Interpretation of karyotyping using
mitogens vs FISH vs SNP-based array
in CLL
Arnon Kater
Dept of Hematology
AMC Amsterdam
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Introduction
Accepted diagnostic workup CLL prior to treatment
• FISH 13q, tris 12, 11q and 17p
• TP53 mutation according to ERIC guidelines
Highly valuable to predict outcome for CIT
Also valuable to predict TKI outcome?
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Number of prior CIT regimens determine Ibrutinib
outcome
ORR†
PFS
P<0.046*
Months
†P<0.05
*HR: 3.108 (95% CI, 0.959 – 10.07)
Brown et al. ASH 2014 Poster #3331
Why? Impact of chemo on DNA stability?
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ASH 2014
Cancer 2015
EFS by FISH
EFS by FISH without CK
EFS by CK
EFS by 17p +/- CK
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Questions
• Should we care about complex cytogenetic abberations?
– according to industry: Yes
• How reproducible is CK using metaphase cytogenetic
analysis following mitogens?
• Can SNP-based array robustly identify patients with a
complex karyotype?
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Chromosome banding analysis
(CBA) in CLL
Cytogenetic analysis in the 1980s-1990s
using TPA revealed chromosome
aberrations in 40-50% of CLL patients
Study
Type of study
Stimulation* Karyotyping (CBA)**
Thompson et al 2015
CBA + FISH
CpG+IL-2
In 90% (56/63) CBA successful
Dicker et al 2006
CBA + FISH
CpG+IL-2
In 80% (106/132) CBA successful
Mayr et al 2006
CBA + FISH
CD40L
In 88% (96/109) CBA successful
In 34% (33/96) structural
rearrangements
Confirmation CD40L results (n=14 cases)
CpG+IL-2
Haferlach et al 2007
CBA + FISH
CpG+IL-2
In 98% (500/506) CBA successful
In 83% (415/500) abnormal karyotypes
Rigolin et al 2012
CBA + FISH
CpG+IL-2
In 36% (30/84) abnormal karyotypes
Put et al 2009
CBA + FISH
TPA
CPG+IL-2
In 38% (82/217) abnormal karyotypes
In 51% (111/217) abnormal karyotypes
Puiggros et al 2012
CBA + FISH + aCGH
TPA
In 31% (22/70) abnormal karyotypes
Van den Neste et al 2007
CBA + FISH
TPA
In 62% (40/65) abnormal karyotypes
CpG oligonucleotides (DSP30) ; IL-2, Interleukin-2
CD40 Ligand ; TPA, 12-O-tetradecanoylphorbol-13-acetate
* Cultured for 72 hours
** Success defined as at least 20 metaphases
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Pitfalls CBA in CLL
• Whole variety of mitogens published: CPG+IL2; CD40L+IL2; LPS;
TPA, never compared
• Success rates highly vary
- Due to previous therapy?
- Outgrowth more aggressive clones?
• If success is a determination of DNA instability than lower rates
of succes are expected at earlier treatment lines
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Principle: Microarray-based Genomic profiling
(large quantity of probes: e.g. 2.7 million)
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Deletion vs Copy neutral loss of heterozygosity (cnLOH)
Uniparental disomy,
i.e: copy neutral LOH
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Array CLL: Example of deletion chromosome 11
deletion
2N
variants
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Example : copy neutral LOH 17p
Heterozygous calls
2n
BBABAA11
Methods
Comparison of Chromosome banding analysis
following mitogens vs SNP-array in well
characterized samples
Phase 1. Compare the different assays on the same samples: Inter-assay comparison
Phase 2. Compare results of the same assays in different labs: Intra-assay comparison
Phase 3. Compare results with clinical outcome
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Karyotyping
(n=30 patients)
• Karyotyping results (chromosome banding analysis)
based on stimulated cultures (e.g. CpG+IL-2)
- 10 cases with complex karyotype without (visible)
del(17p)
- 10 cases with complex karyotype with (visible) del(17p)
• 5-10(?) normal karyotypes (how many?)
Practical issues
• Make use of already existing karyotyping data
Thomson et al, Cancer 2015; 121:3612-21
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SNP-based array
(n=30 patients)
• Microarray analysis on DNA from the same patients
as used for karyotyping
Practical issues
• Make use of stored DNA (if present), or
• Isolate DNA from frozen cell suspensions (cell
culture)
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FISH
(n=30 patients)
• Confirmation of karyotyping results
• Routine CLL FISH-panel for detection of
–
–
–
–
Deletion 13q14
Trisomy 12
Deletion 11q22-23
Deletion 17p
Practical issues
• Make use of already existing FISH-data present in
participating laboratories which did karyotyping, or
• Perform FISH using frozen cell suspensions (cell
culture)
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SNP-array in the Netherlands
• Limit of detection: detection of copy number
abnormalities present in as few as 16% of the cells
• Validated on two different array platforms
- Cytoscan Affymetrix
- HumanOmniExpress12v1.0 Illumina
• Identification of focal deletions and copy neutral
losses of heterozygosity
Stevens-Kroef et al. Molecular Cytogenetics 2014, 7:3
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TP53 mutation analysis
• Perform TP53 analysis on DNA from the same
patients as used for karyotyping (Sanger or NGS)
Practical issues:
• Make use of stored DNA (if present), or
• Isolate DNA from frozen cell suspensions (cell
culture)
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Participating laboratories
• AMC, Amsterdam (Arnon Kater, Clemens Mellink)
• RadboudUMC, Nijmegen (Patricia Groenen, Marian Stevens)
• Laboratori de Citogenètica, Hospital del Mar, Barcelona (Blanca
Sola)
• University Hospital Vall d'Hebron, Barcelona (Fransesc Bosch)
• ?
• ?
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Costs and funding
• SNP array
• FISH
• Sanger TP53
circa 400 euro
circa 200 euro
circa 50 euro
 Janssen has interest in sponsoring
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