FISH – Technical Considerations - San Antonio Society of Pathologists

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FISHing in Pathology
Gopalrao Velagaleti, Ph.D
Director, Cytogenetics Laboratory
Objectives
• Understand the importance of chr. Anomalies in cancer
• Understand the principles of fluorescence in situ hybridization
(FISH)
• Understand different probe strategies
• Understand the limitations of FISH
• Explain the role of Pathologists in obtaining successful FISH
results
• Explain the significance of FISH in Surgical Pathology
• Discuss new developments/testing with FISH
Chromosomal Translocations in
Human Cancer Pathogenesis
 Frequent in Leukemia, Lymphoma & Sarcoma
 Aberrant expression of Oncogenes or Chimeric proteins
 500 recurring cytogenetic abnormalities reported
 fusion genes encode transcriptional factors
 aberrant transcription – key role in oncogenesis
Fluorescence In Situ Hybridization (FISH)
• sensitivity of molecular techniques
• specificity of cytogenetic techniques
• increased resolution
• exceptional tool for diagnosis
• high specificity - limitation
• expensive
• not all gene probes available
Fluorescence In Situ Hybridization (FISH)
http://nhc.batan.go.id/images/400px-FISH_(Fluorescent_In_Situ_Hybridization).jpg
Types of Probes
Dual fusion Probe Strategy
Break-apart Probe Strategy
Tibilette MG. Cytogenet Genome Res 118:229–236 (2007)
Types of Probes
• Dual Fusion Strategy
- gene is not promiscuous
- partner is well characterized
e.g. Alveolar Rhabdomyosarcoma – t(2;13) – PAX3/FKHR
t(1;13) – PAX7/FKHR
Synovial sarcoma – t(X;18) – SYT-SSX1 or SYT-SSX2
• Break-apart Strategy
- gene is promiscuous – many partners
- some partners not known or characterized
e.g. EWS/PNET
– t(11;22) – EWS/ERG
t(7;22) – EWS/ETV1
t(17;22) – EWS/E1AF
t(2;22) – EWS-FEV
inv(22) – EWS/ZSG
Genetic lesions in soft tissue tumors
Tumor
Alveolar Rhabdomyosarcoma
Synovial Sarcoma
Chr. Abn
Genes
Freq
Prog
t(2;13)
PAX3/FKHR
75%
Poor
t(1;13)
PAX7/FKHR
10%
Poor
t(X;18)
SYT-SSX1
65%
Poor
SYT-SSX2
35%
Poor
Congenital/infantile
fibrosarcoma
t(12;15)
ETV6/NTRK3
80%
Good
Clear cell sarcoma
t(12;22)
EWS/ATF1
90%
Poor
Extraskeletal myxoid
chondrosarcoma
t(9;22)
EWS/TEC
75%
Good
t(9;17)
TAF2N/TEC
25%
Mixoid/round cell liposarcoma
t(12;16)
TLS/CHOP
>95%
Alveolar soft part sarcoma
t(X;17)
ASPL/TEF3
>90%
Good
Chang C & Shidham VB. J Mol Diagn 2003, 5:143–154
Genetic lesions in soft tissue tumors
Tumor
EWS Family (EWS/PNET)
Chr. Abn
Genes
Freq
Prog
t(11;22)
EWS/FLI1
85%
Good
t(21;22)
EWS/ERG
5-10%
t(7;22)
EWS/ETV1
<1%
t(17;22)
EWS/EIAF
<1%
t(2;22)
EWS/FEV
<1%
inv(22)
EWS/ZSG
<1%
Desmoplastic small round cell t(11;22)
tumor
EWS/WT1
>95%
Poor
Giant cell fibroblastoma
t(17;22)
COL1A1/PDGFB
Dermatofibrosarcoma
protuberans
t(17;22)
COL1A1/PDGFB
>99%
Good
Chang C & Shidham VB. J Mol Diagn 2003, 5:143–154
Algorithm for soft tissue tumors
Cyto/histo morphology
Round cell
Confirmatory genetic
aberrations
Diagnosis
PAX3/FKHR – 75%
PAX7/FKHR – 10%
Rhabdomyosarcoma
