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

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 141, 2004

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

BTG 2013


Wan TS 141, 2004et al, Cancer Genet Cytogenet 121: 90 – 3, 2000


Cryptic insertion of 141, 2004BCR 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 141, 2004

BTG 2013


Chronic myeloid leukaemia post bmt donor relapse
Chronic myeloid leukaemia 141, 2004post-BMT donor relapse

BTG 2013


Fish some advantages
FISH: some advantages 141, 2004

  • 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 141, 2004 probe for rearrangement

BTG 2013


Characterization of chromosome 11q deletion
Characterization of chromosome 11q deletion 141, 2004

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

BTG 2013


Southern blot hybridization for mll rearrangement
Southern Blot hybridization for 141, 2004MLL rearrangement

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

BTG 2013


Caveats of fish analysis
Caveats of FISH analysis 141, 2004

  • 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 141, 2004

Chronic myeloid leukaemia

BTG 2013



Bcr abl dual colour single fusion translocation probe
BCR-ABL 141, 2004 dual colour single fusion translocation probe



Detection of bcr abl gene fusion by s fish
Detection of 141, 2004BCR-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




Bcr abl dual colour dual fusion translocation probe
BCR-ABL 141, 2004 dual colour dual fusion translocation probe


Bcr abl dual fusion translocation probe
BCR-ABL 141, 2004 dual fusion translocation probe

BTG 2013


Detection of bcr abl gene fusion by d fish
Detection of 141, 2004BCR-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 141, 2004

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

BTG 2013


Variant d fish pattern
Variant D-FISH pattern 141, 2004

BTG 2013


Derivative chromosome 9 9q deletion in cml
Derivative chromosome 9 (9q+) deletion in CML 141, 2004

  • 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


9 141, 2004

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 141, 2004et 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 141, 2004 + 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 141, 2004 + 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 141, 2004

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 141, 2004

  • 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 141, 2004

  • Myeloma

  • CLL

  • Imatinib targets

    • BCR-ABL

    • FIP1L1-PDGFRa fusion

    • PDGFRb rearrangements

  • MDS

    • 5q-

BTG 2013


BTG 2013 141, 2004


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