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

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
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-PDGFRα fusion
– PDGFRβ rearrangements
• MDS
– 5q-

38. BTG 2013