reproducibility and interpretation of karyotyping using
TRANSCRIPT
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
Brown et al. ASH 2014 Poster #3331
Months
*HR: 3.108 (95% CI, 0.959 – 10.07) †P<0.05
ORR†
P<0.046*
PFS
Why? Impact of chemo on DNA stability? 3
ASH 2014
Cancer 2015
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EFS by FISH EFS by CK
EFS by FISH without CK EFS by 17p +/- CK
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
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 CpG+IL-2
In 88% (96/109) CBA successful In 34% (33/96) structural rearrangements Confirmation CD40L results (n=14 cases)
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
Cytogenetic analysis in the 1980s-1990s using TPA revealed chromosome aberrations in 40-50% of CLL patients
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
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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
variants
2N
10
Example : copy neutral LOH 17p
BB- AB- AA-
Heterozygous calls
2n
11
12
Reproducibility and interpretatation of karyotyping using mitogens vs FISH vs
CGH array in chronic lymphocytic leukemia
• 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 AIMS
Study lay out
Group 1: 10 patients* with complex abnormal array profile
Comparison of array and
karyotyping; does karyotyping identify the patients with complex array profile.
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)
Group 2: 10 patients* with complex
abnormal karyotype 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 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
COSTS
n=10
n=10 CLL samples selected in AMC Based on microarray analysis (Agilent 180K oligo)
(blood or bone marrow)
DNA Perform microarray profiling DNA will be provided by AMC €100,-
(isolated from uncultured cells) LAB A Dr Espinet €4000,-
LAB B Radboud umc, Nijmegen, The Netherlands Marian Stevens-Kroef €4000,-
LAB C AMC, Clemens Mellink. data are available
Viable cells Perform cell culture using DSP30/IL2 Viable cells will be provided by AMC €300,-
Perform chromosome banding analysis
LAB X Thessaloniki+ Include FISH analysis for TP53 (+control probe) €3500,-
LAB Y Dr Espinet (optional also FISH) €2500,-
LAB Z subscribe €2500,-
TP53 mutation analysis Perform mutation analysis DNA will be provided by AMC
AMC €1050,-
GROUP 2 Selection based on karyotyping
n=10 samples selected in LabX* Based on chromosome banding analysis after DSP30/IL2 culture COSTS n=10
(blood or bone marrow)
LAB X* Thessaloniki
DNA Perform microarray profiling DNA will be provided by Thessaloniki €150,-
(isolated from uncultured cells) LAB A Dr Espinet €4000,-
LAB B Radboud umc, Nijmegen, The Netherlands Marian Stevens-Kroef €4000,-
LAB C CEITEC Masaryk University, Brno, Czech Republic €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 Center For Scientific Research "Demokritos", Terma Patriarchou Gregoriou, Agia Paraskevi, 15310, Athens, Greece, E-mail: [email protected]€2500,-
LAB Z Dr Espinet (optional also FISH) €2500,-
TP53 mutation analysis Perform mutation analysis DNA will be provided by Thessaloniki
LAB X Thessaloniki €1500,- (maximal)
Group 2: selection based on karyotyping
• 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
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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
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)
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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 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)
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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)
• ?
• ?
26
Costs and funding
• SNP array circa 400 euro
• FISH circa 200 euro
• Sanger TP53 circa 50 euro
Janssen has interest in sponsoring
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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
• 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 AIMS
Study lay out Group 1: 10 patients* with complex
abnormal array profile Comparison of array and
karyotyping; does karyotyping identify the patients with complex array profile.
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)
Group 2: 10 patients* with complex
abnormal karyotype 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 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
COSTS
n=10
n=10 CLL samples selected in AMC Based on microarray analysis (Agilent 180K oligo)
(blood or bone marrow)
DNA Perform microarray profiling DNA will be provided by AMC €100,-
(isolated from uncultured cells) LAB A Dr Espinet €4000,-
LAB B Radboud umc, Nijmegen, The Netherlands Marian Stevens-Kroef €4000,-
LAB C AMC, Clemens Mellink. data are available
Viable cells Perform cell culture using DSP30/IL2 Viable cells will be provided by AMC €300,-
Perform chromosome banding analysis
LAB X Thessaloniki+ Include FISH analysis for TP53 (+control probe) €3500,-
LAB Y Dr Espinet (optional also FISH) €2500,-
LAB Z subscribe €2500,-
TP53 mutation analysis Perform mutation analysis DNA will be provided by AMC
AMC €1050,-
GROUP 2 Selection based on karyotyping
n=10 samples selected in LabX* Based on chromosome banding analysis after DSP30/IL2 culture COSTS n=10
(blood or bone marrow)
LAB X* Thessaloniki
DNA Perform microarray profiling DNA will be provided by Thessaloniki €150,-
(isolated from uncultured cells) LAB A Dr Espinet €4000,-
LAB B Radboud umc, Nijmegen, The Netherlands Marian Stevens-Kroef €4000,-
LAB C CEITEC Masaryk University, Brno, Czech Republic €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 Center For Scientific Research "Demokritos", Terma Patriarchou Gregoriou, Agia Paraskevi, 15310, Athens, Greece, E-mail: [email protected]€2500,-
LAB Z Dr Espinet (optional also FISH) €2500,-
TP53 mutation analysis Perform mutation analysis DNA will be provided by Thessaloniki
LAB X Thessaloniki €1500,- (maximal)
Group 2: selection based on karyotyping
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)