Application of FISH in hematologic malignancies

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  • 1. BTG 2013 Application of FISH in hematologic malignancies Dr Edmond S K Ma Department of Pathology Hong Kong Sanatorium & Hospital
  • 2. 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
  • 3. 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
  • 4. Probes Orange signal: chr 1; Green signal: chr 7 Chromosome enumeration BCR-ABL dual colour dual fusion Locus specific der(9) dic(14;22)der(22) Chromosome painting Multicolour FISH
  • 5. BTG 2013 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
  • 6. From Ma, Wan & Chan. Cancer Reviews Asia-Pacific 2: 131 – 141, 2004
  • 7. BTG 2013 Acute promyelocytic leukaemia (APL) with unusual CG Wan TS et al, Cancer Genet Cytogenet 121: 90 – 3, 2000
  • 8. Wan TS et al, Cancer Genet Cytogenet 121: 90 – 3, 2000
  • 9. Cryptic insertion of BCR at 9q34 in CML Wan TS et al, Leukemia 18: 161 – 2, 2004 D-FISH: 1R2G1F pattern S-FISH ES-FISH D-FISH
  • 10. BTG 2013 Chimerism status by XY-FISH
  • 11. BTG 2013 Chronic myeloid leukaemia post-BMT donor relapse
  • 12. 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
  • 13. BTG 2013 MLL probe for rearrangement
  • 14. BTG 2013 Characterization of chromosome 11q deletion Ma SK et al, Leukemia 16: 953 – 955, 2002
  • 15. BTG 2013 Southern Blot hybridization for MLL rearrangement Ma SK et al, Leukemia 16: 953 – 955, 2002
  • 16. 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
  • 17. BTG 2013 Ph chromosome Chronic myeloid leukaemia
  • 18. From Ma, Wan & Chan. Cancer Reviews Asia-Pacific 2: 131 – 141, 2004
  • 19. BCR-ABL dual colour single fusion translocation probe
  • 20. BTG 2013 Detection of fusion genes by S-FISH
  • 21. 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)
  • 22. Detection of fusion genes by ES-FISH
  • 23. BTG 2013 Detection of fusion genes byES-FISH
  • 24. BCR-ABL dual colour dual fusion translocation probe
  • 25. BTG 2013 BCR-ABL dual fusion translocation probe
  • 26. 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
  • 27. BTG 2013 Three-way Ph translocation *Courtesy of Dr. K. F. Wong, QEH
  • 28. BTG 2013 Variant D-FISH pattern
  • 29. 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
  • 30. 9 der(22) der(9) 22 Derivative chromosome 9 deletion in CML Wan TS et al, J Clin Pathol 56: 471 – 474, 2003 Confirmation: >10% of cells S-FISH Metaphase FISH RT-PCR
  • 31. BTG 2013 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
  • 32. 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
  • 33. 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
  • 34. BTG 2013 BCR-ABL + 9q34 tricolour dual fusion translocation probe BCR-ABL D-FISH fusion fusion der(9) deletion
  • 35. 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
  • 36. BTG 2013 FISH and personalized medicine • Myeloma • CLL • Imatinib targets – BCR-ABL – FIP1L1-PDGFRα fusion – PDGFRβ rearrangements • MDS – 5q-
  • 37. BTG 2013