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

4

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

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

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: pmanola@ipta.demokritos.gr€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

20

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)

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 circa 400 euro

• FISH circa 200 euro

• Sanger TP53 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

• 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: pmanola@ipta.demokritos.gr€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)