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Application of FISH in hematologic malignancies

Dr Edmond S K Ma

Department of Pathology

Hong Kong Sanatorium & Hospital

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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

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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

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

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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

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

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Acute promyelocytic leukaemia (APL) with unusual CG

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

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

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

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Chimerism status by XY-FISH

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Chronic myeloid leukaemia post-BMT donor relapse

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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

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MLL probe for rearrangement

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Characterization of chromosome 11q deletion

Ma SK et al, Leukemia 16: 953 – 955, 2002

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Southern Blot hybridization for MLL rearrangement

Ma SK et al, Leukemia 16: 953 – 955, 2002

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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

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Ph chromosome

Chronic myeloid leukaemia

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

BCR-ABL dual colour single fusion translocation probe

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Detection of fusion genes by S-FISH

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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)

Detection of fusion genes by ES-FISH

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Detection of fusion genes byES-FISH

BCR-ABL dual colour dual fusion translocation probe

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BCR-ABL dual fusion translocation probe

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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

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Three-way Ph translocation

*Courtesy of Dr. K. F. Wong, QEH

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Variant D-FISH pattern

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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

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

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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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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

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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

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BCR-ABL + 9q34 tricolour dual fusion translocation probe

BCR-ABL D-FISH

fusion

fusion

der(9) deletion

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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

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FISH and personalized medicine

• Myeloma

• CLL

• Imatinib targets– BCR-ABL– FIP1L1-PDGFR fusion– PDGFR rearrangements

• MDS– 5q-

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