Application of FISH in hematologic malignancies

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Application of FISH in hematologic malignancies

  1. 1. BTG 2013 Application of FISH in hematologic malignancies Dr Edmond S K Ma Department of Pathology Hong Kong Sanatorium & Hospital
  2. 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. 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. 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. 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. 6. From Ma, Wan & Chan. Cancer Reviews Asia-Pacific 2: 131 – 141, 2004
  7. 7. BTG 2013 Acute promyelocytic leukaemia (APL) with unusual CG Wan TS et al, Cancer Genet Cytogenet 121: 90 – 3, 2000
  8. 8. Wan TS et al, Cancer Genet Cytogenet 121: 90 – 3, 2000
  9. 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. 10. BTG 2013 Chimerism status by XY-FISH
  11. 11. BTG 2013 Chronic myeloid leukaemia post-BMT donor relapse
  12. 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. 13. BTG 2013 MLL probe for rearrangement
  14. 14. BTG 2013 Characterization of chromosome 11q deletion Ma SK et al, Leukemia 16: 953 – 955, 2002
  15. 15. BTG 2013 Southern Blot hybridization for MLL rearrangement Ma SK et al, Leukemia 16: 953 – 955, 2002
  16. 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. 17. BTG 2013 Ph chromosome Chronic myeloid leukaemia
  18. 18. From Ma, Wan & Chan. Cancer Reviews Asia-Pacific 2: 131 – 141, 2004
  19. 19. BCR-ABL dual colour single fusion translocation probe
  20. 20. BTG 2013 Detection of fusion genes by S-FISH
  21. 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. 22. Detection of fusion genes by ES-FISH
  23. 23. BTG 2013 Detection of fusion genes byES-FISH
  24. 24. BCR-ABL dual colour dual fusion translocation probe
  25. 25. BTG 2013 BCR-ABL dual fusion translocation probe
  26. 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. 27. BTG 2013 Three-way Ph translocation *Courtesy of Dr. K. F. Wong, QEH
  28. 28. BTG 2013 Variant D-FISH pattern
  29. 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. 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. 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. 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. 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. 34. BTG 2013 BCR-ABL + 9q34 tricolour dual fusion translocation probe BCR-ABL D-FISH fusion fusion der(9) deletion
  35. 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. 36. BTG 2013 FISH and personalized medicine • Myeloma • CLL • Imatinib targets – BCR-ABL – FIP1L1-PDGFRα fusion – PDGFRβ rearrangements • MDS – 5q-
  37. 37. BTG 2013

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