Application of FISH in hematologic malignancies Dr Edmond S K Ma Department of Pathology Hong Kong Sanatorium & Hospital BTG 2013

Molecular Cytogenetics The utilization of techniques based on fluorescence in-situ hybridization in which DNA probes are labelled with different fluorochromes to map one or more specific regions of the genome Bridges cytogenetics and molecular genetics Techniques: FISH CGH 24-colour karyotyping (M-FISH / SKY) Array CGH BTG 2013

Any role for FISH in the post-genomic era? Manageable by routine diagnostic laboratories Answer to specific clinical questions Practical advantages Numerical abnormality Multiple fusion partners Breakpoint heterogeneity Applicable to many specimen types BTG 2013

Probes Chromosome enumeration Locus specific Multicolour FISH Orange signal: chr 1; Green signal: chr 7 BCR-ABL dual colour dual fusion Multicolour FISH Chromosome painting der(9) dic(14;22)der(22)

FISH as an investigative tool in haematological malignancies Detection of numerical and structural abnormalities in interphase and metaphase cells Characterization of marker chromosomes Detection of cryptic translocation Usually detected by CG Not usually detected by CG Lineage involvement by the neoplastic clone Disease monitoring after treatment Chimerism study post-sex-mismatched BMT BTG 2013

From Ma, Wan & Chan. Cancer Reviews Asia-Pacific 2: 131 – 141, 2004

Acute promyelocytic leukaemia (APL) with unusual CG Wan TS et al, Cancer Genet Cytogenet 121: 90 – 3, 2000 BTG 2013

Wan TS et al, Cancer Genet Cytogenet 121: 90 – 3, 2000

Cryptic insertion of BCR at 9q34 in CML D-FISH: 1R2G1F pattern D-FISH S-FISH ES-FISH Wan TS et al, Leukemia 18: 161 – 2, 2004

Chimerism status by XY-FISH BTG 2013

Chronic myeloid leukaemia post-BMT donor relapse BTG 2013

FISH: some advantages Genetic abnormality measurable in dividing and non-dividing cells Covers CG failure Covers mature B-cell disorders Applicable to many specimen types Applicable to heterogeneous breakpoints or multiple translocation partners Quantitative Standardization Nomenclature (ISCN), criteria for interpretation and proficiency testing BTG 2013

MLL probe for rearrangement BTG 2013

Characterization of chromosome 11q deletion Ma SK et al, Leukemia 16: 953 – 955, 2002 BTG 2013

Southern Blot hybridization for MLL rearrangement Ma SK et al, Leukemia 16: 953 – 955, 2002 BTG 2013

Caveats of FISH analysis No global view of chromosomal complement Requires clinicopathological or prior cytogenetics information Issues related to analytical sensitivity and probe specificity Susceptibility to artifacts Cannot detect minute aberrations (< 20 kb) Aneuploidy versus amplification BTG 2013

Chronic myeloid leukaemia Ph chromosome Chronic myeloid leukaemia BTG 2013

From Ma, Wan & Chan. Cancer Reviews Asia-Pacific 2: 131 – 141, 2004

BCR-ABL dual colour single fusion translocation probe

Detection of fusion genes by S-FISH BTG 2013

Detection of BCR-ABL gene fusion by S-FISH Accurate for metaphase FISH Problem of false positive (~ 4%) Normal cutoff range 10% (Dewald et al, Cancer Genet Cytogenet 71: 7; 1993) 7% (Cox Froncillo et al, Ann Hematol 73: 113; 1996) BTG 2013

Detection of fusion genes by ES-FISH

Detection of fusion genes byES-FISH BTG 2013

BCR-ABL dual colour dual fusion translocation probe

BCR-ABL dual fusion translocation probe BTG 2013

Detection of BCR-ABL gene fusion by D-FISH Normal range for 500 interphase nuclei  4 nuclei ( 0.8%) Buño et al, Blood 92: 2315; 1998 Monitor response to therapy Normal cutoff for 6,000 nuclei = 0.079% Residual disease level 7 - 53 nuclei (0.117 - 0.883 %) Dewald et al, Blood 91: 3357; 1998 BTG 2013

Three-way Ph translocation *Courtesy of Dr. K. F. Wong, QEH BTG 2013

Variant D-FISH pattern BTG 2013

Derivative chromosome 9 (9q+) deletion in CML Occurs in ~ 15% of cases Deletion of reciprocal ABL-BCR fusion gene At the time of Ph translocation Correlates with a poor prognosis Sinclair et al. Blood 95: 738 - 743, 2000 Huntly et al. Blood 98: 1732 - 1738, 2001 Partly overcome by imatinib Huntly et al. Blood 102: 2205 – 2212, 2003 BTG 2013

Wan TS et al, J Clin Pathol 56: 471 – 474, 2003 Derivative chromosome 9 deletion in CML 9 der(22) der(9) 22 Confirmation: >10% of cells S-FISH Metaphase FISH RT-PCR Wan TS et al, J Clin Pathol 56: 471 – 474, 2003

Atypical BCR-ABL interphase D-FISH patterns Primo et al, 2003 83% typical 17% atypical Wan et al, 2003 Among 46 CML Typical = 44 (95%) Atypical = 2 Lisa Siu (QEH, 2008) Among 22 CML Typical = 17 (77%) ABL-BCR deletion = 2 ABL deletion = 2 BCR deletion = 1 BTG 2013

BCR-ABL + 9q34 tricolour dual fusion translocation probe Normal cell: 2 G + 2 O/aqua Ph+ cell: 1 G + 1 O/aqua + 1 G/O fusion + 1 G/O/aqua fusion der(9) deletion cell: 1 G + 1 O/aqua + 1 G/O fusion False+ cell: 1 G + 1 O/aqua + 1 G/O/aqua fusion BTG 2013

BCR-ABL + 9q34 tricolour dual fusion translocation probe Normal cell: 2 G + 2 O/aqua Ph+ cell: 1 G + 1 O/aqua + 1 G/O fusion + 1 G/O/aqua fusion der(9) deletion cell: 1 G + 1 O/aqua + 1 G/O fusion False+ cell: 1 G + 1 O/aqua + 1 G/O/aqua fusion BTG 2013

der(9) deletion BCR-ABL D-FISH BCR-ABL + 9q34 tricolour dual fusion translocation probe fusion fusion BTG 2013

Clinical use of interphase FISH in risk stratification CLL 13q-, 11q-, 17p-, +12 Myeloma High-risk cytogenetic markers t(4;14) t(14;16) del(17)p/p53 chromosome 1q gain Coupled with cell sorting or immunofluorescence BTG 2013

FISH and personalized medicine Myeloma CLL Imatinib targets BCR-ABL FIP1L1-PDGFRa fusion PDGFRb rearrangements MDS 5q- BTG 2013

BTG 2013