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

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Presentation on theme: "BTG 2013 Application of FISH in hematologic malignancies Dr Edmond S K Ma Department of Pathology Hong Kong Sanatorium & Hospital."— Presentation transcript:

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

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9 Cryptic insertion of BCR at 9q34 in CML Wan TS et al, Leukemia 18: 161 – 2, 2004 D-FISH: 1R2G1F pattern S-FISHES-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 nuclei ( %) –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: , 2000 –Huntly et al. Blood 98: , 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

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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 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

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

36 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

37 BTG 2013 FISH and personalized medicine Myeloma CLL Imatinib targets –BCR-ABL –FIP1L1-PDGFR fusion –PDGFR rearrangements MDS –5q-

38 BTG 2013


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