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

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application of fish in hematologic malignancies

Application of FISH in hematologic malignancies

Dr Edmond S K Ma

Department of Pathology

Hong Kong Sanatorium & Hospital

BTG 2013

molecular cytogenetics
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

any role for fish in the post genomic era
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

probes
Probes

Chromosome enumeration

Locus specific

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)

fish as an investigative tool in haematological malignancies
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

acute promyelocytic leukaemia apl with unusual cg
Acute promyelocytic leukaemia (APL) with unusual CG

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

BTG 2013

slide9

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

fish some advantages
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

characterization of chromosome 11q deletion
Characterization of chromosome 11q deletion

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

BTG 2013

southern blot hybridization for mll rearrangement
Southern Blot hybridization for MLL rearrangement

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

BTG 2013

caveats of fish analysis
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

ph chromosome
Ph chromosome

Chronic myeloid leukaemia

BTG 2013

detection of bcr abl gene fusion by s fish
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

detection of bcr abl gene fusion by d fish
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

BTG 2013

three way ph translocation
Three-way Ph translocation

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

BTG 2013

derivative chromosome 9 9q deletion in cml
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

BTG 2013

slide30

9

22

der(9)

der(22)

Derivative chromosome 9 deletion in CML

Confirmation:

>10% of cells

S-FISH

Metaphase FISH

RT-PCR

Wan TS et al, J Clin Pathol 56: 471 – 474, 2003

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

Atypical BCR-ABL interphase D-FISH patterns

BTG 2013

slide33

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

slide34

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

slide35

der(9) deletion

BCR-ABL D-FISH

BCR-ABL + 9q34 tricolour dual fusion translocation probe

fusion

fusion

BTG 2013

clinical use of interphase fish in risk stratification
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

fish and personalized medicine
FISH and personalized medicine
  • Myeloma
  • CLL
  • Imatinib targets
    • BCR-ABL
    • FIP1L1-PDGFRa fusion
    • PDGFRb rearrangements
  • MDS
    • 5q-

BTG 2013

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