Application of fish in hematologic malignancies
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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

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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

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

BTG 2013


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

  • 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


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


Acute promyelocytic leukaemia (APL) with unusual CG

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

BTG 2013


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


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


Chimerism status by XY-FISH

BTG 2013


Chronic myeloid leukaemia post-BMT donor relapse

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

BTG 2013


MLL probe for rearrangement

BTG 2013


Characterization of chromosome 11q deletion

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

BTG 2013


Southern Blot hybridization for MLL rearrangement

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

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

BTG 2013


Ph chromosome

Chronic myeloid leukaemia

BTG 2013


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


BCR-ABL dual colour single fusion translocation probe


Detection of fusion genes by S-FISH

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)

BTG 2013


Detection of fusion genes by ES-FISH


Detection of fusion genes byES-FISH

BTG 2013


BCR-ABL dual colour dual fusion translocation probe


BCR-ABL dual fusion translocation probe

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

BTG 2013


Three-way Ph translocation

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

BTG 2013


Variant D-FISH pattern

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

BTG 2013


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


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


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


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


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

  • 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

  • Myeloma

  • CLL

  • Imatinib targets

    • BCR-ABL

    • FIP1L1-PDGFRa fusion

    • PDGFRb rearrangements

  • MDS

    • 5q-

BTG 2013


BTG 2013


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