Anna Bofin

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Molecular Cytology
Anna M Bofin
Associate Professor of Pathology
Department of Laboratory Medicine, Children’s and Women’s Health
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«Fine needle aspiration is a
mechanicophysical tumour cell
enrichment procedure»
Professsor Bjørn Hagmar
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What is molecular cytology?
The in situ localization of selected molecules…
Utilization of advanced technologies that allow
for the detection, analysis and quantification
of molecules in their natural environment,
such as cells, tissues, organs, embryos and
tumors….
…in diagnostics and research.
AMB May 2012
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Why molecular cytology?
 Primary diagnostics
 Prognostic information
 Predictive information
Determine
treatment strategies
 Monitoring disease
 Diagnose recurrence
 Research
AMB May 2012
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Cytology – Whole interphase cells
 A complete cell membrane
 A full set of chromosomes
 All the cytoplasm




Air-dried
Cell suspension
Snap-frozen
Fixed
Henrietta Lacks’ ‘”Immortal’ Cells” – The Smithsonian Magazine 2010
AMB May 2012
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Whole cells
 Exfoliated cells
Urine, serous fluids, expectorat, vaginal cytology
 Brushed, scraped or washed from a surface
Bronchial cytology, cervical cytology, skin scrape
cytology, urinary bladder
 Fine needle cytology
Fine needle aspiration (FNA), fine needle sampling (FNS)
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Techniques
 In Situ Hybridization
 Immunocytochemistry
 PCR / RT-PCR
 Flow cytometry
 cDNA microarray
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The molecular biological pathway
FISH/CISH/PCR
PCR
ICC/IHC
http://fajerpc.magnet.fsu.edu
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“…any cell with a nucleus can be
examined using (F)ISH techniques”
JK Blancato, BR Haddad
Medical Cytogenetics
(Ed HFL Mark) 2000 pp 147
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Fluorescence in situ hybridisation
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Applications
 Aneuploidy
 Gene copy number
 Structural breakpoint analysis
 Translocation
 Microdeletion
 Gene mapping
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ISH probes
Metaphase
 Whole chromosome probes
 Alpha satellite/centromere probes
 Unique sequence probes
Lodish et al Molecular Cell Biology 2005
Interphase
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Alpha satellite/centromere probe
 Repetitive sequences
 Near chromosome centromeres
 Chromosome specific
 Detect aneuploidy
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Unique sequence probes (locus
specific probes)
 Target regions NOT repeated in the genome
 Chromosome deletions
 Oncogenes – c-myc; HER2; EGFR
 Telomeric and subtelomeric probes
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Amplification patterns
Double minutes = extrachromosomal amplification
MYCN FISH in neuroblastoma
Interphase
Metaphase
www.utoronto.ca/.../cytogenetics
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Amplification patterns
Homogeneous staining regions = intrachromosomal amplification
HER2 FISH in breast cancer
Interphase
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HER2/chromosome 17 FISH
Looking for gene amplification - breast cancer
 FNA cytology
 Ratio 2:2 = 1.0
 No gene amplification
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HER2/chromosome 17 FISH
Looking for gene amplification - breast cancer
 FNA cytology
 Ratio 2:>15=7.5
 High grade
gene amplification
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Morphology in the fluorescence microscope
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Morphology in the fluorescence microscope
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Morphology in the fluorescence microscope
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Morphology in the fluorescence microscope
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HER2 TOP2A Chromosome17
Brystkreft
BRCA1
HER2
TOP2A
TP53
Bofin, et al Cytopathology 2003;14:314-319
Ingen
TOP2A
genamp
amp amp
HER2 amp/TOP2A
nonamp
HER2/TOP2A
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Translocations
Locus specific probes
Diagnostics
Prognostication
Follow-up
Leukemia –CML brc/abl translokasjon
Lymphoma –Mantle cell lymphoma t(11;14)(q13;q32)
Sarcoma – synovial sarcoma - t(x;18)
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Fusion and split translocations
Red and green co-localise
to detect a known translocation
Red and green separate to
detect a break without the need
to know the translocation partner
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Translocations - fusions
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Translocations - Splits
Bishop R Bioscience Horizons 2010;3:85-95
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Screening for disease or relapse
Multicolour FISH
Cancer of the urinary bladder – urine cytology
Copy number changes in ch. 3, 7,
or 17 in at least 4 cells
OR
Loss of 9p21 in at least 12 cells
Ferras S et al Cancer Cytopathol 2009 Feb 25;117(1):7-14.
