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

2. 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

3. 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

4. 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)

5. 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

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

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

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

9. 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

10. Chimerism status by XY-FISH
BTG 2013

11. Chronic myeloid leukaemia post-BMT donor relapse
BTG 2013

12. 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

13. MLL probe for rearrangement
BTG 2013

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

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

16. 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

17. Chronic myeloid leukaemia
Ph chromosome
Chronic myeloid leukaemia
BTG 2013

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

19. BCR-ABL dual colour single fusion translocation probe

20. Detection of fusion genes by S-FISH
BTG 2013

21. 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

22. Detection of fusion genes by ES-FISH

23. Detection of fusion genes byES-FISH
BTG 2013

24. BCR-ABL dual colour dual fusion translocation probe

25. BCR-ABL dual fusion translocation probe
BTG 2013

26. 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
BTG 2013

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

28. Variant D-FISH pattern
BTG 2013

29. 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
BTG 2013

30. 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

31. 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

33. 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

34. 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

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

36. 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

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

38. BTG 2013