Post on 02-Jan-2016
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BTG 2013
Application of FISH in hematologic malignancies
Dr Edmond S K Ma
Department of Pathology
Hong Kong Sanatorium & Hospital
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
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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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