consolidation were given 10 maintenance
blocks of alternating chemotherapy, including two
additional 2-month blocks of imatinib. Therefore,
this study used a more intensive maintenance therapy compared with the GIMEMA and GMALL studies.14,15 Thirty patients were included in this study and
compared with 21 historic controls. Out of 29 assessable patients, 21 (72%) were in CR after induction
chemotherapy vs 6/21 (29%) in controls (p=0.003).
Five additional CR were obtained after salvage with
imatinib and four after salvage with additional
chemotherapy in the control group. Overall survival
(OS) was 66% at 1 year vs 43% in the control group
(p=0.005). The 1-year relapse-free survival was 58%
vs 11% (p=0.0003).16 Therefore, the use of imatinib
during the consolidation and maintenance phases in
elderly patients with Ph+ ALL appears to improve
outcome, including OS, but there was no apparent
plateau on the Kaplan-Meier survival curves, mainly
because of relapse.
Combination therapy with imatinib and chemotherapy
Several clinical trials conducted mainly in younger
adult patients with newly diagnosed BCR-ABL-positive ALL assessed the efficacy and feasibility of different imatinib-chemotherapy combination regimens.
In a prospective phase II study conducted by the
Japan Adult Leukemia Study Group (JALSG), 80
patients with newly diagnosed BCR-ABL-positive
ALL received imatinib in combination with induction therapy, followed by alternating cycles of imatnib and intensive consolidation chemotherapy.17
Remission induction therapy resulted in complete
remission (CR) in 96% of patients, resistant disease
was observed in one patient and early death in two
patients. Polymerase chain reaction negativity for
BCR-ABL transcripts in bone marrow samples was
obtained in 71% of patients. The profile and incidence of severe toxicity were not different from
those associated with the historic chemotherapyalone regimen. Twenty patients relapsed after a
median CR duration of 5.2 months. Allogeneic
hematopoietic stem-cell transplantation (HSCT) was
performed in 49 patients, 39 of whom underwent
transplantation during their first CR. The 1-year
event-free and OS rates were estimated to be 60%
and 76% respectively. These were significantly better than those for the historical control group treated
with chemotherapy alone (p<.0001). Remarkably,
the probability for OS at 1 year was not lower for
those patients who underwent allogeneic HSCT
(73%) as opposed to those who did not (85%).
Therefore, their study not only shows that imatinibcombined regimen is effective and feasible for newly
diagnosed BCR-ABL-positive ALL, but suggests that
it may achieve outcome results comparable to allo
SCT. The relatively short period of observation must
however be taken into account. Encouraging survival data were also reported by Thomas et al. in a
study combining imatinib with hyperCVAD
chemotherapy. The CR rate was 96% in patients
with active disease, and bcr-abl transcripts became
undetectable by RT-PCR in 5 patients after hyperCVAD plus imatinib and in an additional 12 patients
after allogeneic SCT.18
Parallel administration of imatinib with induction
and consolidation chemotherapy was studied by the
Spanish PETHEMA group, with a CR rate approaching 90%.19 Overall, these studies yielded no evidence
of unexpected toxicities related to the addition of
imatinib, and subsequent TBI-based stem cell transplantation did not appear to be adversely affected by
preceding imatinib therapy.
Two different schedules of imatinib-chemotherapy combinations were explored in sequential
patients cohorts who were treated within a recent
prospective multicenter GMALL trial including 92
patient with newly diagnosed Ph+ALL.20 While imatinib and chemotherapy were administered on an
alternating schedule in the first cohort of patients,
the second cohort received imatinib parallel to
induction and early consolidation chemotherapy,
and then without interruption until SCT.
Coadministration of imatinib and induction cycle 2
(INDII) resulted in a CR rate of 95% and PCR negativity for BCR-ABL in 52% of patients, compared to
19% in patients in the alternating treatment cohort
(p=0.01). This indicates greater antileukemic efficacy
of a treatment strategy in which imatinib is started
earlier and given parallel to chemotherapy. In the
concurrent cohort, grade III/V cytopenias and transient hepatotoxicity required interruption of induction in 87% and 53% of patients respectively.
However, duration of induction was not prolonged
when compared to patients receiving chemotherapy
alone. No imatinib-related severe hematologic or
non-hematologic toxicities were noted with the
alternating schedule. This confirmed the PETHEMA
data19 showing that parallel administration does not
significantly increase toxicity. Importantly, both
schedules of imatinib help SCT in CR1 in the majority (77%) of patients, compared with just above
60% in a historic control group.
The imatinib dosage most commonly used in the
above studies ranged from 400 to 600 mg, partly
because of concerns regarding toxicity when combined with intensive chemotherapy. A clinical trial
evaluating a strategy based on high-dose imatinib
(800 mg per day) combined with a less intensive
chemotherapy regimen consisting of vincristine and
dexamethasone (DIV induction regimen) enrolled 31
patients with Ph+ lymphoid leukemias (18 relapsing
or refractory Ph+ ALL and 13 lymphoid blast crisis of
CML).21 Complete remission was obtained in 28 out
of 30 assessable patients, and the CR rate in patients
older than 55 years was 90%. Median time to neutrophil recovery was 21 days. Six out of 31 patients
developed fungal infections, possibly related to dexamethasone. Vincristine-induced neuropathy was
noted in 14 patients, with no evidence of additional
toxicity in older patients. Nine out of 19 patients
below 55 years underwent allogeneic SCT. Given
the favourable balance between efficacy and toxicity, the authors proposed investigating the use of this
DIV regimen as front-line therapy in elderly patients.
While the former studies initiated imatinib during
induction and independent of the response to
chemotherapy, the Group for Research on Adult
Acute Lymphoblastic Leukemia (GRAAPH) conducted a study in which imatinib was first administered
after induction with HAM (mitoxantrone with intermediate-dose cytarabine) consolidation in good
early responders (corticosensitive and chemosensitive ALL) or earlier during the induction course in
combination with dexamethasone and vincristine in
poor early responders (corticoresistant and/or
chemoresistant ALL).22 Imatinib was then continuously administered until stem cell transplantation
(SCT). Forty-five patients with newly diagnosed Ph+
ALL were treated, with overall CR and BCR-ABL
real-time quantitative polymerase chain reaction
(RQ-PCR) negativity rates of 96% and 29% respectively. All of the 22 CR patients (100%) with a donor
actually received allogeneic SCT in first CR. At 18
months, the estimated cumulative incidence of
relapse, disease-free survival, and overall survival
were 30%, 51%, and 65% respectively, comparing
favorably with results obtained in the pre-imatinib
LALA-94 trial.4
Overall, the results obtained in this GRAAPH
study are very consistent in tems of the overall
response rate, and in confirming the value of a combined approach with imatinib and chemotherapy.
Differences between the study results mostly relate
to outcome, both in patients who underwent SCT
and those who did not. This shows the need to evaluate the role of allogeneic SCT in the context of different pre-transplant regimens.
Stem cell transplantation after front-line imatinib
As described above, incorporation of imatinib into
chemotherapy regimens prior to SCT appears to be a
useful strategy for managing the time to allogeneic
SCT and making SCT in CR1 easier for a great majority of Ph+ALL patients. Post-transplant outcome of
29 patients previously treated with an imatinibbased strategy was reported by Lee et al.23 At the time
of enrollment, 23 patients (79%) had achieved a CR
while 3 patients were refractory to both imatinib and
chemotherapy. Relapse prior to SCT was significantly less frequent in the imatinib group than in the historic control (3.5% vs 42.3%, p=0.002). This allowed
allogeneic SCT in first CR in 86% of the 29 patients,
a significantly greater proportion than in the historic
control group. With a median follow-up duration of
25 months after SCT, the 3-year estimated probabilities of relapse, non-relapse mortality and disease-free
survival were 3.8%, 18.7% and 78.1% respectively.
Acute transplant-related toxicity and mortality were
not different in the two groups. These results indicate overall superior survival with pre-transplant
imatinib therapy.
The question whether imatinib therapy may compromise the outcome of subsequent SCT is very
important in advanced Ph+ leukemias in which SCT
offers the best option for cure. The effect of prior
exposure to imatinib on transplant-related mortality
was retrospectively analyzed by Deininger et al. in 21
patients with Ph+ ALL and 70 patients with CML
who had received imatinib before SCT.24 At the time
of SCT, 40% of ALL patients had active disease compared to 84% and 95% prior to imatinib, and 44% of
CML patients were in accelerated phase or blast crisis. At 24 months, estimated transplant-related mortality was 44% and estimated relapse mortality 24%.
No unusual organ toxicities were observed.
Compared to historic controls, previous imatinib
treatment did not influence overall survival, progression-free survival or non-relapse mortality, although
there was a trend towards higher relapse mortality
and significantly less chronic graft-versus-host disease. Overall, there was no evidence that imatinib
negatively affects major outcomes after SCT, suggesting that imatinib before SCT is safe.
Imatinib after stem cell transplantation
Detection of minimal residual disease (MRD) after
SCT is associated with a high probability of relapse.
Starting Imatinib in the setting of MRD may lower
this high relapse rate. In a prospective multicenter
GMALL study, 27 Ph+ALL patients received Imatinib
on detection of MRD after SCT. Bcr/abl transcripts
became undetectable in 14 of 27 patients (52%), after
a median of 1.5 months (earlyCRmol). All patients who
achieved an earlyCRmol remained in remission for the
duration of Imatinib treatment, 3 patients relapsed
after Imatinib was discontinued. Failure to achieve
PCR negativity soon after starting Imatinib predicted
relapse. This occurred in 12 out of 13 patients (92%)
after a median of 3 months. Disease-free survival
(DFS) in earlyCRmol patients is 91±9% and 54±21% after
12 and 24 months respectively, compared with 8±7%
after 12 months in patients remaining MRD-positive
(p=0.0001).25 Therefore, approximately half of
Ph+ALL patients who receive Imatinib for MRD positivity after SCT experience prolonged DFS which
can be anticipated by the rapid achievement of a
molecular CR. Continued detection of bcr/abl transcripts after 2-3 months on Imatinib identifies
patients who will ultimately experience relapse and
in whom additional or alternative antileukemic treatment should be started.26-27
Acquired resistance to imatinib
Given the remarkably high CR rate achieved with
imatinib-based therapy and its good tolerability in
newly diagnosed Ph+ALL patients, acquired resistance to imatinib is the principal cause of treatment
failure.28,29 Enhanced drug efflux, insufficient serum
levels or BCR-ABL independence due to secondary
transforming events are theoretical causes of drug
failure that have not been confirmed in Ph+ALL.
Pharmakokinetic resistance has been shown to cause
the substantial rate of meningeal leukemia in
patients with Ph+ALL receiving Imatinib without
adequate CNS-directed prophylaxis, due to insufficient penetration of the blood brain barrier by imatinib.28 Prophylactic intrathecal CNS prophylaxis is
therefore an essential element of any imatinib-based
treatment strategy for Ph+ALL or CML-LBP.
Mutations in the tyrosine kinase domain (TKD) of
BCR-ABL have been the focus of studies on resistance and are found in the majority of Ph+ALL
patients who relapse while on imatinib,33-37 Two
recent reports addressing the frequency and distribution in patients with advanced or de novo Ph+ALL,
demonstrated TKD mutations in up to 80% of
patients with acquired resistance, with predominance of p-loop mutations known to confer high
Numerous point mutations in the KD of BCR-ABL
that impair imatinib binding to varying degrees have
been identified as a major mechanism of acquired
resistance in patients with chronic myeloid leukemia
(CML).28,29 Data on BCR-ABL mutations in patients
with Ph+ALL or lymphoid blast crisis of CML are
more limited. Two studies of patients with advanced
Ph+ lymphoid leukemias identified 5 different KD
mutations in 14 of the 17 evaluted patients with
acquired resistance to imatinib.31,32 Greater quantities
of the E255K/V p-loop mutation, which occurred in
6 of 9 patients (67%) following their treatment with
imatinib was suggested by one of these reports31 but
not by the other.32 However, all point mutations
arose at positions within the KD that are known to
be important for drug binding and to give significant
resistance to imatinib in vitro.33-35 This demonstrated
that different mutations within the BCR-ABL KD
can be responsible for refractoriness of Ph+ lymphoid leukemias to imatinib, and also suggested that
KD mutations may be a frequent mechanism of, or
contributory factor to, acquired imatinib resistance
during salvage therapy with imatinib.
