Interpretation of karyotyping using
mitogens vs FISH vs SNP-based array
in CLL
Arnon Kater
Dept of Hematology
AMC Amsterdam
1
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?
2
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?
3
ASH 2014
Cancer 2015
EFS by FISH
EFS by FISH without CK
EFS by CK
EFS by 17p +/- CK
4
Questions
• Should we care about complex cytogenetic abberations?
– Different groups demonstrated prognostic value
– Never been introduced in official guidelines (e.g. CCMO)
– 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?
5
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
6
Pitfalls CBA in CLL
• Whole variety of mitogens published: CPG+IL2; CD40L+IL2; LPS;
TPA; head to head comparisons lacking
• Success rates 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
7
Principle: Microarray-based Genomic profiling
(large quantity of probes: e.g. 2.7 million)
8
Deletion vs Copy neutral loss of heterozygosity (cnLOH)
Uniparental disomy,
i.e: copy neutral LOH
9
Array CLL: Example of deletion chromosome 11
deletion
2N
variants
10
Example : copy neutral LOH 17p
Heterozygous calls
2n
BBABAA11
Reproducibility and interpretatation of
karyotyping using mitogens vs FISH vs
CGH array in chronic lymphocytic
leukemia
12
STUDY AIMS
• comprehensive review of the literature related to karyotyping, FISH, and
microarray profiling in CLL will be established (as proposed by B.
Espinet/A. Puiggros).
• Find out if there is a superior technique / mitogen to perform
karyotyping.
• Test the reproducibility (and diagnostic yield) of chromosome banding
analysis using mitogens DSP30/IL2.
• Test the reproducibility and diagnostic yield of microarray profiling in
identifying patients with a complex karyotype (as revealed by CBA).
• The values of complex karyotype and complex array profile in predicting
outcome will be evaluated in larger study group
Study lay out
Group 1: 10 patients* with complex
abnormal array profile
Group 2: 10 patients* with complex
abnormal karyotype
Comparison of array and
karyotyping; does karyotyping
identify the patients with
complex array profile.
Comparison of karyotyping and array;
does array identify the patients
with a complex karyotype.
Reproducibility of microarray by
testing on other array platforms
/labs (Lab A, Lab B and Lab C)
Reproducibility of microarray by
testing on other array platforms
/labs (Lab A, Lab B and Lab C)
Reproducibility of karyotyping by
testing on different labs (Lab X, Y
and Z)
Reproducibility of karyotyping by
testing on different labs (Lab X, Y
and Z)
* The TP53 mutation status and 17p status is known or will be determined
Group 1: selection based on microarray
GROUP 1 Selection based on microarray profiling
n=10 CLL samples selected in AMC
Based on microarray analysis (Agilent 180K oligo)
(blood or bone marrow)
COSTS
n=10
DNA
(isolated from uncultured cells)
Perform microarray profiling
DNA will be provided by AMC
LAB A
Dr Espinet
LAB B
Radboud umc, Nijmegen, The Netherlands Marian Stevens-Kroef
LAB C
AMC, Clemens Mellink. data are available
€100,€4000,€4000,-
Viable cells
Perform cell culture using DSP30/IL2
Viable cells will be provided by AMC
Perform chromosome banding analysis
LAB X
Thessaloniki
+ Include FISH analysis for TP53 (+control probe)
LAB Y
Dr Espinet (optional also FISH)
LAB Z
subscribe
€300,-
TP53 mutation analysis
Perform mutation analysis
AMC
€3500,€2500,€2500,-
DNA will be provided by AMC
€1050,-
Group 2: selection based on karyotyping
GROUP 2 Selection based on karyotyping
n=10 samples selected in LabX*
(blood or bone marrow)
LAB X*
Based on chromosome banding analysis after DSP30/IL2 culture
COSTS n=10
Thessaloniki
DNA
(isolated from uncultured cells)
Perform microarray profiling
DNA will be provided by Thessaloniki
LAB A
Dr Espinet
LAB B
Radboud umc, Nijmegen, The Netherlands Marian Stevens-Kroef
LAB C
CEITEC Masaryk University, Brno, Czech Republic
€150,€4000,€4000,€4000,-
Viable cells
Perform cell culture using DSP30/IL2
Viable cells will be provided by lab subcribe
€200,Perform chromosome banding analysis
LAB X
Thessaloniki
+ Include FISH analysis for TP53 (+control probe)
€3500,- (in case not done yet)
LAB Y
Dr Kalliopi Manola, Laboratory of Health Physics, Radiobiology & Cytogenetics, National
€2500,- Center For Scientific Re
LAB Z
Dr Espinet (optional also FISH)
€2500,-
TP53 mutation analysis
Perform mutation analysis
LAB X
Thessaloniki
DNA will be provided by Thessaloniki
€1500,- (maximal)
• Grant support from Janssen (approx €55.000)
• Groups discussion Saturday 16.00
(VIP registration desk)
Discussion points (definition of complexity)
• A karyotype will be defined as complex when
≥3 chromosomal aberrations are observed
(structural and/or numerical) (Baliakas et al
2014).
• An array profile will be defined as complex
when ≥3 copy number aberrations > 5 Mb
are observed.
