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

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


Application of fish in hematologic malignancies

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


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


Application of fish in hematologic malignancies

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


Application of fish in hematologic malignancies

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

Chimerism status by XY-FISH

BTG 2013


Chronic myeloid leukaemia post bmt donor relapse

Chronic myeloid leukaemia post-BMT donor relapse

BTG 2013


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


Mll probe for rearrangement

MLL probe for rearrangement

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


Application of fish in hematologic malignancies

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


Bcr abl dual colour single fusion translocation probe

BCR-ABL dual colour single fusion translocation probe


Detection of fusion genes by s fish

Detection of fusion genes by S-FISH

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 fusion genes by es fish

Detection of fusion genes by ES-FISH


Detection of fusion genes byes fish

Detection of fusion genes byES-FISH

BTG 2013


Bcr abl dual colour dual fusion translocation probe

BCR-ABL dual colour dual fusion translocation probe


Bcr abl dual fusion translocation probe

BCR-ABL dual fusion translocation probe

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


Variant d fish pattern

Variant D-FISH pattern

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


Application of fish in hematologic malignancies

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


Application of fish in hematologic malignancies

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


Application of fish in hematologic malignancies

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


Application of fish in hematologic malignancies

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


Application of fish in hematologic malignancies

BTG 2013


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