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)

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 TumorChr. AbnGenesFreqProg Alveolar Rhabdomyosarcomat(2;13)PAX3/FKHR75%Poor t(1;13)PAX7/FKHR10%Poor Synovial Sarcomat(X;18)SYT-SSX165%Poor SYT-SSX235%Poor Congenital/infantile fibrosarcoma t(12;15)ETV6/NTRK380%Good Clear cell sarcomat(12;22)EWS/ATF190%Poor Extraskeletal myxoid chondrosarcoma t(9;22)EWS/TEC75%Good t(9;17)TAF2N/TEC25% Mixoid/round cell liposarcomat(12;16)TLS/CHOP>95%Good Alveolar soft part sarcomat(X;17)ASPL/TEF3>90% Chang C & Shidham VB. J Mol Diagn 2003, 5:143–154

Genetic lesions in soft tissue tumors TumorChr. AbnGenesFreqProg EWS Family (EWS/PNET)t(11;22)EWS/FLI185%Good t(21;22)EWS/ERG5-10% 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 tumor t(11;22)EWS/WT1>95%Poor Giant cell fibroblastomat(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 Confirmatory genetic aberrations Cyto/histo morphologyDiagnosis RhabdomyosarcomaDSRCTEWS/PNET Round cell PAX3/FKHR – 75% PAX7/FKHR – 10% EWS/FLI1 – 85% EWS/ERG – 10% EWS/ETV1 EWS/EIAF EWS/FEV EWS/WT1 – 95%Congenital fibrosarcomaSynovial sarcoma Spindle cell ETV6/NTRK3 – 80%SYT/SSX1 - 65% SYT/SSX2 – 35% Chang C & Shidham VB. J Mol Diagn 2003, 5:143–154

Algorithm for soft tissue tumors Confirmatory genetic aberrations Cyto/histo morphologyDiagnosis Epithelioid EWS/ATF1 – 90%Clear cell sarcomaMyxoid liposarcomaExtraskeletal myxoid chondrosarcoma Myxoid TLS-CHOP – 95% EWS/CHOP EWS/TEC – 75% Chang C & Shidham VB. J Mol Diagn 2003, 5:143–154

EWSR – major contributor EWS (chromosome 22q12) FLI1 (chromosome 11q24) ERG (chromosome 21q22) ETV1 (chromosome 7p22) EIAF (chromosome 17q21) FEV (chromosome 2q33) ZSG (chromosome 22q22) Ewing/PNET DSRCT WT1 (chromosome 11p13) Clear cell sarcoma ATF1 (chromosome 12q13) Extraskeletal myxoid chondrosarcoma TEC (chromosome 9q22-23) Myxoid/round cell liposarcoma CHOP (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) Time from excision to fixation Duration of fixation Volume of tissue to volume of fixative Thickness of the section FIXATIVEOPTIMAL FISH RESULTS 10% NBF>6 to 48 hours Alcoholic Formalin>6 to 48 hours Zinc Formalin>6 to 48 hours Davidson’s AFANONE PreferNONE Bouin’sNONE A. Babic et al. / Methods 52 (2010) 287–300

FISH – Technical Considerations 3 hours 6 hours 24 hours 1 hour NBF Alcoholic F A. Babic et al. / Methods 52 (2010) 287–300 Bouin’s – 1 hour Prefer – 1 hour

FISH – Technical Considerations 0 minutes 2 hours 4 hours 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 CASEBCL2 Break-apartBCL2 Dual fusion 10/150 (0%)0/400 (0%) 20/215 (0%)4/400 (1%) 340/150 (27%)14/200 (7%) 41/200 (1%)0/200 (0%) 540/130 (31%)5/306 (2%) 630/150 (20%)15/400 (4%) 730/200 (15%)4/104 (4%) 830/160 (19%)4/100 (4%) 9 53/263 (20%)7/127 (6%) Tibilette MG. Cytogenet Genome Res 118:229–236 (2007) Validation ?????

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.

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. FISH – Technical Considerations 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 B = homozygous 9p del (low grade papillary tumor) D = polysomy( high grade tumor) UROVYSION: bladder cancer 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 Halling KC & Kipp BR, Human Pathology (2007) 38, 1137–1144

New Developments – Solid Tumors F = Trisomy 7 G & H = polysomy (tumor) Biliary tract malignancy 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 Halling KC & Kipp BR, Human Pathology (2007) 38, 1137–1144

New Developments – Solid Tumors J = Tetrasomy K = polysomy (tumor) Lung cancer 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% Halling KC & Kipp BR, Human Pathology (2007) 38, 1137–1144

New Developments – Solid Tumors M = Homozygous 9p deletion O = polysomy (tumor) Barrett’s esophagus Barrett’s esophagus - ~125 times higher risk for carcinoma FISH – low vs high grade dysplasia & adenocarcinoma Sensitivity and specificitylow-grade dysplasia – 70% & 89% high-grade dysplasia – 84% & 93% adenocarcinoma – 94% & 93% Halling KC & Kipp BR, Human Pathology (2007) 38, 1137–1144 N = Isolated gain of 8q

New Developments – Solid Tumors 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 NORMALHemizygous deletionHomozygous deletion PTEN = red; MBPR1A = green; FAS = yellow; CEP 10 = aqua Sircar et al., J Pathol 2009; 218: 505–513 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

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