EWS/WT1 – 95%
DSRCT
EWS/FLI1 – 85%
EWS/ERG – 10%
EWS/ETV1
EWS/EIAF
EWS/FEV
EWS/PNET
ETV6/NTRK3 – 80%
Congenital fibrosarcoma
SYT/SSX1 - 65%
SYT/SSX2 – 35%
Synovial sarcoma
Spindle cell
Chang C & Shidham VB. J Mol Diagn 2003, 5:143–154
Algorithm for soft tissue tumors
Cyto/histo morphology
Confirmatory genetic
aberrations
Diagnosis
Epithelioid
EWS/ATF1 – 90%
Clear cell sarcoma
TLS-CHOP – 95%
EWS/CHOP
Myxoid liposarcoma
EWS/TEC – 75%
Extraskeletal myxoid
chondrosarcoma
Myxoid
Chang C & Shidham VB. J Mol Diagn 2003, 5:143–154
EWSR – major contributor
FLI1
(chromosome 11q24)
EWS
(chromosome 22q12)
ERG
(chromosome 21q22)
ETV1
(chromosome 7p22)
EIAF
Ewing/PNET
(chromosome 17q21)
FEV
(chromosome 2q33)
ZSG
(chromosome 22q22)
WT1
(chromosome 11p13)
ATF1
DSRCT
(chromosome 12q13)
Clear cell sarcoma
TEC
Extraskeletal myxoid
chondrosarcoma
CHOP
Myxoid/round cell
liposarcoma
(chromosome 9q22-23)
(chromosome 12q13)
The role of “Pathologist”
 Integral part of the team – success of FISH results
 Appropriate sample collection, storage and transport
 Expertise in identifying tumor and areas of interest
 Direction – morphological/histological information
FISH – Technical Considerations
Factors affecting FISH assays (FFPE tissues)
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Time from excision to fixation
Duration of fixation
Volume of tissue to volume of fixative
Thickness of the section
FIXATIVE
OPTIMAL FISH RESULTS
10% NBF
>6 to 48 hours
Alcoholic Formalin
>6 to 48 hours
Zinc Formalin
>6 to 48 hours
Davidson’s AFA
NONE
Prefer
NONE
Bouin’s
NONE
A. Babic et al. / Methods 52 (2010) 287–300
FISH – Technical Considerations
6 hours
3 hours
24 hours
Prefer – 1 hour
Bouin’s – 1 hour
Alcoholic F
NBF
1 hour
A. Babic et al. / Methods 52 (2010) 287–300
FISH – Technical Considerations
2 hours
4 hours
0 minutes
0 minutes
Delay in fixation
Khoury et al., Modern Pathology (2009) 22, 1457–1467
FISH – Technical Considerations
Arch Pathol Lab Med—Vol 131, January 2007
FISH – Technical Considerations
a, b = fragility of nuclei due to necrosis on tissue sections
c = autofluorescence not removed from tissue sections
Tibilette MG. Cytogenet Genome Res 118:229–236 (2007)
FISH – Technical Considerations
CASE
BCL2 Break-apart
BCL2 Dual fusion
1
0/150 (0%)
0/400 (0%)
2
0/215 (0%)
4/400 (1%)
3
40/150 (27%)
14/200 (7%)
4
1/200 (1%)
0/200 (0%)
5
40/130 (31%)
5/306 (2%)
6
30/150 (20%)
15/400 (4%)
7
30/200 (15%)
4/104 (4%)
8
30/160 (19%)
4/100 (4%)
9
53/263 (20%)
7/127 (6%)
Validation ?????
Tibilette MG. Cytogenet Genome Res 118:229–236 (2007)
FISH – Technical Considerations
Probe validation/localization should be confirmed by:
Scoring of a minimum of 5 metaphase cells to verify that each probe hybridizes to the
appropriate chromosome target(s) and to no other chromosomes. Care should be taken
in evaluation of potential probe contamination, as the contaminating probe may be
present in a dilute concentration, thus hybridizing more weakly than the probe of interest.
