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
Chromosome painting
BCR-ABL dual colour dual fusion
Multicolour FISH 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
S-FISH
D-FISH:
1R2G1F pattern
D-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

Derivative chromosome 9 deletion in CML
9
22 der(9) der(22)
Wan TS et al , J Clin Pathol 56: 471 – 474, 2003
Confirmation:
>10% of cells
S-FISH
Metaphase FISH
RT-PCR

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
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-PDGFR a fusion
– PDGFR b rearrangements
• MDS
– 5q-
BTG 2013

BTG 2013