Using denaturing high-performance liquid chromatography and sequencing, Soverini et al. screened
for ABL kinase domain mutations in 370 patients
with Ph+ lymphoid leukemias with hematologic or
cytogenetic resistance to imatinib.38 Mutations were
found in 83% of lymphoid blast crisis/Ph+ acute
lymphoblastic leukemia (ALL) patients. P-loop and
T315I mutations were particularly frequent in Ph+
ALL and advanced-phase CML patients. Amino acid
substitutions at seven residues (M244V, G250E,
Y253F/H, E255K/V, T315I, M351T, and F359V)
accounted for 85% of all resistance-associated
mutations.31,32
The very short median time to progression
observed in patients with advanced Ph+ALL treated
with imatinib in the early phase II studies suggested
the possibility that KD mutations might already be
present before imatinib therapy. While BCR-ABL
KD mutations were not detected in patients with
chemotherapy-resistant acute lymphoid leukemia
before imatinib treatment when a direct sequencing
approach was used,31,32 it was subsequently demonstrated that low-level KD mutations are present at
least in a small proportion of imatinib-naïve Ph+
ALL.39 As the proportion of mutant alleles at the start
of imatinib therapy were low, highly sensitive detection techniques are required to detect low-level
mutant clones.
Pfeifer et al. investigated the prevalence and distribution pattern of BCR-ABL TKD mutations in pretherapeutic leukemic samples and bone marrow
samples collected throughout imatinib-based therapy from patients with newly diagnosed Ph+ ALL,
and in leukemic samples from relapsed patients by
means of highly sensitive denaturing HPLC
(WAVE™) and allel-specific oligonucleotide PCR
(ASO-PCR) and by cDNA sequencing.40 Remarkably, TKD mutations were detected in a minor subpopulation of leukemic cells in 40% of newly diagnosed and imatinib-naïve patients. At relapse, the
dominant cell clone had an identical mutation in
90% of cases, the overall prevalence of mutations at
relapse was 80%, with predominance of p-loop
mutations. Thus, BCR-ABL mutations giving highlevel imatinib resistance are present in a substantial
proportion of patients with de novo Ph+ ALL and
eventually lead to relapse. This provides a rationale
for the front-line use of kinase inhibitors active
against these BCR-ABL mutants.
Second generation ABL-kinase inhibitors
Several novel ABL kinase inhibitors have recently
entered the clinical testing phase.41,42 Nilotinib is a
selective, aminopyrimidine which is 30 times more
potent in vitro than imatinib and active against most
tested imatinib resistant Bcr-Abl mutations. The
safety and efficacy of nilotinib administered at a
dose of 400 mg bid was evaluated in 34 patients
with Ph+ALL who had relapsed after or were refractory to imatinib. Chromosomal abnormalities other
than Ph+ were noted in 35% and extramedullary
involvement in 9% of Ph+ALL patients. Complete
responses were reported in 2 (6%) patients. The
main reason for discontinuing nilotinib was disease
progression. The most frequent Grade 3 or 4 adverse
events occurring in patients with Ph+ALL were
thrombocytopenia in 3 (9%) patients, and 2 (6%)
patients each had neutropenia, blood bilirubin
increased, ALT elevation and bone pain. These clinical phase II data therefore show clinical activity and
an acceptable safety and tolerability profile in
patients with imatinib resistant relapsed/refractory
Ph+ALL, suggesting that trials in front-line treatment
are warrented.43
Dasatinib is a novel, oral, multi-targeted kinase
inhibitor of BCR-ABL and SRC. Its usefulness in the
treatment of Ph+ALL was explored in an open
label, multi-center, global phase-II study which
included 46 relapsing patients with Ph+ALL. These
patients had previously been treated with
chemotherapy including imatinib, and 37% of
them had received a stem cell transplant. The starting dose of dasatinib was 70 mg twice daily (BID).
In the 40 patients with baseline mutation data, imatinib-resistant BCR-ABL mutations were observed
in 78%, one with T315I. The overall complete
hematologic response rate was 35%. The major
hematologic response (MHR) in the 31 patients
with baseline mutations was 45%. The median
duration of MHR was 11 months and the median
progression-free survival was 3.7 months. Grades 3
and 4 thrombocytopenia occurred in 13% and
67%, respectively and grades 3 and 4 neutropenia
occurred in 27% and 52% of patients, respectively.
Most frequent non-hematologic toxicities included
diarrhea, nausea and pleural effusion. Overall,
these data show significant clinical efficacy in this
prognostically poor group of patients, but also
emphasize the need to employ these second generation kinase inhibitor at earlier stages of Ph+ALL
rather than as salvage therapy.44
Acknowledgments
Supported by the Competence Network Acute and
Chronic Leukemias, BMBF grant 01GI9971, the National
Genome Research Network (NGFN) and the Adolf Messer
Foundation.
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Detection of minimal residual disease in adult patients
with acute lymphoblastic leukemia: methodological
advances and clinical significance
M. Brüggemann
T. Raff
S. Böttcher
S. Irmer
S. Lüschen
C. Pott
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2007;1:168-174
lthough 80%-95% of adult patients
with acute lymphoblastic leukemia
(ALL) achieve complete clinical
remission with current treatment protocols,1-4 the majority of them ultimately
relapse. Relapses are caused by residual
malignant cells that are undetectable by
standard diagnostic techniques.5 With the
development of more sensitive techniques
for the detection of malignant cells, the
presence of minimal residual disease
(MRD) in patients in complete clinical
remission has clearly been demonstrated.
It is important to determine whether such
sensitive MRD detection has clinical significance.6-13 Therefore, different techniques for sensitive and specific MRD
quantification have been established in
the past 15 years.
A
MRD techniques
Each approach is characterized by
advantages and limitations, mainly related
to its sensitivity and specificity (see Table
1, reviewed in5,14-18), which should be taken
into account when large scale multicenter
clinical MRD studies are planned.
Immunological analysis
Flow cytometry represents a rapid and
reliable option for investigating MRD in
the vast majority of ALL patients. The characteristic immunophenotype of residual
ALL cells is identified via multi-colour flow
cytometry. The main problem is the discrimination of malignant cells from normal
lymphoid precursors resembling ALL cells.
In T-ALL, discrimination is facilitated by
the fact that early T-cell development takes
place in the thymus and therefore detection
of (TdT+ and/or CD34+) T-precursors in
the bone marrow or blood signifies the
presence of T-ALL cells.15 By contrast, in
precursor-B-ALL the sensitive distinction of
residual ALL from normal precursor-B-cells
is not easy, particularly in regenerating
bone marrow during or after therapy when
benign B-precursors can account for up to
5% of all leucocytes.19 The unambiguous
| 168 |
identification is based on aberrant
immunophenotypes with qualitative (e.g.
expression of CD66c, CD13, CD33, NG-2,
CD21) or quantitative (e.g. underexpression of CD10, CD38, CD45 and overexpresssion of CD58) differences in expression patterns compared to benign B-precursors.15,20-23 Using 4-colour flow cytometry,
leukemia-associated immunophenotypes
can be identified in about 90% of B-precursor and more than 95% of all T-ALL
patients24 reaching a detection limit of 10–3
to 10–4.9,22,25-27 However, immunophenotypic
shifts occur;20,28 therefore preferably two
different aberrant immunophenotypes
should be monitored to prevent false negativity. New immunophenotypic tools like
6- to 8-colour flow cytometry, new fluorochromes, improved analysis facilities,
and characterisation of additional
leukemia-associated antigens will improve
sensitivity and specificity of this
method.22,29 Recently, EuroFlow, a European
network of Flow laboratories started to
develop standardized ≥6-colour flow cytometric strategies for fast, accurate and sensitive quantification of low level MRD.
PCR analysis of clonal immunereceptor gene
rearrangements
During early T- and B-cell development
immunoglobulin (Ig) and T-cell receptor
(TCR) genes rearrange and form fingerprint-like sequences at their junctional
regions. As the different subtypes of ALL
represent clonal expansions of the malignant counterpart of early lymphoid development, almost all ALLs harbour clonal Ig
and/or TCR gene rearrangements allowing
for DNA based MRD quantification in
more than 90% of all ALL patients. Clonal
Ig rearrangements can be identified in
>95% of all precursor B-ALL but also in
about 10% to 15% of T-ALL. The large
majority of T-ALL display clonally
rearranged TCR genes which also occur as
cross-lineage rearrangements in about
90% of precursor B-ALL.16,30-35 Clonality can
be assessed using primers annealing to
conserved regions of the V-, D- and J-segments of the
different immunereceptor genes. With the use of fluorescent dye labeled primers PCR-products can be
easily detected by automated fluorescence fragment
analysis (GeneScanning) with a size-discrimination
down to 1 basepair. By this consensus primer – PCR
approach a clonal rearrangement can be detected with
a sensitivity of one single tumour cell in the background of 20 to 1,000 polyclonal lymphoid cells.36-38 To
obtain a higher level of sensitivity, DNA sequencing
of the junctional regions is required in order to design
tumour-specific primers and probes. Thanks to the
development of real-time quantitative (RQ)-PCR
techniques, precise quantification is possible during
the early exponential phase of PCR amplification.
This technology eliminates variations during late
post-exponential phases of PCR reaction and during
post-PCR manipulation of samples. A major problem
when using rearranged immunereceptor genes as
MRD-PCR targets is the possibility of oligoclonality
and continued rearranging during the course of therapy and during follow-up that can lead to false negative PCR results.30,39-41 Therefore two immunereceptor
gene PCR targets should usually be used for Ig/TCR
based MRD analyses. Several RQ-PCR approaches
have been developed for almost all Ig/TCR gene
rearrangements.17,42-47 Standardization of analysis and
data interpretation are the subject of the European
Study Group on MRD detection in ALL (ESG) made
up of 32 experienced PCR laboratories spread over
Europe, Israel, Singapore, and Australia.48 Currently,
most ALL trials that implement MRD analysis apply
Ig/TCR based assays since for the moment these represent the most standardized and sensitive techniques.
PCR analysis of chromosomal translocations
Structural chromosomal aberrations that lead to
characteristic fusion genes are ideal PCR targets
because they are highly specific and can reach excellent sensitivities of 10–4 to 10–6.49 In ALL most assays
analyze specific fusion gene transcripts via reverse
transcriptase (RT)-PCR analysis, but some chromosome aberrations are also detected on DNA level.50,51
These targets allow a rapid and relatively cheap
MRD quantification because the same set of reagents
can be used for different patients. The disadvantages
are the possibility of false positivity due to cross-contamination which is difficult to recognize, since
leukemia-specific fusion gene RT-PCR products are
not patient-specific. Also, RNA is rather unstable and
different stability of fusion gene transcripts and control gene transcripts may result in unreliable MRD
quantification.52 The main problem is that only about
30-40% of precursor B-ALL (BCR-ABL, E2A-PBX1,
MLL-AF4, TEL-AML1) and about 10-20% of T-ALL
(particularly SIL-TAL1, CALM-AF10) patients have
specific PCR detectable chromosome aberrations.16
Standardized approaches have been developed particularly for the quantification of BCR-ABL fusion
gene transcripts in Philadelphia chromosome positive
(Ph+) ALL.53,54 MRD quantification in this ALL subgroup is important not only after stem cell transplantation but also for monitoring treatment with tyrosine kinase inhibitors.
Although similar MRD results can be obtained by
flow cytometry and PCR analysis, MRD levels and
sensitivity may differ in a significant proportion of
cases, emphasizing that these techniques are not simply exchangeable.26,55-58
Table 1. Characteristics of MRD techniques in acute lymphoblastic leukemia.16,18
Applicability
PrecursorB-ALL
T-ALL
Immunophenotyping
80-95%
>95%
PCR analysis of chromosomal
translocations
30-40%
10-20%
PCR analysis of clonal
Ig/TCR gene rearrangements
>90%
~95%
Advantages
Disadvantages
fast
quantitative with information on benign cells
cell viability can be determined
immunophenotypic shift
background of benign cells
limited sensitivity using 3- to 4-colour
flow cytometry
fast
high sensitivity (10-4-10-6)
stable targets
relatively cheap
cross-contamination of PCR products
instability of RNA
differences in expression levels possible
applicable only for a minority of patients
high sensitivity (10-4 -10-5)
stability of DNA
applicable for almost all ALL patients
time consuming
relatively expensive
loss of markers due to clonal evolution
Clinical significance of MRD in ALL
Prognostic value of MRD in adult ALL
Several large-scale studies in childhood ALL have
demonstrated that MRD analysis by molecular or
highly sensitive immunological methods can predict
clinical outcome and that this MRD information is
independent of classic prognostic factors at diagnosis
such as age, sex, white blood cell count, immunophenotype and chromosome aberrations.6-13
Results of MRD evaluation in adult ALL generally
confirmed the findings obtained in paediatric ALL.59-63
However, frequency of MRD positivity tends to be
higher in adult disease, probably reflecting the higher in vivo drug resistance.
Comparable to the result of paediatric trials, high
level MRD after induction treatment is associated
with a high risk of relapse in adult ALL. In the
GMALL MRD study on 196 standard-risk ALL
patients, MRD levels ≥10–4 after start of consolidation
treatment was detected in about 25% of cases and
was associated with an extremely poor prognosis (3year disease-free survival (DFS) 12%)59 (Figure 1).