Discussion points
• Evaluation of value of complex karyotype /
complex array profile in predicting outcome.
• Perform Literature overview
• Study larger number of cases (with clinical
follow up data)
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
20
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
21
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)
22
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)
23
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
24
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)
25
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)
• ?
• ?
26
Costs and funding
• SNP array
• FISH
• Sanger TP53
circa 400 euro
circa 200 euro
circa 50 euro
 Janssen has interest in sponsoring
27
Beste Arnon en Clemens,
Hierbij enkele slides die je kunt
gebruiken bij de ERIC meeting.
Juni 2016
Comparison of karyotyping with
microarray-based profiling in CLL
Marian Stevens-Kroef
Clemens Mellink
Arnon Kater
STUDY AIMS
• Find out if there is a superior technique / mitogen to perform
karyotyping. comprehensive review of the literature related to
karyotyping, FISH, and microarray profiling in CLL will be established (as
proposed by B. Espinet/A. Puiggros).
• Test the reproducibility (and diagnostic yield) of chromosome banding
analysis using mitogens DSP30/IL2.
• Test the reproducibility and diagnostic yield of microarray profiling in
identifying patients with a complex karyotype (as revealed by CBA).
• The values of complex karyotype and complex array profile in predicting
outcome will be evaluated in larger study group
Study lay out
Group 1: 10 patients* with complex
abnormal array profile
Group 2: 10 patients* with complex
abnormal karyotype
Comparison of array and
karyotyping; does karyotyping
identify the patients with
complex array profile.
Comparison of karyotyping and array;
does array identify the patients
with a complex karyotype.
Reproducibility of microarray by
testing on other array platforms
/labs (Lab A, Lab B and Lab C)
Reproducibility of microarray by
testing on other array platforms
/labs (Lab A, Lab B and Lab C)
Reproducibility of karyotyping by
testing on different labs (Lab X, Y
and Z)
Reproducibility of karyotyping by
testing on different labs (Lab X, Y
and Z)
* The TP53 mutation status and 17p status is known or will be determined
Group 1: selection based on microarray
GROUP 1 Selection based on microarray profiling
n=10 CLL samples selected in AMC
Based on microarray analysis (Agilent 180K oligo)
(blood or bone marrow)
COSTS
n=10
DNA
(isolated from uncultured cells)
Perform microarray profiling
DNA will be provided by AMC
LAB A
Dr Espinet
LAB B
Radboud umc, Nijmegen, The Netherlands Marian Stevens-Kroef
LAB C
AMC, Clemens Mellink. data are available
€100,€4000,€4000,-
Viable cells
Perform cell culture using DSP30/IL2
Viable cells will be provided by AMC
Perform chromosome banding analysis
LAB X
Thessaloniki
+ Include FISH analysis for TP53 (+control probe)
LAB Y
Dr Espinet (optional also FISH)
LAB Z
subscribe
€300,-
TP53 mutation analysis
Perform mutation analysis
AMC
€3500,€2500,€2500,-
DNA will be provided by AMC
€1050,-
Group 2: selection based on karyotyping
GROUP 2 Selection based on karyotyping
n=10 samples selected in LabX*
(blood or bone marrow)
LAB X*
Based on chromosome banding analysis after DSP30/IL2 culture
COSTS n=10
Thessaloniki
DNA
(isolated from uncultured cells)
Perform microarray profiling
DNA will be provided by Thessaloniki
LAB A
Dr Espinet
LAB B
Radboud umc, Nijmegen, The Netherlands Marian Stevens-Kroef
LAB C
CEITEC Masaryk University, Brno, Czech Republic
€150,€4000,€4000,€4000,-
Viable cells
Perform cell culture using DSP30/IL2
Viable cells will be provided by lab subcribe
€200,Perform chromosome banding analysis
LAB X
Thessaloniki
+ Include FISH analysis for TP53 (+control probe)
€3500,- (in case not done yet)
LAB Y
Dr Kalliopi Manola, Laboratory of Health Physics, Radiobiology & Cytogenetics, National
€2500,- Center For Scientific Re
LAB Z
Dr Espinet (optional also FISH)
€2500,-
TP53 mutation analysis
Perform mutation analysis
LAB X
Thessaloniki
DNA will be provided by Thessaloniki
€1500,- (maximal)
Discussion points (1)
• Number of viable frozen cells to be send for karyotyping (>10 X 106)
• Amount of DNA to be send for genomic array (>500 ng)
• If FISH (17p/TP53) already done it has not be repeated
• If TP53 mutation analysis is already done this has not be repeated.
• Is sanger sequencing (minimal sensitivity 10%) OKE, or should we
apply for next generation sequencing?
• Number of cases for study technical issues (karyotyping, mitogen,
array)
Discussion points (definition of complexity)
• A karyotype will be defined as complex when
≥3 chromosomal aberrations are observed
(structural and/or numerical) (Baliakas et al
2014).
• An array profile will be defined as complex
when ≥3 copy number aberrations > 5 Mb
are observed.
Discussion points
• Evaluation of value of complex karyotype /
complex array profile in predicting outcome.
• Perform Literature overview
• Study larger number of cases (with clinical
follow up data)