One of the following methods should be used to determine chromosomal localization:
inverted DAPI, sequential G-/R-/or Q- to FISH or other banding method;
use of a cell line containing the region of chromosome of interest as an
independently identifiable target on a solid stained chromosome (e.g., structural
rearrangements, trisomy, etc.);
other methods that localize the probe at a level of resolution appropriate to the
intended chromosome target.
FISH – Technical Considerations
Assay sensitivity and reportable ranges must be set in each laboratory based on the
following database collection and analyses and/or statistical analyses. Results from samples
used to establish reportable ranges should not be reported as test results.
Database collection must be specific for an intended tissue type or cell
population.
The normal database should consist of an adequate number of cells from a group
of control individuals (as determined by the director) who do not have abnormalities
involving the target (and control) probes. Acceptable normal databases should include
at least 500 nuclei each from 20 control samples or 200 nuclei each from 30 control
samples. When possible, an abnormal database should be established.
Biannual (twice per year) calibration or continuous quality monitoring is required to ensure
that assay analytical sensitivity and specificity remain at the levels established during initial
validation.
New Developments – Solid Tumors
UROVYSION: bladder cancer
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•
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FDA approved for bladder cancer monitoring
FDA approved for assessing hematuria for bladder cancer
More sensitive than BTA for detection of recurrent tumors
Overall higher sensitivity than cytology (42% vs 75%)
Lower specificity than cytology (93% vs 85%)
Detection of recurrent urothelial carcinoma before morphologic evidence
B = homozygous 9p del (low grade papillary tumor)
D = polysomy( high grade tumor)
Halling KC & Kipp BR, Human Pathology (2007) 38, 1137–1144
New Developments – Solid Tumors
Biliary tract malignancy
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Malignant vs benign pancreatobiliary strictures - difficult
Sensitivity of routine cytology is low (6-66%)
On FISH – polysomy and trisomy
Polysomy PPV (positive predictive value) – 100% for tumor
Trisomy 7 PPV – 80% without primary sclerosing cholangitis
only 30% with PSC
F = Trisomy 7
G & H = polysomy (tumor)
Halling KC & Kipp BR, Human Pathology (2007) 38, 1137–1144
New Developments – Solid Tumors
Lung cancer
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•
•
•
Centrally located tumors – Cytology sensitivity 68%
Peripheral tumors – 45% for brushings & 28% in washings
On FISH – polysomy and tetrasomy
Comparison of Cytology, FISH & Cytology + FISH
Bubendorf et al., 55%, 52% & 76%
Halling et al.,
54%, 72% & 76%
J = Tetrasomy
K = polysomy (tumor)
Halling KC & Kipp BR, Human Pathology (2007) 38, 1137–1144
New Developments – Solid Tumors
Barrett’s esophagus
• Barrett’s esophagus - ~125 times higher risk for carcinoma
• FISH – low vs high grade dysplasia & adenocarcinoma
• Sensitivity and specificity low-grade dysplasia – 70% & 89%
high-grade dysplasia – 84% & 93%
adenocarcinoma – 94% & 93%
M = Homozygous 9p deletion
N = Isolated gain of 8q
O = polysomy (tumor)
Halling KC & Kipp BR, Human Pathology (2007) 38, 1137–1144
New Developments – Solid Tumors
Malignant mesothelioma
Malignant mesothelioma
Homozygous 9p deletion
Papillary mesothelial hyperplasia
Negative for 9p deletion
Takeda et al., Pathology International 2010; 60: 395–399
New Developments – Solid Tumors
Prostate cancer
NORMAL
Hemizygous deletion
Homozygous deletion
PTEN = red; MBPR1A = green; FAS = yellow; CEP 10 = aqua
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PTEN deletion – common in hormone refractory prostate cancer
PTEN deletion – pre-invasive prostatic intraepithelial neoplasia
PTEN deletion – grade and stage progression of prostate cancer
PTEN homozygous deletion – hormonal escape
Sircar et al., J Pathol 2009; 218: 505–513
SUMMARY
• Reviewed the FISH methodology
• Reviewed different probe strategies
• Reviewed applications of FISH in solid tumors
• Role of Pathologists – success of FISH
• Technical aspects and limitations of FISH
• Recent advances in FISH
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