This agrees with findings of Mortuza and colleagues60 who investigated 85 adult patients with Blineage ALL. DFS for patients with detectable MRD
3-5 months and 6-9 months after diagnosis, was 11%
and 0% respectively, compared to 74% and 80% in
MRD-negative patients. Similarly, Brisco et al.64 analyzed MRD in 27 adults using PCR techniques. Eight
out of 11 patients (73%) with MRD >10–3 relapsed
compared to 6/16 (38%) with MRD <10–3 after the
end of induction (day +22 to +68). In an immunophenotypic study on 102 adolescent and adult ALL
patients Vidriales and colleagues62 demonstrated the
high predictive value of day +35 MRD. However
relapse rate was about 50%, even in patients with
MRD levels <0.05%. Therefore, MRD after induction
in adult ALL has a high discriminative value. But also
patients with low or undetectable MRD at this timepoint show considerable relapse rates.59,62,64 In contrast, early MRD quantification during induction (day
+11 or day +14) identifies patients with a very rapid
molecular response and an excellent prognosis (3year DFS 90-92%), in line with reports on childhood
ALL. Therefore, the same MRD status achieved after
a different period of time results in different prognoses. The combined information on MRD kinetics
identifies those patients with a rapid tumour clearance and a favourable outcome from those with persistent disease and a particularly high relapse rate.
Few MRD studies focussed on T-ALL patients65,66
who account for about 20% of adult ALL cases.
Immunophenotypic data suggest the relapse-predicting value of MRD also for this patient group.66 MRD
positivity before consolidation (MRD+: 38% of
patients), before the third reinduction (MRD+: 34%),
and before reinduction cycle 6 (MRD+: 17%) was
associated with a 2-year relapse rates of 81.5%,
54.5% and 50.0% respectively, while MRD negativity at those time-points predicted a more favorable
outcome (relapse rate 38.9%, 15.8% and 16.4%).66
By contrast, preliminary PCR data of the UKALL
study group did not provide conclusive results and, in
particular, failed to predict outcome on the basis of
discrete testing time-points,65 whereas MRD was
highly predictive in B-cell precursor ALL.60 Within the
GMALL trial, percentages of MRD positivity and
prognostic impact did not significantly differ in
patients with T-lineage ALL compared with B-lineage ALL.59 However, only standard risk patients, and
therefore only the immunophenotypic subgroups of
cortical T-ALL, pre-B and c-ALL, were investigated.
Systematic and prospective analyses on differences
in MRD kinetics depending on immunophenotypes
have still not been carried out for adult ALL.
In Ph+ ALL, RT-PCR analysis of BCR-ABL fusion
transcript is a useful tool for MRD detection and is
traditionally used to monitor disease after bone marrow/stem cell transplantation (BMT/SCT). It
appears, that evidence of MRD after allogeneic SCT
in Ph+ patients is a poor prognostic sign.67,68
Wassmann et al.69 investigated the effect of imatinib
to lower the probability of relapse in this setting and
started imatinib treatment after re-conversion to
MRD-positivity after SCT within a phase II trial.
BCR-ABL transcripts became undetectable by both
quantitative and nested RT-PCR in 15/29 (52%)
patients. This was associated with a sustained remission whereas MRD persistence 6-10 weeks after start
of Imatinib treatment correlated with an almost certain relapse. In addition, Pane et al.70 demonstrated
that early quantification of residual disease before
SCT is a prognostic parameter in Ph+ ALL. Leguay et
al. presented data from the GRAAL AFR03 study71 in
which imatinib combined with high dose
chemotherapy improved molecular remission before
transplantation and led to an improved outcome.
MRD as indicator of impending relapse
Outcome in adult patients with relapsed ALL is
extremely poor, even if a second remission is
achieved.72 Molecular detection of an imminent
relapse may help to initiate salvage therapy before
hematological relapse with a lower tumour burden
and therefore possibly improve treatment results.
The power of MRD monitoring as an indicator of an
imminent relapse was prospectively evaluated in 105
MRD negative patients after consolidation treatment
within the GMALL trials.61 From a total of 28 out of
105 patients (27%) who converted to MRD positivity, 17/28 (61%) have so far relapsed with a median
time to relapse of 9.5 months after MRD conversion.
100
100
80
80
DFS (%)
DFS (%)
60
40
20 day+11
p(trend)<0.001
0
0
1
2
3
4
5
60
40
20 week+16
p<0.001
0
0
1
Years
MRD
negative
<10-2
≥10-2
3-year DFS (95% CI)
91.7 (76.1-100)
51.9 (21.4-82.4)
33.1 (18.7-47.5)
2
3
4
Years
MRD
negative/<10-4
≥10-1
3-year DFS (95%)
65.5 (53.9-77.1)
12.2 (0.0-27.4)
Figure 1. Probability of disease-free survival (DFS) according to MRD results during induction (day +11)and after start of
consolidation (week 16) in adults treated according to the GMALL 06/99 or 07/03 protocol (see for details59)
In 15 of those patients, MRD was detectable within
the quantitative range of PCR in hematological
remission. Thirtheen of these patients (89%)
relapsed after a median interval of 4.1 months. Of the
77 continuously MRD negative patients, only 5 (6%)
have relapsed. Therefore, conversion to quantifiable
MRD positivity during early postconsolidation was
highly predictive of subsequent relapse. In the ongoing GMALL 07/03 trial, salvage treatment should be
started at the time of re-occurrence of quantifiable
MRD.
olds. Precise MRD thresholds for risk-group assignment have to be defined for each treatment protocol.
Treatment blocks before the critical MRD sampling.
time-points cannot be changed because this directly
influences the prognostic value and discriminative
thresholds.73
3. preferably at least two time-points should be
chosen for MRD-based risk stratification because
confirmation of the MRD result by the second
improves accuracy, particularly when MRD levels are
around the discriminative threshold.
Requirements for an MRD based risk stratification
Treatment options for MRD-based risk-groups
In both childhood and adult ALL, MRD quantification can be used to evaluate the treatment response
and therefore allow the identification of low-risk
(LR) and high-risk (HR) patients who may benefit
from therapy reduction or therapy intensification
respectively. Several considerations are important
when MRD information from existing treatment
protocols is developed into new clinical treatment
protocols:16
1. the choice of the MRD technique, because different techniques may differ in sensitivity, optimal
sampling time-point and applicability in multicenter
trials. A switch-over between different MRD techniques should be avoided unless proof is provided
that the MRD results are fully identical. High sensitive MRD techniques (at least 10–4) are required for
accurate recognition of LR patients. However, if only
HR patients need to be identified, less sensitive but
cheaper and more rapid methodologies like
GeneScanning of Ig/TCR gene rearrangements and 3to 4-colour flow cytometry may be suitable;
2. the optimal sampling time-points and thresh-
Patients with a low risk of relapse might benefit
from reduction of treatment intensity in order to
avoid overtreatment. Overall shortening of treatment
duration or omission of intensification cycles are possible options. The decision depends on the overall
treatment intensity and on the time-point of MRDbased risk-stratification. Although earlier studies
without maintenance therapy beyond the first year
of treatment gave inferior results,74,75 this approach
might be justified in MRD LR patients. By contrast,
MRD HR patients may benefit from any kind of
treatment intensification, including SCT, intensification cycles and experimental approaches. However,
it is known that patients with high MRD levels
before SCT76,77 are at high risk of relapse, so that
attempts to minimize MRD levels before transplantation can be considered.71 For patients with an intermediate MRD course a third risk group can be
defined. These intermediate risk patients, along with
patients with suboptimal MRD assays that do not
fulfil technical requirements for MRD-based risk
assessment, should probably receive standard treat-
ment according to the experience of the individual
study.75 In these cases, MRD information at later
time-points could identify MRD re-occurrence before
clinical relapse allowing for an early salvage treatment.61
The relative sizes of MRD-based risk groups differ
from those that were obtained in childhood trials. In
pediatric patients, MRD-defined LR-groups make up
40% to 90% of patients, whereas only 5% to 15%
belong to the MRD-HR-groups.6,7,13,22 In trials on adult
ALL, the MRD-HR-group is larger and the MRD-LRgroup smaller, probably reflecting the differences in
biology of the disease.59
Based on the results of the MRD studies protocol
committees of several clinical trials on MRD in adult
ALL patients included MRD into treatment stratification. Within the ongoing GMALL 07/03 trial patients
with MRD levels consistently <10–4 after induction
treatment and undetectable MRD after end of consolidation treatment form the MRD-LR group.
Patients with persistent MRD levels >10–4 after induction (day +71) and/or during consolidation are allocated to the MRD-HR group.75,78 In MRD-LR patients,
maintenance treatment is not given after the first
year of therapy while the MRD-HR-group is eligible
for allogeneic SCT. Bassan et al.63 describe an MRDoriented therapy for all t(4;11)neg/t(9;22)neg ALL
patients. MRD positive patients (defined as MRD
>10–4 before induction-consolidation cycle 6 and
MRD positivity before cycle 8) are allocated to allogeneic or autologous SCT, whereas MRD negative
patients receive standard maintenance regardless of
classic risk factors. Four-year DFS was 76% in MRD
negative patients compared with only 24% in the
MRD positive group despite treatment intensification. The PETHEMA ALL-AR-03 trial79 focuses on
high risk t(9;22)neg ALL and does not perform SCT in
first complete remission in cases of standard cytologic response and MRD <0,05% after early consolidation. By contrast, patients with a slow cytologic
response and/or MRD >0,05% after early consolidation later receive allogeneic SCT. Preliminary results
indicate that prognosis of HR patients with adequate
response to induction and adequate clearance of
MRD does not worsen if allogeneic SCT is avoided.
Conclusions
MRD has been proven to be an independent prognostic factor in childhood and adult ALL.
Quantitative PCR and flow cytometry are the most
widely used techniques at the moment for MRD
quantification although each has advantages and disadvantages that must be carefully considered. Precise
MRD levels and optimal sampling time-points must
also be defined for each treatment protocol before
MRD-based risk stratification can be implemented.
Whether or not patient outcome can be improved by
integrating MRD into treatment decisions is currently the subject of several clinical trials.
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Acute Myeloid Leukemia
Targeting critical pathways in leukemia stem cells
S. Anand
W-I. Chan
B.T. Kvinlaug
B.J.P. Huntly
Department of Hematology,
University of Cambridge, Cambridge
Institute for Medical Research,
Hills Road, Cambridge, UK
2007;1:175-182
ecently, many cancers have been
demonstrated to be completely
dependent upon a small population
of putative cancer stem cells for their continued growth and propagation.1 The existence of cancer stem cells was first demonstrated in acute myeloid leukemia (AML),
by Dick and colleagues in Toronto.2,3
Leukemic blasts from patients with a spectrum of phenotypic subtypes of AML
(M0-M7, excluding M3, by the FAB classification) were flow sorted into an immature fraction which was CD34+ and CD38–
and a more mature fraction which was
both CD34+ and CD38+. Following xenotransplantation into immunocompromised non-obese diabetic-severe combined immunodeficiency (NOD-SCID)
mice, which are known to support the
engraftment of normal hematopoietic
cells, it was demonstrated that only the
immature (CD34+/CD38–) fraction could
transfer the leukemia. This putative SCID
leukemia initiating cell (SL-IC, equivalent
to the leukemia stem cell or LSC) population shares a surface phenotype (CD34+/
CD38–) with the stem cell population
which regenerates normal human
hematopoiesis in NOD-SCID mice, the
SCID repopulating cell (SRC). Based on
these findings, the authors proposed that
AML arranges itself as a developmental
hierarchy and that the normal hematopoietic stem cell was the likely target cell for
transformation.3 Subsequently, LSCs have
also been demonstrated in acute lymphoblastic leukemia (ALL),4 and cancer
stem cells have been demonstrated in a
number of solid organ tumours such as
breast,5 CNS tumors,6 prostate7 and colon8,9
suggesting that the majority of malignancies are dependent upon such a compartment.
Survival rates for the majority of
patients with AML have not improved
much in the last twenty years,10 and any
improvement is probably due to the risk
stratification and changes in treatment of
young patients with favourable cytogenet-
R
ics, along with overall improvements in
general supportive care.11 Two drugs, an
anthracycline and cytosine arabinoside
(Ara-C), have provided the basis of AML
therapy for this entire period,11 although
newer agents are currently under investigation.12 Therefore, most patients with
AML continue to die following relapse of
their disease and it is the LSC compartment that is responsible for both relapse
and the development of resistance to therapy that often accompanies it. This suggests that current AML therapeutic regimens, which often lead to complete morphological remissions in patients, preferentiality target bulk leukemia cells and
tend to spare the LSC compartment
(Figure 1). Leukemia stem cells therefore
represent the critical targets for eradication of leukemia. Unfortunately, little is
known of the biology of these cells.
Particularly important for any therapeutic
strategy is to recognise that the targeted
pathway should have efficacy against
LSCs but preferentially spare normal
hematopoietic stem cell (HSC) function.
Recently in vitro and in vivo assays which
can assess both leukemic and normal
hematopoietic stem cell function have
been developed (see later), allowing for
the development of such therapies.13 This
article will outline current knowledge and
suggest how leukemia stem cells might be
targeted for future therapeutic gain.