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Chromosome copy number
Urinary cytology
FISH
Chromosomes 3,7,17
Locus 9p21
E Berggren/A Bofin
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Pap Urothelial carcinoma WHO grade 2-3 FISH
Pap Urothelial carcinoma, WHO grade 3 FISH
E Berggren/A Bofin
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Pap Urothelial carcinoma, WHO grade 3 (FISH)
Pap Cis,/Urothelial carcinoma, WHO grade 2 FISH
E Berggren/A Bofin
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Cytogenetic analyses
Karyotyping FISH
 Lipoma
 Synovial sarcoma
t(X;18) (SYT;SSX)
 Rhabdomyosarcoma
Cohen IJ et al Synovial sarcoma of bone delineated by spectral karyotyping.Lancet. 1997 ;350(9092):1679-80
Croes R et al Adult sclerosing rhabdomyosarcoma: cytogenetic link with embryonal rhabdomyosarcomaVirchows
Arch. 2005 Jan;446(1):64-7
Sandberg AA Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors: lipoma. Cancer Genet
Cytogenet. 2004 Apr 15;150(2):93-115
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FISH in the path lab
 Contributes to diagnosis
 Surveillance in patient follow-up
 Prognostic and predictive information
 Determining treatment strategies
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FISH - pros and cons
 Simpler protocol – minimal pretreatment
 No formalin-fixation artefacts
 Whole nuclei - no truncation artefacts
 Little “background” fluorescence
 Recognisable morphology compared to routine
stained smears
 Cytology not always available
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Artefacts/Problems
Smearing artefacts
Non-specific background fluorescence
Low stringency washing
Cross-hybridisation
Poorly prepared probe
Probe too small
Amplification techniques (TSA)
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Immunocytochemistry
 Cell smears
 Fine needle aspirates
 Liquid-based cytology
 Smears
 Cell suspension
 Snap frozen
 Cell blocks – «pseudohistology»
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Immunocytochemistry
Cervix
 Liquid-based cytology
HSIL
 MIB1
 p16INK4a
Sahebali et al
Immunocytochemistry in liquid‐based cervical cytology: Analysis of clinical use following a
cross‐sectional study International Journal of Cancer 2005;118:1254-1260
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Immunocytochemistry
Breast carcinoma
Diff Quick – atypical ductal cells
Oestrogen receptor
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Immunocytochemistry
Breast carcinoma
 Reliability of a negative result
 Snap freezing
 Fixative/air-drying
 The epitope may become slightly deformed resultiong in a false
neg. Result
Cuthbert et al Cytopathology 1990;1:339-47
Sauer T, Beraki E, Jebsen P. Anal Quant Cytol Histol 1998;20:122-6
 A negative ER on FNA should prompt ER on tissue
biopsy
Lea D, Bofin A. Er hormonundersøkelse av cytologisk materiale
i brystkreftsvulster til å stole på? Bioingeniøren 2011:3;6-12
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Immunocytochemistry
Malignant melanoma
Atypical melanocytes
Immunocytochemistry:HMB45
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Painful subcutaneous tumour
S100 protein
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Painful subcutaneous tumour
Neurofibroma
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Large tumour in the shoulder
FNA smear
B-cell lymphoma
LCA
CD20
CD99
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Soft tissue tumour in a child
FNA smear
MGG
Desmin
Alveolar
rhabdomyosarcoma
Lisa Walaas, Rikshospitalet, Oslo
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ICC - pros and cons
 Simple procedure
 air-dried smears
 LBC
 Cell blocks
 Provides additional information in inoperable patients
 Enables pre-operative chemotherapy
 Not always enough material for multiple ICC’s
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Polymerase Chain Reaction(PCR)
Finding small sequences of DNA and making them “visible”
DNA
mRNA converted to cDNA by means of reverse transcriptase
5’
DNA is amplified:
5’
DNA
3’
3’
5’
DNA
3’
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RT-PCR (reverse transcriptase)
Fluorochrome
Probe
Quencher
DNA
DNA
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Real time RT-PCR
 Relative quantification
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B-RAF mutation in thyroid FNA
 Diagnostics
 Determining treatment strategy
 B-REF mutation detection in papillary carcinoma (45 %)
 BRAF-mutation in a follicular-like neoplasm indicates a
follicular variant of a papillary carcinoma AND total
thyroidectomy rather than a diagnostic
hemithyroidectomy
Canadas-Garre et al Ann Surg 2012 May;255(5):986-92
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PCR- pros and cons
 Costly, perhaps complicated procedure
 Requires a reasonable amount of material
 Quantitation or relative quantitation of small amounts
of RNA/DNA
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Flow cytometry
Flow cytometry is a method allowing the analysis of of
cells or particles suspended and separated in a fluid.
 The fluid flows past a focused laser beam.
 The cells are usually labelled using fluorescent probes
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
which bind to specific cell associated molecules
Phenotypic, biochemical and molecular
characteristics of the individual cells
Visualised as a scatter chart or graph
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Flow cytometry
Rudbeck laboratory, University of Uppsala
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Flow cytometry
 FNS/FNA from lymph nodes/lymphoid tissue
 Particularly in non-Hodgkins lymphoma
 Requires a certain number of cells
 No morphology – keep some material back for
microscopy
Zeppa et al Cytopathology 2010;21:300-310
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Quality assurance/control
Follow procedure – particularly posthybridisation
washes
Qualified readers
National and international QA programmes
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Take home message
Think special tests - Always