Targeting leukemia stem cells
The importance of specifically targeting
LSCs whilst relatively sparing normal
HSC function has already been emphasised. Unfortunately, our knowledge of
LSC biology and even normal HSC function is relatively limited. To specifically
target the LSC we must therefore improve
our knowledge of the basic biology of
these cells and of potential differences
between normal and leukemic stem cells.
Already, we know of differences in surface
antigen expression and current strategies
for therapeutically exploiting this are
underway (see below). The primary functions of the
HSC are to maintain both the stem cell compartment
and adequate numbers of the terminally differentiated effector cells of the blood. The control of these
functions is complex and involves the interaction
between cell autonomous programmes within the
HSC and cell non-autonomous signals from the stem
cell niche. However, the number of potential cell fate
decisions available to a stem cell are relatively few. A
stem cell may quiesce, self-renew, differentiate or
apoptose (Figure 2) and it is the delicate balance
between these interconnected processes which
allows us to dramatically respond throughout life to
stresses such as severe infection and bleeding where
increased numbers of mature effector cells are
required without depleting the stem cell reserve.
Basically, the same cell fate decisions are available to
leukemia stem cells, although the processes controlling the decisions are dysregulated (Figure 2).
Targeting these processes, based upon a differential
reliance of LSCs and normal HSCs on specific pathways, may allow for preferential eradication of the
LSC compartment. A list of such potential targets is
summarised in Table 1.
Targeting differences in surface phenotype
Although both the LSC in AML and normal HSCs
express CD34+ but not CD38–, there are differences
between the LSC and HSC surface phenotype.
Whilst the normal HSC is lin–, CD 34+/38–/90+/123– /lo
/117+/71+/HLA-DR– , the majority of AML LSC are
lin–, CD34+/38–/90–/123+/117–/71+/HLA-DR– 2,3,14,15-17
(see Figure 3, panel A). Recent therapeutic success
with monoclonal antibodies and immunoconjugate
therapy, for example in CD20+ lymphoproliferative
disorders with Rituximab (Mabthera®) and in AML
with gemtuzumab ozogamicin (GO, Myelotarg®),
have demonstrated the potential efficacy of targeting
cells based on their surface phenotype. Therefore,
the differences already noted between the AML LSC
and normal HSC make this an attractive proposition
in AML therapy. Interestingly, a recent report suggests that the majority of AML LSC actually express
CD33,18 the target of GO. Unfortunately however,
CD33 also appears to be expressed on the majority of
HSC.18 This suggests that GO may not be a specific
agent targeting AML stem cells, but it may explain
the high complete remission rate seen with this agent
and the occasional cases of prolonged cytopenia fol-
Table 1. Potential LSC targets.
Potential LSC targets
Comments
CD123 (IL-3 receptor α)
Present on most LSC but absent or only present at low levels on normal HSC.
A fusion immunoconjugate of the cognate ligand of the receptor, IL-3, to the Diptheria toxin,
DT388IL3 is currently in clinical trials.
The AP-1 transcriptional pathway
Both JunB and c-Jun are AP1 transcription factors.
48
The AP-1 complex consists of either a Jun-Jun homodimer or a Jun-Fos heterodimmer. T
his complex regulates the expression of multiple genes essential for cell proliferation, differentiation and apoptosis.
c-Jun appears to direct myelomonocytic differentiation whilst JunB is a stem cell tumour suppressor.
Decreased expression of both c-Jun and JunB in AML appears to contribute
to the leukemic phenotype and restoration of their levels may annul this phenotype.
mTOR
The mammalian target of rapamycin is a downstream signaling component of the PI3K-AKT
29,30,70
pathway and mediates cellular responses to stresses such as DNA damage and nutrient deprivation
through phosphorylation of substrates such as p70S6K and 4EBP. The PI3K-AKT pathway is constitutively
activated in AML and recent mouse studies suggest that PI3K activity may have a role in the self-renewal of LSC.
Inhibitors of mTOR, such as Rapamycin, exist and are currently in randomized trials for AML
NF-κB
Nuclear Factor kappa B is a dimeric transcription family formed by combination of 5 members which share a
related DNA binding element. NF-kB controls gene expression of a number of targets in response
to stimuli such as cytokines, growth factors, stress stimuli, and viral and bacterial infection.
It is frequently dysregulated in cancer and is constitutively activated in the AML LSC compartment
where it appears to be anti-apoptotic, but not the normal HSC compartment. NF-κB is retained in an
inactive form under basal conditions by the inhibitor of kB (I-κB) family of proteins, which sequester
the inactive dimers in the cytoplasm. Most stimuli activate NF-κB by stimulating the I-κB kinase (IKK)
family of proteins, which phosphorylate the I-κB proteins and target them for ubiquitination and
degradation in the proteasome. Therapies which target this pathway, such as Parthenolide,
proteasome inhibitors and IKK inhibitors are currently in clinical trials.
57-60
CD44
CD44 is a cell surface glycoprotein involved in cell/cell and cell/matrix interactions,
and is widely expressed on a number of tumours. CD44 is present on AML LSC and ligation
of this receptor with an activating antibody (H90) appears to direct differentiation and loss of LSC activity.
67,68
Reference
20
A
A
Current
Chemotherapy
Recurrence of disease
B
Targeted
LSC
HSC :
CD34+/38-/
CD90+/123-/Io/117+
CD 71+/HLA-DR-
HSC
LSC
Normal
Leukeamic
LSC :
CD34+/38-/
CD90-/123+/117+
CD 71+/HLA-DR-
Tumor involution
Targeted
LSC +
Conventional
therapy
Disease remission
B
Tumor debulking
Leukemia stem cell
Non-clonogenic
leukemia cell
HSC
Figure 1. Targeting the leukemia stem cell in AML therapy.
(A) Current therapies for AML usually result in a significant
decrease in leukemic burden, often a complete morphological response. However, the majority of patients
relapse. This suggests that current therapies kill the nonclonogenic leukemic cells while sparing the LSC compartment. (B) Specifically targeting the LSC compartment,
either alone or more probably in combination with conventional AML therapy, should lead to more durable remissions and improved survival in AML.
CD123/ IL-3
receptor α
Monoclonal Ab/
Immunoconjugate
i.e. IL3-Diptheria
toxin
Normal progeny
LSC
Leukemic progeny
C
HSC
Quiescence
Differentiation
Normal progeny
Niche
D
LSC
Self-renewal
Leukemic progeny
Apoptosis
Figure 2. Cell fate decisions available to a leukemia stem
cell. A leukemia stem cell and its specific niche are shown.
Similarly to a normal HSC, intrinsic cell-autonomous cues
and extrinsic interactions between the LSC and the niche
determine the fate of the LSC. In addition, there are the
same, limited numbers of cell fate decisions available to
the LSC: to quiesce, to differentiate, to self-renew or to
apoptose. Targeting differences in these processes
between the LSC and the HSC compartment may help to
eradicate leukemia stem cells (see text).
lowing GO treatment.19
CD123, the IL3α receptor is expressed on most
LSCs but not, or only at low levels, on HSCs16,18, its
expression being upregulated on later normal
myeloid progenitors. To take advantage of this, and
selectively target AML LSCs, a specific fusion of IL3
and a diphtheria toxin (DT388IL3) has been generated by Hogge, Frankel and colleagues.20 Preclinical
studies demonstrated in vitro sensitivity of AML-
HSC
Tumor
involution
Normal progeny
Figure 3. Targeting the AML LSC surface phenotype. (A)
Differences exist between the surface phenotype of the
normal HSC and the LSC (shown in red in the figure).
Where there is expression of an antigen on an LSC but not
an HSC (i.e. for CD123, the IL3α receptor), the use of a
monoclonal antibody or immunoconjugate specific to this
antigen will target the LSC but not the normal HSC compartment. Targeting this compartment will lead to selective LSC depletion or eradication (B). Even if, as is the case
with CD123, the antigen is expressed later during normal
ontogeny (where the MoAb or immunoconjugate will target
a more committed normal progenitor cell, in this instance
a myeloid progenitor cell), this will simply lead to transient
cytopenias, with the progenitor compartment eventually
regenerated by the normal HSC. However, targeting the
LSC should help to eradicate the leukemia, as decrease or
preferentially eradication of the LSC compartment will prevent recurrence of AML post-therapy (C and D).
colony forming cells (CFC), AML-long-term culture
initiating cells (LT-CIC) and AML-suspension culture
initiating cells (SC-IC) to DT388IL3, whereas normal
bone marrow showed no change in LT-CIC, minimal
change in SC-IC and some decrease in CFC, but to a
lesser degree than in AML.20 In addition, ex vivo treatment of AML cells with DT388IL3 dramatically
decreased the engraftment of disease in NOD-SCID
mice, and in vivo administration to NOD-SCID recipients of AML cell inoculums was demonstrated to be
safe, significantly decreased AML engraftment and in
50% of cases eradicated the disease completely.20
Furthermore, normal bone marrow cells treated ex
vivo with DT388IL3 showed no difference in engraftment in NOD-SCID mice compared to untreated
controls20 (Figure 3 panels B-D). Further toxicity studies have since shown an acceptable side effect profile
in Cynomolgus monkeys,21 and DT388IL3 is currently
being assessed in a phase I/II clinical trial in patients
with relapsed or refractory AML.22 In this poor prognostic grouping, preliminary data suggests efficacy in
clearing marrow blasts with acceptable toxicity and
further dose escalation is planned.22
Targeting self-renewal/quiescence in LSCs
Unlimited self-renewal potential is one of the hallmarks of cancer,23 but is also a defining characteristic
of normal stem cells. Currently, the mechanisms of
self renewal in malignant cells are poorly understood.
Pathways such as the HOX gene,24,25 WNT/βCatenin,26,27 Notch,28 PTEN,29,30 Hedgehog31 and BMI132,33 pathways have all been found to be mutated in
human cancers and these same pathways are also
implicated in the maintenance of normal stem
cells.29,30,33-38 Although their involvement in normal
stem cell self-renewal makes these pathways less
attractive candidates, the finding that they are frequently mutated or aberrantly activated suggests that
they may function differently in malignant and normal stem cells, and thus may be selectively targeted
in LSCs. In addition, β-catenin has recently been
shown to be dispensable for hematopoiesis in a
mouse model39 and two recent reports have demonstrated that constitutive Wnt signaling actually
impairs normal HSC function.40,41 Overall, these data
suggest as an example that the WNT/β-Catenin pathway, known to be constitutively activated in AML,15
may potentially be beneficially targeted in the LSC
compartment.42
Recent evidence has demonstrated that certain
leukemia-associated fusion oncogenes, such as
MOZ-TIF2,43 MLL-ENL44 and MLL-AF945 may alter
the properties of committed murine myeloid progenitors and confer self-renewal when retrovirally
expressed within this compartment. This ultimately
leads to the generation of AML from the committed
myeloid progenitor compartment in bone marrow
transplant recipient mice. Existing evidence in
humans also suggests that a progenitor compartment
may be transformed to generate leukemia.4,27 Overall,
these data challenge the previously held belief which
suggested the HSC was the only target for transformation in acute leukemias. However, the ability to
alter self-renewal of murine committed myeloid progenitors was shown to be missing in another representative and fully transforming leukemia-associated
oncogene, BCR-ABL.43 As the oncogenes MOZ-TIF2,
MLL-ENL and MLL-AF9 are fusions of transcriptionally active proteins, whereas BCR-ABL encodes a
constitutively activated tyrosine kinase, this suggests
that MOZ-TIF2, MLL-ENL and MLL-AF9 (re)-establish a transcriptional programme which leads to selfrenewal. Using this technical platform, it should be
possible to identify the transcriptional programmes
associated with this self-renewal, using expression
analysis following the expression of these oncogenes
in committed myeloid progenitors. This in turn may
identify both known and novel genes and pathways
that are commonly activated to mediate self-renewal
in AML. Furthermore, examination of the reliance of
normal HSC on these pathways may then identify
pathways differentially used by the HSC and LSC
compartments that can be therapeutically targeted. A
self-renewal signature has recently been demonstrated by Armstrong and colleagues for MLL-AF9 using
such an approach, confirming the importance of the
Hoxa cluster and suggesting newer candidate selfrenewal genes such as Mef2c.45
Another transcriptional pathway which appears to
alter self-renewal in myeloid malignancies is that
associated with the AP-1 transcription factor JunB.
JunB is a known transcriptional regulator of
myelopoiesis and also a tumour suppressor gene.46 In
further experiments, enforced expression of JunB has
been shown to cause a decrease in the size of the
stem cell compartment in mice and targeted deletion
of JunB in HSCs led to an increase in the size of the
long-term (LT) HSC and granulocyte monocyte progenitor (GMP) compartments. Furthermore, these
mice developed a myeloproliferative disorder with
some progressing to develop acute leukemia.47 In this
study, JunB was also shown to regulate LT-HSC self
renewal, and its loss was associated with an increase
in proliferation of the HSC compartment and a
decrease in apoptosis of HSCs. JunB loss has also
been associated with leukemic self-renewal in
AML.48 Using a murine model of AML induced by a
graded reduction of the PU.1 myeloid regulatory
transcription factor, Rosenbauer, Tenen and colleagues demonstrated that JunB and the related factor c-Jun were critical target genes, in turn downregulated by the reduction in PU.1. In addition, the
association between low PU.1 and low JunB expression was confirmed from expression analysis of
human AML samples, and was particularly marked
when expression within the LSC compartment
(CD34+/CD38–) was analysed. Lastly, restoration of
JunB expression in murine leukemic PU.1 knockdown cells inhibited their clonogenic efficiency in
vitro and their ability to transfer leukemia in vivo.48
Overall, these data demonstrate that modulation of
the JunB transcriptional pathway may reduce selfrenewal in leukemia stem cells, and suggest this
pathway as a therapeutic target.
Quiescence and cell cycle entry are very tightly
controlled in normal HSC to help self-renewal and
prevent depletion of the stem cell pool. The PI3KAKT-FoxO pathway, mediated through effectors
such as p21, seems to be central to this cell cycle control.29,30,49,50 Two recent reports demonstrate that constitutive activation of the PI3K pathway through targeted deletion of PTEN, the major lipid phosphatase
attenuating PI3K signaling, results both in HSC
depletion and in the development of acute leukemia
in mice.29,30 Following deletion of PTEN, HSC numbers were seen to briefly rise, with increased entry
into cell cycle. However, this was followed by a
marked reduction in HSC number over time. This
quantitative decrease was also accompanied by qualitative defects in stem cell and hematopoietic reconstitution, as confirmed by competitive and non-competitive bone marrow transplant assays. The mice
also demonstrated an initial myeloproliferative phase
that was quickly followed by acute leukemia of both
lymphoid and myeloid phenotype, that was transplantable to secondary recipients.29,30 Deregulation of
the mTOR pathway downstream of AKT is a prominent consequence of PTEN inactivation. Inhibition of
this pathway is possible and the authors used the
inhibitor, rapamycin, to examine the effects on both
leukemogenesis and normal HSC self-renewal. Not
only did rapamycin restore normal cell cycle characteristics, normal number and reconstitution function
to PTEN-/- HSC, its administration to PTEN-/- mice prevented the development of leukemia. In adoptive
transfer experiments, rapamycin also depleted the
number of leukemia-initiating cells and, importantly,
rapamycin was shown to prolong survival even
when administered to mice with established
leukemia. This important study therefore demonstrates in a mouse model that drugs may target selfrenewal/quiescence in leukemia stem cells without
eliminating normal bystander HSC. Furthermore, it
identifies genes which promote stem cell quiescence
as amenable targets, because although normal stem
cells require quiescence as a defense mechanism,
adopting quiescence may be detrimental to LSCs.
The PI3K pathway is one of the major signaling
pathways downstream of oncogenic tyrosine kinases, and as such, is constitutively activated in human
AML.51 mTOR inhibitors such as rapamyin (sirolimus) and its derivatives (temsirolimus, CCI-779,
everolimus, RAD001 and AP23573) have already
been demonstrated to have efficacy against AML cell
lines and patient samples.52,53 These studies not only
showed a decreased survival of AML blasts, due partly to induction of apoptosis,52 but also demonstrated
a loss of clonogenic potential of AML blasts whilst
sparing normal hematopoietic progenitors.53 This
supports the suggestion made above in mice that
rapamycin targets LSC self-renewal whilst sparing
self-renewal of normal HSC. mTOR inhibitors have
also been shown to synergise with Ara-C and
Etoposide, with the latter combination dramatically
decreasing engraftment of AML in NOD-SCID
mice.52,54 Furthermore, rapamycin induced clinically
significant responses in 4 out of 9 patients with poor
risk AML.49 Overall, these data suggest that mTOR
inhibitors may target both self-renewal and survival
mechanisms in AML. Further phase I and II studies of
mTOR inhibitors, alone and in combination with
standard chemotherapy regimens, are currently
underway to test these hypotheses.55,56
Targeting apoptosis in LSCs
The NF-κB pathway has been shown to be activated in cancer, where it mediates growth and proliferation signals, evasion of apoptosis and tumor invasion and metastasis. Furthermore, inhibition of NFκB induces apoptosis in several malignant cell
types.57 NF-κB was first demonstrated by Jordan and
colleagues to be constitutively active in primary
AML CD34+ cells, but not normal CD34+ cells. They
further demonstrated that the proteosome inhibitor
MG-132 could downregulate NF-κB activity in primary AML cells and led to apoptosis in CD34+ AML
cells but not in normal CD34+ cells.58 This work was
subsequently extended to demonstrate that pretreatment of AML cells with MG-132 and the
anthracycline idarubicin selectively prevented the
engraftment of AML in NOD/SCID mice, but did
not affect the ability of normal marrow to engraft.59
Use of a dominant negative inhibitor of NF-κB (IκB)
also downregulated NF-κB activity and induced a
p53-regulated apoptotic response in primary AML
cells, confirming the importance of the NF-κB pathway in this response.
The NF-κB pathway has also more recently been
targeted using parthenolide. Parthenolide (PTL) is a
sesquiterpine lactone and is the major active component in feverfew. This is herbal medicine which has
been used to treat headache and rheumatoid arthritis for centuries and has recently been demonstrated
to have antitumour activity.60 Jordan and colleagues
have demonstrated that PTL preferentially targets
the LSC compartment, both in vitro and in vivo, while
sparing the normal HSC compartment. This selection process was not demonstrated when AML and
normal CD34+ cells were treated with Ara-C. PTL
was also demonstrated to inhibit NF-κB, and the
level of inhibition of NF-κB and activation of p53
correlated with the induction of apoptosis.60 PTL
also increased the levels of reactive oxygen species
(ROS) in AML cells and the induction of apoptosis
also seemed to be dependent upon the oxidative
changes, as pretreatment of AML cells with the
antioxidant N-acetylcysteine (NAC) completely
abolished the effects of PTL. These data suggest that
PTL acts selectively through a number of pathways,
through inhibition of NF-κB, but also through activation of p53 and the induction of ROS. Importantly,
this study also suggests that AML LSC may be more
sensitive to alterations in their oxidative environment than normal HSC, presenting another potential
pathway for therapeutic targeting. Unfortunately,
the solubility of PTL prevents its clinical use but
chemical modification to improve this are underway. Other inhibitors of IκB kinase (which phosphorylates and targets IκB for destruction, and whose
inhibition would in turn decrease NF-κB activity) are
also entering clinical trials.61
Directing differentiation in LSC
Another important characteristic of cancer is the
failure of normal differentiation. A differentiation
block of varying degrees is a main feature of AML.
The paradigm of differentiation therapy has been
demonstrated in acute promyelocytic leukemia
(APML), associated with certain rearrangements of
the retinoic acid receptor alpha (RARA), where pharmacological doses of all-trans retinoic acid (ATRA)
cancel the differentiation block and induces myeloid
differentiation. It is not known if this specifically targets the LSC or a later leukemic progenitor.
However, it is known that ATRA treatment needs to
be combined with standard chemotherapy or
arsenic trioxide to maximise the likelihood of cure.
This suggests that at least some LSCs fail to differentiate when exposed to ATRA.
The differentiation block seen in AML probably
reflects repression of gene programmes associated
with differentiation, either through loss of function
mutations to myeloid master-regulators such as PU.1
and C/EBPα, or through repression of these masterregulators by leukemia-associated fusion oncogenes
such as AML1-ETO and PML-RARA.62,63 These
fusion oncogenes are known to involve corepressors
and histone deacetylases in an inhibitory complex
leading to gene silencing. This suggests that histone
deacetylation inhibitors (HDACi) may cancel these
properties, restoring gene expression. Some degree
of success has been shown in the treatment of cutaneous T-cell lymphomas, however, there are concerns over the specificity of these agents64 and clinical trials continue. A future alternative might be to
re-establish the PU.1 and C/EBPα programmes
through direct peptidomimetics or peptidomimetics
of downstream effectors such as c-Jun.48 A more likely alternative would be through small molecular
inhibition of, or systemic siRNA delivery against
leukemia-associated fusion proteins. However this is
not yet technically possible, but advances suggest
that in the future this may be an option for certain
transcription factors.65,66
Differentiation is a complex process driven by specific transcriptional programmes started through cell
intrinsic cues in stem and progenitor cells and also
via external stimuli from the stem cell niche. A mediator of this stem cell/niche interaction, the CD44
surface antigen, is another potential target for differentiation of LSCs. CD44 is a glycoprotein that functions as a cell adhesion molecule through interaction
with matrix ligands such as hyaluron. Ligation of
CD44 in vitro was shown to restore differentiation to
AML primary cells from subtypes M1-M567 and a
recent report by Dick and colleagues demonstrates
that targeting CD44 with an activating monoclonal
antibody (H90) led to eradication of human AML
LSCs in an in vivo NOD/SCID transplantation
assay.68 This effect appeared to be due to a combination of induction of differentiation, with concomitant loss of SL-IC (LSC) activity, and a decreased SLIC homing efficiency to the stem cell niche. These
effects were considerably more apparent for the LSC
compartment than the normal HSC compartment.
These findings are also confirmed by a simultaneous
report of the requirement for CD44 in a mouse
model of CML. This demonstrated that the BCRABL+ LSC compartment was dependent upon CD44
to a much greater degree for homing and engraftment than the normal HSC compartment.69 Overall,
these data suggest that in vivo disruption of the
CD44-mediated LSC/niche interaction may induce
commitment at the expense of self-renewal.
Another important conclusion is that the leukemic
process does not totally annul niche requirements of
the LSC. This could lead to a completely new therapeutic approach, that of targeting the LSC/niche
interaction. Further studies defining the biology of
this interaction and how it differs from normal
HSC/niche requirements may also lead to future
therapeutic benefit.
Conclusions
There is much room for improvement in the current treatment of acute myeloid leukemia. Recent
studies have demonstrated the importance of
leukemia stem cells in treatment failure associated
with AML and have looked at the biology of these
rare and elusive cells. Importantly, these new studies
suggest that there are some identifiable differences
between normal HSCs and LSCs. These could be
used in therapy. These studies also suggest that some
existing therapeutic agents may already have selective activity against the LSC compartment. In particular, it will be important in future studies to understand more about the biology of LSCs and better
define the differences from normal HSC biology.
This should identify potential molecular targets for
prospective preclinical and clinical evaluation. The
existence and critical importance of LSC also suggests that we need to assess newer therapeutics in a
different way. Until now, these response assays have
mainly measured proliferation as an end point and
may therefore have missed activities against the LSC
compartment. As detailed above, in vitro and in vivo
assays do exist to measure LSC activity. Using these
assays will identify agents to test against the LSC
compartment in vivo in clinical trials. The availability
of such agents, probably used in combination with
current AML therapy to target the total tumour load,
should make an important impact on the outlook and
survival of patients with AML.
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Clinical use of molecular markers in adult
K. Mrózek
P. Paschka
G. Marcucci
S.P. Whitman
C.D. Bloomfield
Division of Hematology and
Oncology, Department of Internal
Medicine, Comprehensive
Cancer Center, The Ohio State
University, Columbus, Ohio, USA
2007;1:183-192
A
B
S
T
R
A
C
T
It is generally accepted that specimens from all newly diagnosed patients with acute
myeloid leukemia (AML) should be subjected to cytogenetic analysis. The results are
then used to determine prognosis and often therapeutic approaches. Increasingly,
testing for submicroscopic molecular genetic alterations is also performed. Several
gene mutations and changes in gene expression have been shown to represent prognostic factors and/or potential targets for therapy in patients categorized in specific
cytogenetic subsets. Until now, these molecular genetic alterations have been most
important to determine prognosis of patients with core-binding AML, i.e., those having either t(8;21)(q22q;22) or inv(16)(p13q22)/t(16;16)(p13;q22), and of patients with
a normal karyotype, the single largest cytogenetic group with approximately 45% of
adults with AML. Gene-expression profiling has also been shown to be a useful tool
for the classification and, to some extent, prognosis of AML. In this article we provide
a brief overview of the most important molecular genetic alterations with established
or potential clinical significance in adult AML.
ytogenetic and molecular genetic
studies have revealed that acute
myeloid leukemia (AML) is a
genetically heterogeneous disease.1-3 More
than 200 structural and numerical aberrations have been recognized as recurring in
AML.1 Cytogenetic findings at diagnosis
have been among the most important
independent prognostic factors for complete remission (CR), relapse risk and
overall survival.4-10 However, there is a
continuous increase in the amount of data
on the prognostic role of molecular genetic alterations within cytogenetically
defined groups of AML patients. In this
article, we will briefly summarize recent
publications about those molecular genetic alterations that contribute to prognosis
of AML patients with specific cytogenetic
findings such as t(8;21)(q22q;22) and
inv(16) (p13q22)/t(16;16)(p13;q22), chromosome aberrations characteristic of corebinding factor (CBF) AML,11 and patients
with a normal karyotype (Table 1).
Together these cytogenetic groups
account for about 60% of adults with
AML under the age of 60.
C
Mutations of KIT as molecular genetic
prognostic factors in core-binding factor (CBF)
AML with t(8;21) and inv(16)/t(16;16)
Among adults with de novo AML, t(8;21)
and inv(16)/t(16;16) are found in 7% and
8% of patients respectively.7 At the molecular level, both chromosome aberrations
lead to rearrangements involving genes
encoding different subunits of CBF,12-14
This is a transcription factor involved in
the regulation of normal hematopoiesis.15
In t(8;21), the RUNX1 (AML1) gene encoding subunit α of CBF is fused to the
RUNX1T1(ETO) gene,12 and in inv(16)/
t(16;16) the CBFB gene, encoding subunit
β of CBF, is fused to the MYH11 gene.13
Protein products of the CBF fusion genes
act as dominant negative inhibitors of normal hematopoiesis and contribute to
leukemogenesis.15-17
The introduction of higher doses of
cytarabine (HiDAC) as consolidation
therapy has considerably improved the
outcome for AML patients with t(8;21)
and inv(16)/t(16;16).18 It appears that
HiDAC is more effective in the setting of
repetitive cycles than in one cycle.19,20
Table 1. Clinically relevant genes mutated and/or overexpressed in CBF AML and CN AML.
Gene symbol
Gene name
Chromosome
location
KIT
v-kit Hardy-Zuckerman 4 feline sarcoma
viral oncogene homolog
FLT3
fms-related tyrosine kinase 3
13q12
MLL
myeloid/lymphoid or mixed-lineage leukemia
(trithorax homolog, Drosophila)
11q23
CEBPA
CCAAT/enhancer binding protein
(C/EBP), alpha
NPM1
nucleophosmin
(nucleolar phosphoprotein B23, numatrin)
5q35
WT1
Wilms tumor 1
11p13
BAALC
brain and acute leukemia gene, cytoplasmic
8q22.3
ERG
v-ets erythroblastosis virus E26
oncogene like (avian)
21q22.3
MN1
meningioma (disrupted in balanced
translocation) 1
4q11-q12
19q13.1
22q11
Because of the involvement of subunits of CBF at
the molecular level and favorable response to treatment, patients with t(8;21) are often combined with
those with inv(16)/t(16;16) into one favorable-risk
prognostic category of AML. However, despite similarities, patients with t(8;21) differ from those with
inv(16)/t(16;16) in many pretreatment features and
clinical outcome.21-23 The relatively high CR rates of
85% to 89% are similar in both cytogenetic
groups.21-23 But lower CR probability was associated
with hepatomegaly only in inv(16)/t(16;16) patients,
while higher BM blasts and, unexpectedly, nonwhite race were associated only in t(8;21) patients.22
There was no significant difference in either
relapse risk or overall survival (OS) did not differ significantly between t(8;21) and inv(16)/t(16;16) groups
in univariable analyses.21-23 However, the OS of
patients with t(8;21) was significantly shorter than
the OS of those with inv(16)/t(16;16) after adjusting
for age, log[WBC], and log[platelets].22 This seems to
be related to a different response to salvage treatment. Patients with t(8;21) have had a significantly
shorter survival after relapse than inv(16)/t(16;16)
patients in three large, independent studies.21-23
Also, two studies have shown that the presence of
a secondary +22 was a favorable prognostic factor for
relapse risk in inv(16)/t(16;16) patients.21,22 By contrast, a possible interaction between secondary chromosome aberrations and race was observed in t(8;21)
patients in one study.22 Nonwhite patients with secondary aberrations other than del(9q) had shorter OS
than patients with t(8;21) as a single abnormality or
those with a secondary del(9q). By contrast, OS of
white patients with t(8;21) was not influenced by
secondary aberrations.22 Since approximately 50% of
all CBF AML patients are still not cured with contemporary chemotherapy,22 it is important to identify
patients who are likely to fail current standard therapy early, if possible at diagnosis. They can then be
treated with novel investigational or more aggressive
therapies, e.g., stem cell transplantation (SCT).
Recent studies have shown that mutations in the
KIT gene, which encodes a member of the type III
receptor tyrosine kinase (RTK) family,24 may be the
first molecular prognostic marker in CBF AML predicting a less favorable outcome (Table 2).
Importantly, the abnormal KIT protein encoded by
the mutated KIT gene represents a potential therapeutic target. KIT mutations have been reported in
20-45% of CBF AML cases,25-27 although some studies
report a lower frequency.28-29 This variable incidence
of KIT mutations can be partly explained by different
sizes of patient cohorts studied, a variety of techniques used to detect mutations, and differences in
regions of the KIT gene covered by mutational analyses. The existence of potential geographic and ethnic
variation in the frequency of KIT mutations still hasn’t been evaluated. In CBF AML, KIT mutations
mainly cluster within the activation loop in the
kinase domain encoded by sequences of exon 17 and
in exon 8 that encodes an extracellular part of the
receptor. But mutations in other regions have also
been reported.26,30,31
The adverse prognostic significance of KIT mutations in patients with t(8;21) at diagnosis has been
well documented. Different KIT mutations analyzed as one group and mutations in exon 17 have
both been associated with inferior OS,26,28,29 eventfree survival (EFS),28,29 relapse incidence,26 relapsefree survival (RFS),29 and cumulative incidence of
relapse (CIR).27
The prognostic impact of KIT mutations in AML
with inv(16)/t(16;16) is less clear. In one study, KIT
mutations in exon 8 increased the relapse rate, but
not OS,25 but had no prognostic significance in two
other smaller series.26,29 The most recent, study was
performed on a relatively large group of inv(16)
patients, all of whom were similarly treated on
Cancer and Leukemia Group B protocols including
higher cytarabine doses for consolidation.27 This
study showed that the presence of any KIT mutation
(involving both exon 8 and exon 17) led to a higher
CIR. Notably, the difference in CIR was primarily
caused by KIT mutations in exon 17.27 Patients with
exon 17 mutations had more than 6 times a higher
risk of relapse than those without KIT mutations. In
multivariable analyses, KIT mutations, both those in
exon 8 and exon 17, impacted negatively on OS after
adjusting for sex.27
Table 2. Molecular genetic alterations affecting clinical outcome of AML patients with core binding AML and cytogenetically normal AML.
Cytogenetic group
Molecular genetic
alteration
Frequency
%
Prognostic significance
t(8;21)(q22;q22)
Mutations of
KIT
12-47
Patients with KIT mutations, especially those with mutations
in exon 17 that encodes the activation loop in the kinase
domain of KIT, have lower OS, EFS, RFS, relapse incidence
and CIR compared with patients with wild-type KIT.
inv(16)(p13q22)/
t(16;16)(p13;q22)
Mutations of
KIT
22-47
Patients with KIT mutations in exon 8 had worse RR than
patients with wild-type KIT in one study.
25
Patients with KIT mutations in exon 17 had higher CIR, and OS
after adjusting for sex in one study.
27
Normal karyotype
Normal karyotype
FLT3-ITD
MLL-PTD
28-33
5-11
References
26-29
Two relatively small studies did not detect any prognostic
impact of KIT mutations.
26,29
Patients with FLT3-ITD have significantly shorter CRD, DFS and OS
than patients without FLT3-ITD.
52, 53,
57, 60
FLT3-ITD-positive patients with either no expression of a FLT3
wild-type allele or a high FLT3 mutant to FLT3 wild-type allele ratio
have particularly poor prognosis.
51, 56
Patients with MLL-PTD have a significantly shorter
remission duration than patients without MLL-PTD.
No difference in DFS and OS between patients with and without
80-82
MLL-PTD undERGoing intensive treatment that included
autologous SCT in one recent study.
84
Normal karyotype
Mutations of
CEBPA
15-19
Patients with CEBPA mutations have CRD and OS significantly
longer than patients with the wild-type CEBPA gene.
60, 61,
87
Normal karyotype
Mutations of
NPM1
45-64
Patients with NPM1 mutations and no FLT3-ITD have significantly
better CR rates, EFS, RFS, DFS, and OS than patients without
NPM1 mutations and FLT3-ITD.
NPM1 mutations do not seem to significantly affect poor prognosis
of patients with FLT3-ITD.
55, 64,
92
Normal karyotype
Mutations of
WT1
10
Patients with WT1 mutations and FLT3-ITD fail to achieve a
CR with standard induction chemotherapy
Normal karyotype
Overexpression of
BAALC
NA
Patients with high expression of the BAALC gene have
significantly worse CR rates and shorter DFS, EFS and OS
than patients with low expression of the BAALC gene.
Normal karyotype
Overexpression of
ERG
NA
Patients with high expression of the ERG gene in blood have
significantly shorter OS and higher CIR than patients with low
expression of the ERG gene.
100,
101
Normal karyotype
Overexpression of
MN1
NA
Patients with high expression of the MN1 gene have significantly
shorter OS and RFS and higher RR than patients with low
expression of the MN1 gene.
105
94
60, 62,
97
OS, overall survival; EFS, event-free survival; RFS, relapse-free survival; CIR, cumulative incidence of relapse; RR, risk of relapse; CRD, complete remission duration;
DFS, disease-free survival; SCT, stem cell transplantation; CR, complete remission; NA, not applicable.
When activated by binding of its ligand, KIT RTK
manages biologic processes like cell proliferation, differentiation, and survival. Importantly, KIT mutations lead to a constitutive activation of the receptor.
This makes the abnormal KIT protein a potential target for TK inhibitors. However, it is essential to
determine the exact type of KIT mutation in each
patient because of the differential sensitivity to TK
inhibitors. The first generation TK inhibitor imatinib
shows in vitro activity against the variants of exon 8
mutations tested so far32-35 and against exon 17 mutations involving codon N822,31,36 but not against
mutants involving codon D816.37 The latter mutations may be successfully targeted with other compounds, such as dasatinib,38 tandutinib (MLN518)39
and midostaurin (PKC412).28,35,40 In the future, testing
for the presence of KIT mutations might guide therapeutic decisions at diagnosis,. Future clinical trials are
necessary to investigate the usefulness of TK
inhibitors as part of therapy administered to patients
with CBF AML.
Molecular markers in cytogenetically normal AML (CN-AML)
Around 45% of adults with AML are cytogenetically normal (CN) at presentation.5-8 Their outcome is
varied, with usually 20-40% of patients being longterm survivors.1,5-8,41 Consequently, considerable
research is on-going to identify molecular markers
that predict outcome and can serve as therapeutic
targets. Using molecular genetic techniques, such as
RT-PCR, global gene-expression profiling and/or
direct sequencing, recurring molecular alterations of
prognostic significance are increasingly being identified in CN-AML.42-44 These include gene mutations
and overexpression of single genes (Tables 1 and 2).
Recently, global gene-expression profiling has been
undertaken to identify gene expression signatures
associated with important molecular markers in CNAML patients.45-47
Mutations of the FLT3 gene
The FLT3 gene encodes a membrane-bound protein of the class III RTK family. It is involved in regulation of proliferation, differentiation and apoptosis
of hematopoietic cell progenitors.48 Internal tandem
duplications (ITDs) of the FLT3 gene occur within the
juxtamembrane domain (exons 14 and 15). The
duplications can vary in length from 3 to over 400
nucleotides but always create an in-frame transcript.
This is translated into a constitutively activated protein which, ligand-independent, promotes the aberrant proliferation and survival of leukemic blasts.49
FLT3-ITDs are detected in 28-33% of CN-AML
patients.50-57 Further 5-14% of CN-AML patients carry
missense mutations in exon 20 of FLT3, i.e., within
the activation loop of the tyrosine kinase domain
(FLT3-TKD).52,55-57 FLT3-TKDs also promote constitutive phosphorylation of the FLT3 protein and disruption of normal hematopoiesis. Point mutations in the
juxtamembrane domain have also been reported,
although not frequently.58,59
Multiple studies have demonstrated the impact of
FLT3 mutations on the clinical outcome of CN-AML
patients. FLT3-ITD has been found to be an independent prognostic factor for complete remission
duration (CRD),60,61 CIR,62 and OS.60,62 This is particularly true for FLT3-ITD-positive patients whose
blasts do not express the FLT3 wild-type (WT)
allele,51 or have a FLT3 mutant/FLT3-WT allele ratio
higher than the median value.56 The prognostic significance of the FLT3-TKD in the absence of the FLT3ITD remains controversial,52,63 as does the high-level
overexpression of FLT3-WT.
Optimum treatment for CN-AML patients with
FLT3-ITD mutations is unclear. Both allogeneic SCT64
and autologous peripheral blood SCT in first CR65-67
have been reported to overcome the adverse prognostic effect of FLT3-ITD. However, other groups
have found that the outcome of FLT3-ITD-positive
patients is still worse than that of patients without
FLT3-ITD, even in the setting of SCT.51,68
The constitutively activated FLT3 protein is an
attractive therapeutic target for small-molecule TK
inhibitors (e.g., midostaurin, lestaurtinib or tandutinib). As single agents these compounds have shown
limited benefit in relapsed or refractory patients.69-73
However, inhibition of FLT3 autophosphorylation
has been shown in responding patients.71-73 This has
led to the current evaluation of these TK inhibitors in
combination with chemotherapy in newly diagnosed
patients. Other approaches currently in pre-clinical
studies include FLT3 antibody therapy,74 which is predicted to also target overexpressed FLT3-WT, and
inhibitors of downregulatory pathways such as 17allylamino-17-demethoxygeldanamycin (17-AAG),
an inhibitor of the molecular chaperone heat shock
protein 90.75-77
Mutations of the MLL gene
The MLL gene is a homeotic regulator that encodes
a nearly 430-kilodalton protein with histone lysine 4
methyltransferase activity. This protein regulates
HOX gene expression during hematopoietic stem cell
development.78 ALU-mediated recombination within
the MLL gene generates a partial tandem duplication
(PTD) spanning exons 5 through 11 or, less often,
exons 5 through 12.79,80 MLL-PTD occurs in about 8%
of adult de novo CN-AML,80-82 and it was the first
molecular defect identified as an adverse prognostic
factor in CN-AML.80,83 It has usually been reported to
adversely impact CRD, but not OS.80-82 Recent data
indicate improved outcome in younger adults treated
with autologous SCT in first CR.84
The MLL-WT transcript is not expressed in AML
blasts with the MLL-PTD.85 Transcriptional reactivation of the MLL-WT allele occurs in response to DNA
methyltransferase (DNMT) and/or histone deacetylase (HDAC) inhibitors and is associated with
enhanced sensitivity to cell death. Therefore, pharmacologic reversal of MLL-WT silencing by
demethylating agents and histone deacetylase
inhibitors should be investigated in CN-AML
patients with the MLL-PTD.85
Mutations of the CEBPA gene
The CEBPA gene encodes the C/EBPα protein, a
member of the family of basic region leucine zipper
(bZIP) transcription regulators involved in granulopoiesis.86 CEBPA mutations occur in 15-19% of CNAML.60,61,87 They confer significantly longer CRD and
OS.60,61
Mutations of the NPM1 gene
NPM1 encodes nucleophosmin, a nucleus-cytoplasm shuttling protein, involed in preventing nucleolar protein aggregation, regulation of ribosomal protein assembly and their nucleocytoplasmic transport,
the initiation of centrosome duplication and the regulation of the p53 and Arf tumor-suppressor pathways.88 Its exact role in oncogenesis is controversial.
Nucleophosmin is most prominent in the nucleus,
but in patients with mutated NPM1, nucleophosmin
shows cytoplasmic expression that may interfere
with its normal functions.89
NPM1 mutations are found in 45-64% of CN-AML
patients,55,64,90-92 and usually predict outcome only in
the context of other markers. Coexistence of NPM1
mutations with MLL-PTD and CEBPA mutations is
rare, but about 40% of NPM1 mutated patients are
also FLT3-ITD-positive. The poor outcome of
patients with FLT3-ITD is relatively unaffected by
the presence or absence of NPM1 mutations.
However, among patients without FLT3-ITD, those
with NPM1 mutations have a significantly better
response to induction therapy, disease-free survival
(DFS), RFS, EFS and OS.55,64,92
Mutations of the WT1 gene
WT1 encodes a zinc finger DNA-binding protein
that continually shuttles between the nucleus and
cytoplasm.93 Depending on cellular context, it can
also be involved in transcriptional activation or
repression. Its role in hematopoiesis and leukemogenesis is not well established, although it has been
suggested that impairment of WT1 protein function
could promote stem cell proliferation and induce a
block in differentiation.94 In a recent study of CNAML, WT1 mutations were found in 7 out of 70
patients, and in 6 of them it coexisted with FLT3ITD. None of the 5 patients with WT1 mutations and
FLT3-ITD treated with curative intent achieved a CR
with standard induction chemotherapy.94 This agrees
with results of an earlier study which was not
restricted to CN-AML.95
Overexpression of the BAALC gene
The BAALC gene encodes a protein with no
homology to known proteins or functional domains.
BAALC expression is mostly detected in hematopoietic precursors and neuroectoderm-derived tissues.
High expression has been found in AML, acute lymphoblastic leukemia (ALL) and chronic myelogenous
leukemia (CML) in blast crisis, but not in chronicphase CML or chronic lymphocytic leukemia (CLL).96
High expression of BAALC mRNA in CN-AML
predicts adverse clinical outcome, including primary
resistant disease, shorter DFS, OS, EFS and higher
CIR.60,62,97 BAALC expression seems to be particularly
useful in predicting outcome in CN-AML patients
without FLT3-ITD and CEBPA mutations.60 It has
been suggested that patients with high BAALC
expression might benefit from allogeneic SCT.62
Overexpression of the ERG gene
ERG is one of over 30 members of the ETS gene
family. Most of these are down-stream nuclear targets of signal transduction pathways regulating and
promoting cell differentiation, proliferation and tissue invasion.98,99 In CN-AML high ERG expression
adversely impacts on CIR and EFS.100,101 For OS, an
interaction between expression of ERG and BAALC
has been observed. The adverse impact of high ERG
expression on OS was only observed in patients with
low BAALC expression.100,101
Overexpression of the MN1 gene
The MN1 gene is a transcriptional co-activator,102
which was initially found rearranged in patients with
meningioma with t(4;22)(p16;q11)103 and AML with
t(12;22)(p13;q11~12).104 Mn1 null mice die shortly
after birth as the result of a secondary cleft palate,
suggesting that this gene plays an important role in
normal bone development.102 Recently, overexpression of MN1 was found to be an independent unfavorable prognostic factor for OS and RFS in CNAML.105 These results await corroboration. Further
evidence of these results must still be provided.
Interrelation of molecular genetic markers
It has been suggested that at least two somatic
mutations with different consequences cooperate
with each other to cause AML, since one mutation
alone is not enough to transform a normal cell into a
leukemic blast. Class I mutations are those in the sig-
nal transduction pathways (e.g., FLT3-ITD, FLT3TKD) that provide a proliferation stimulus, and class
II mutations occur in genes encoding hematopoietic
transcription factors (e.g., CEBPA or RUNX1) that
impair cell differentiation.106,107
Because different mutations and changes in gene
expression can occur in the same AML patient, it is
important to evaluate the prognostic impact of the
interaction between molecular alterations. For
instance, NPM1 mutations predict better outcome
mainly in the absence of FLT3-ITD,55,64,92 and the level
of ERG expression identifies subsets of patients with
differing prognoses within the subset of patients
with NPM1 mutations but not FLT3-ITD.101 Clearly,
prospective investigation of prognostic interactions
among many genetic lesions is needed to design a
clinically relevant prognostic classification of CNAML.
Gene-expression profiling in AML
Gene-expression profiling (GEP) has been shown
to be a useful tool for the classification of leukemias.
Golub et al.108 were the first to show that AML and
ALL could be distinguished on the basis of characteristic gene-expression signatures. More recently,
Haferlach et al.109 reported the ability of GEP to correctly identify cytogenetic subsets of AML with
t(15;17), t(8;21), and inv(16), CLL, and pro-B-cell ALL
(pro-B-ALL) with translocations involving 11q23
with 100% specificity and 100% sensitivity. A similar specificity (i.e. 99.7%) with a lower degree of sensitivity was achieved for the diagnosis of CN-AML,
AML with translocations involving 11q23, AML with
complex karyotype, pre-B- and T-ALL and CML.109
Within adult AML, distinct gene-expression patterns have been shown to be associated with specific cytogenetic and molecular alterations.45,46,109 Using
unsupervised hierarchical analysis, two groups
reported clustering to be driven by the presence of
specific karyotypes (i.e., t(15;17), t(8;21), inv(16), normal cytogenetics)45,46 and genetic mutations (i.e.
CEBPA) or abnormal oncogene expression (i.e.
EVI1).46 Both unsupervised and supervised approaches were able to identify specific gene signatures associated with the aforementioned karyotypes and/or
molecular aberrations (including also del(7q)/-7 and
FLT3 mutations). But it is of interest that no distinct
gene-expression patterns were found to identify
patients with other molecular or cytogenetic
rearrangements (e.g. MLL-PTD or trisomy 8) from
those without these aberrations.45
Within specific cytogenetic categories, GEP has
also helped identify novel biologic and prognostic
subgroups. In the study by Bullinger et al.,45 CN-AML
patients predominantly clustered into two distinct
subclasses. The presence or absence of FLT3 muta-
tions and the FAB morphologic subtypes (M1/M2
versus M4/M5) were different between the two.
Patients in these subclasses had a significantly different OS. Prognostic significance of these clusters has
recently been confirmed by Radmacher et al.47 in an
independent set of patients, using a different
microarray platform. Cluster analysis confirmed the
prognostic impact of the Bullinger gene-expression
signature for OS and DFS. Also, Radmacher et al.47
developed a class prediction algorithm that identified
a signature-based classifier for outcome prediction.
Subgroups of patients with significantly different OS
and DFS were identified by this outcome classifier
which seemed strongly associated with the FLT3ITD. However, the classifier for dichotomized outcome classes had only modest predictive accuracy,
with OS and DFS of about 60% of patients being
accurately predicted. Furthermore, although the classifier showed some ability to identify a subset of
patients with poor outcome among patients without
FLT3-ITD,47 other classifiers which can more precisely predict outcome of CN-AML patients are needed.
Although in a recent study of pediatric patients,110
commonalities between t(8;21) and inv(16) geneexpression signatures were found, GEP has been
repeatedly shown to accurately identify these two
cytogenetic subgroups in adult patients.45,46 Within
each cytogenetic group of adult patients distinct
molecular subgroups were identified45,46 whose biologic and prognostic significance is under evaluation.
Similarly, although distinct gene-expression signatures could identify patients with FLT3 or NPM1
mutations from those carrying the corresponding
WT alleles,45,111 these patients appear to segregate in
several clusters. This perhaps reflects the presence of
these genomic abnormalities in different cytogenetic
groups and/or the overlapping of these with other
molecular markers in AML.45,46,112
Gene-expression profiling seems to be useful in
AML classification. But this approach also has limitations. Several studies have shown that the differentiation stage of the lineage, reflected by the FAB classification, might direct the unsupervised clustering,
which may prevent clear analyses.113 Furthermore,
inconsistencies between data obtained using different microarray platforms have been detected.113,114
Finally, at least in CN-AML, only a moderate predictive accuracy for outcome prediction has been reported.47 However, by taking a general view of the molecular heterogeneity of AML, gene-expression profiling
may help to examine pathogenetic mechanisms and
therefore provide new understanding of tumor biology and identify novel therapeutic targets. Geneexpression profiling by itself will probably nor be
enough to show the whole pathobiologic nature of
AML. The integration with other genomic technolo-
gies, such as high-throughput mutational analyses
and proteomic approaches, will be necessary to take
on this important challenge.113
11.
Conclusions
Several molecular markers with prognostic significance within particular cytogenetic groups of AML
have been and continue to be identified. These
molecular markers will probably guide future therapies both as prognostic factors and targets for specific therapeutic intervention. It is, however, important
to understand complex interactions among various
mutations and changes in gene expression. Studies
investigating all known prognostic molecular alterations concurrently to determine their relative impact
on patients’ prognosis are ongoing, especially in CNAML. It is hoped that cytogenetic and molecular
genetic analyses will allow accurate prediction of the
response to therapy and the tailoring of treatment to
specific genetic lesions acquired by the leukemic
blasts, and that this will result in an improved clinical
outcome for AML patients.
Acknowledgements
Supported in part by National Cancer Institute, Bethesda,
Maryland grants CA77658, CA101140 and CA16058,
and the Coleman Leukemia Research Foundation.
12.
13.
14.
15.
16.
17.
18.
19.
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Management of elderly patients with
H. Dombret
E. Raffoux
L. Degos
From the Department
of Clinical Hematology
Saint-Louis Hospital,
University Paris,
Paris, France
2007;1:193-199
dvances in the treatment of
younger patients with acute
myeloid leukemia (AML) have
been obtained with intensified treatments
such as high-dose chemotherapy or allogeneic stem cell transplantation. However,
AML is predominantly a disease of the
elderly and these options are not appropriate for patients over 50 to 60 years old. In
older patients, even the advantages
offered by standard intensive chemoteraphy are still open to discussion because of
excessive toxicity and short duration of
response. Factors related to age, including
poor performance status (PS), pharmacodynamic changes, and organ dysfunctions,
may negatively impact on treatment tolerance.1-5 Factors related to disease biology,
including more frequent prior myelodysplastic syndrome (MDS), expression of a
multidrug resistance (MDR) phenotype,
and unfavorable karyotype, may lower
the response rate and response duration.1-5
In a recent retrospective American survey,
the outcome of elderly patients with AML
was very poor with a median survival of 2
months and a 2-year survival rate of 6%.
Only a minority of patients underwent
chemotherapy within two years after
AML diagnosis.6 The proportion of elderly
patients treated intensively is probably
slightly higher in European countries than
in the United States, but does not exceed
30 to 40% even when considering only de
novo AML patients. The eligibility of older
patients with AML for standard chemotherapy must be appropriately defined.
This is particularly important because
many of the new agents and therapeutic
strategies being developed in AML are
only proposed for these so-called unfit
patients. However, some of these new
agents may also benefit fit patients when
combined with standard chemotherapeutic agents. This has led to a great deal of
current research in these patients.
A
Standard intensive chemotherapy
Objectives and results of the recent
prospective randomized trials using intensive chemotherapy in older patients with
AML are summarized in Table 1. As indicated, study objectives were essentially
hematopoietic growth factors (HGF) (9
studies),8-11,14-16,18,19,22 anthracyclines or induction regimen (5 studies),7,12,13,17,18,25 postremission therapy (5 studies),7,12-15,20,21,25 alltrans retinoic acid (1 study),20,21 MDR modulation (1 study),23 and interleukin-2 (IL-2)
maintenance (1 study).24
Response to intensive induction
Induction death (ID)
Compared to earlier reports,26 CR rates
have only slightly improved during the last
15-year period and are now 50-60% (Table
1). This is much lower than in younger
patients. A reduction in ID rate (now usually 10-20%) has been progressively
observed. This reduction may be related to
a more rigorous selection of patients, but
other positive factors may have played a
role. The state of health of people aged
around 70 years of age has improved and
these patients present less frequent or
severe
pretreatment
comorbidities.
Advances in supportive care, notably antifungal therapies, may have also contributed. Surprisingly, all studies which
tested the ability of granulocyte or granulocyte-macrophage colony-stimulating factor (G-CSF and GM-CSF) to reduce ID rate
gave negative results, even if both factors
were able to significantly reduce the duration of chemotherapy-induced neutropenia without affecting response and
response duration. This means that the
role of G-CSF and GM-CSF in elderly AML
management is not clearly defined,
although HGF administration may reduce
hospitalization duration, a particular problem for these older patients.
Resistant disease
Resistant disease after intensive induction (25 to
45%) is now the main cause of failure in older
patients. Compared to younger patients, higher resistance disease (RD) rates are also observed when considering only patients with AML-nk, suggesting that
the trend towards more unfavorable cytogenetics is
not the only determining factor for the higher resistance rates observed in older patients. We have already
mentioned the more frequent expression of a MDR
phenotype, which has been shown to influence the
outcome independently of unfavorable cytogenetics.27
Poor-prognosis internal tandem duplications of the
FLT3 gene do not appear to be more frequent in older
patients.28-30 The incidence of good-prognosis NPM1
mutations has not yet been fully defined in older
patients and might play a role. In a gene expression
profile study, unsupervised clustering of samples from
170 older patients with AML (median age, 65 years)
identified a cluster of 24 patients associated with
NMP1 mutation and a relatively favorable outcome.31
Interestingly, the highest rate of RD was observed in a
cluster of 22 patients with notable MDR gene expression. Finally, genome-wide analysis using comparative
genomic hybridization (CGH) or single nucleotide
polymorphism (SNP) arrays which are currently being
performed in large cohorts of pediatric, younger, and
older AML patients will probably identify those with
cryptic abnormalities associated with resistance to
standard chemotherapy. At present, most attempts to
Table 1. Response to intensive induction chemotherapy (period 1990-2006).
Study
(period)
Study
objectives
Patients
(N)
Median age
(years)
Secondary
AML (%)
Normal or favorable
cytogenetics* (%)
CR rate
(%)
ID rate
(%)
RD rate
(%)
EORTC-HOVON7
(1986-1993)
Anthracycline
Post-remission
489
68
10
38
42
18
40
AMLCSG8
(1990-1992)
HGF
173
71
0
49
59
17
24
ECOG9
(1990-1992)
HGF
117
64
0
NA
52
NA
NA
EORTC-HOVON10
(1990-1994)
HGF
318
68
22
48
56
13
31
GOELAMS11
(1992-1994)
HGF
240
66
0
58
62
16
22
MRC 12,13
(1990-1998)
Induction regimen
Post-remission
1311
66
23
55
55
19
26
CALGB14,15
(1990-1993)
HGF
Post-remission
388
69
0
NA
53
25
22
SWOG16
(1992-1994)
HGF
211
68
24
NA
46
19
35
SWOG17
(1995-1998)
Induction regimen
328
67
23
36
38
17
45
ECOG18
(1993-1997)
Anthracycline
HGF
348
68
NA
50
42
17
41
HGF
110
77
0
NA
65
12
23
ATRA
Post-remission
242
66
16
NA
33
13
54
HGF
722
68
22
50
55
14
31
HOVON-MRC23
(1997-1999)
MDR modulation
419
67
25
56
51
15
34
CALGB24
(1998-2002)
IL-2 maintenance
669
71
27
NA
46
NA
NA
ALFA25
(1999-2006)
Anthracycline
Post-remission
416
72
15
53
57
10
33
Swedish group19
(1992-1999)
AMLSG20,21
(1997-2003)
EORTC-GIMEMA22
(1995-2001)
* Percentage is given in patients with adequate cytogenetic study; HGF: hematopoietic growth factor; MDR: multidrug resistance; CR: complete remission; ID: induction
death; RD: resistant disease; NA: not available.
Table 2. Cytogenetic risk subsets.
Younger AML studies*
Older AML studies
MRC
(N= 1065 patients)
CALGB
(N= 635 patients)
AMLSG4
(N= 361 patients)
HOVON41
(N=293 patients)
t(8;21)
inv(16)/t(16;16)
t(8;21)
inv(16)/t(16;16)
t(8;21)
inv(16)/t(16;16)
inv(16)/t(16;16)
t(8;21)
inv(16)/t(16;16)
Other
Other
Other
Normal
t(8;21)
11q abns
+8, +11
Normal
-Y
7q-
Complex
–7, 3q abns.
±
-5, 5q-, 7q+8, t(6;9), t(9;22)
9q, 11q, 20q, 21q, 17p abns.
Complex
Complex
Rare aberrations
Other
Other
13
Favorable
(Low-risk)
Intermediate
(Standard-risk)
Unfavorable
(High-risk)
5
* According to MRC, SWOG/ECOG, and CALGB classifications for younger adults42; abns: abnormalities.
significantly reduce the rate of RD after intensive
induction have failed. Studies evaluating variations of
or additions to the standard 3+7 regimen (daunorubicin, 45-60 mg/m2 for 3 days; cytarabine, 100-200
mg/m2 CI for 7 days) were essentially negative. MDR
modulation using either PSC-833 or zosuquidar did
not increase the response rate.23,32,33 Some results were
observed in studies testing HGF with the aim of priming AML blasts in the cell cycle during chemotherapy.10,11,18,19,22 But although HGF priming has been reported to be beneficial in younger patients with standardrisk AML,34,35 results were more heterogeneous in older
patients.36 No recommendation on G-CSF or GM-CSF
priming may therefore be made in elderly AML
patients. A very interesting recent study which awaits
confirmation showed increased CR rate, longer eventfree and overall survival when all-trans retinoic acid
(ATRA) was added to standard ICE chemotherapy.20
Finally, the addition of fludarabine to cytarabine and
G-CSF failed to show any significant improvement.37
Selection of eligible patients
There are no standardized selection criteria for
intensive chemotherapy. As a consequence, selection bias may vary from one study to another, making valuable comparisons difficult. In patients
enrolled in prospective trials, trial eligibility criteria
are an important first selection step usually including favorable PS, the absence of organ failure, severe
uncontrolled infection, psychiatric disease, or central nervous system involvement. However, most
investigators would agree that patients are also
more subjectively selected according to their physiological age and associated comorbidities. Their
own willingness to receive or not receive intensive
chemotherapy is also an important factor. Usually,
intensive treatment is not offered to patients aged
80 years or more. Very recently, a refined comorbidity index, namely the Hematopoetic Cell
Transplantation Comorbidity Index (HCTCI), has
also been used in elderly patients with AML.38-40
Even if HCTCI was identified as an independent
prognostic factor for outcome, a larger evaluation of
the sensitivity and specificity of each HCTCI item
by itself is needed to more precisely define AML
patients unlikely to benefit from intensive therapy
because of unacceptable toxicity. Some AML features such as cytogenetics and prior MDS also influence this decision. This can be seen from the variations observed in the proportion of patients with
secondary AML or AML with a normal or favorable
karyotype enrolled in the trials listed in Table 1. In
four recent studies evaluating the prognostic value
of cytogenetics exclusively in older patients, some
important differences or uncertainties may be noted
(Table 2).4,5,13,41 It seems, however, that the optimal
classification for older patients should clearly differ
from the classification(s) used in younger patients
(Table 2).42 First, core binding factor (CBF) leukemias
which represent the favorable subset in younger
patients are much less frequent in older patients and
appear to be also less favorable.3,13,41 It is even
unclear whether all these CBF-AMLs are to be associated with a better prognosis than AMLs with a
normal karyotype (AML-nk) in older patients.3,4,13 At
the other end of the spectrum, due to relatively low
numbers of patients and the relatively poor outcome in general, the unfavorable subset is difficult
to define. Only very unfavorable features such as
complex karyotype always identify patients with a
significantly worse outcome (Table 2).
Incorporation of more recently developed agents
Emerging agents for older AML treatment were
reviewed last year by S. Amadori and R. Stasi in the
EHA 2006 Education Program.43 Because of their
predominant hematologic toxicity, some of these
agents are candidates for combination with standard chemotherapeutic agents in the context of an
intensive treatment. Some others may be combined
with intensive chemotherapy to sensitize AML
blasts to chemotherapy-induced damage. Gemtuzumab ozogamicin (GO) is approved in the US for
the treatment of patients with CD33-positive AML
in first relapse aged 60 years or older who are not
considered to be candidates for standard chemotherapy. In a sequential front-line Phase II study,
GO has been used as single agent before standard
intensive chemotherapy.44 Several cooperative
groups are currently investigating the role of GO
administered concomitantly to first line chemotherapy, mainly in younger adults. The first very interesting results came last year from the British MRC
Phase III AML-15 study.45 The addition of intermediate-dose GO (3 mg/m2 for one dose) to the first
and third courses of chemotherapy significantly
reduced the risk of relapse and prolonged diseasefree survival (DFS) without a significant advantage
in terms of overall survival (OS), at least with the
present follow-up. Some patients aged 60 years or
more were included in this study and seemed to tolerate the combined treatment well. However, larger
specific studies are needed for these patients. GO is
less effective in AML blasts expressing a MRD phenotype. This might make it less beneficial in older
patients. Clofarabine is a next generation purine
nucleoside analog approved for the treatment of
children with refractory or relapsing acute lymphoblastic leukemia. In adults, interesting results
have been recently reported in patients with AML.
In patients eligible for intensive therapy, clofarabine
has been combined with intermediate-dose cytarabine,46 anthracycline with or without cytarabine,47
or anthracycline and GO.48 Larger front-line studies
are needed to confirm the promising potential of
these combined therapies, especially in older
patients. Cloretazine is a novel sulfonylhydrazine
alkylating agent which has recently been reported
to be associated with significant efficacy and modest extrahematologic toxicity when administered as
a single agent to older patients with previously
untreated AML.49 Intensive induction combining
cloretazine with usual chemotherapeutic agents
may be another option that could be tested.
Liposomal daunorubicin might also be of interest in
older AML patients, as recently reported in a phase
III study from the Italian GIMEMA group.50
Bortezomib, FLT3 inhibitors, or bcl2 antisens,51 may
also be good candidates to combine with standard
chemotherapeutic agents, to sensitize AML blasts to
chemotherapy-induced apoptosis.
Post-remission therapy and relapses
Consolidation chemotherapy
There is no confirmed post-remission strategy in
elderly patients with AML once CR has been
reached using standard intensive induction. Highdose consolidation courses, with or without highdose cytarabine, are usually too toxic to benefit
most of these patients, as a large number of them
may not receive all programed treatment because of
acquired comorbidities. On the other hand, beneficial effects associated with prolonged therapy with
lower doses of chemotherapy have been reported in
elderly patients but not in younger patients.7,52 In the
ALFA-9803 study, CR patients randomized to
receive a consolidation therapy with six relatively
mild cycles as out-patients had a longer DFS and OS
than those randomized to receive one intensive
induction-like cycle.25 The neeed for re-hospitalization, transfusions, and intravenous antibiotics was
significantly less in those treated as out-patients.
Interesting large, cooperative group studies testing
prolonged maintenance with new agents such as
GO, tipifarnib, or 5-azacitidine have been carried
out or are ongoing. However, results are not yet
available.
Allogeneic stem cell transplants

n o i t

Transcription

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