Interpretation of karyotyping using mitogens vs FISH vs SNP-based array in CLL. Arnon Kater Dept of Hematology AMC Amsterdam

Transcription

1 Interpretation of karyotyping using mitogens vs FISH vs SNP-based array in CLL Arnon Kater Dept of Hematology AMC Amsterdam 1

2 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

3 Number of prior CIT regimens determine Ibrutinib outcome PFS ORR P<0.046* Months *HR: (95% CI, ) P<0.05 Brown et al. ASH 2014 Poster #3331 Why? Impact of chemo on DNA stability? 3

4 ASH 2014 Cancer 2015 EFS by FISH EFS by CK EFS by FISH without CK EFS by 17p +/- CK 4

5 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

6 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 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 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

7 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

8 Principle: Microarray-based Genomic profiling (large quantity of probes: e.g. 2.7 million) 8

9 Deletion vs Copy neutral loss of heterozygosity (cnloh) Uniparental disomy, i.e: copy neutral LOH 9

10 Array CLL: Example of deletion chromosome 11 deletion 2N variants 10

11 Example : copy neutral LOH 17p 2n Heterozygous calls BB- AB- AA- 11

12 Reproducibility and interpretatation of karyotyping using mitogens vs FISH vs CGH array in chronic lymphocytic leukemia 12

13 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

14 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

15 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 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 Thessalon + 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,-

16 Group 2: selection based on karyotyping 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 Thessalon + Include FISH analysis for TP53 (+control probe) 3500,- (in case not done ye LAB Y Dr Kalliopi Manola, Laboratory of Health Physics, Radiobiology & Cytogenetics, 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)

17 Grant support from Janssen (approx ) Groups discussion Saturday (VIP registration desk)

18 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.

19 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)

20 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

21 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:

22 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

23 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

24 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

25 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

26 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

27 Costs and funding SNP array FISH Sanger TP53 circa 400 euro circa 200 euro circa 50 euro Janssen has interest in sponsoring 27

28 Beste Arnon en Clemens, Hierbij enkele slides die je kunt gebruiken bij de ERIC meeting. Juni 2016

29 Comparison of karyotyping with microarray-based profiling in CLL Marian Stevens-Kroef Clemens Mellink Arnon Kater

30 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

31 Group 1: 10 patients* with complex abnormal array profile Study lay out Group 2: 10 patients* with complex abnormal karyotype 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) 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

32 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 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 Thessalon + 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,-

33 Group 2: selection based on karyotyping 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 Thessalon + Include FISH analysis for TP53 (+control probe) 3500,- (in case not done ye LAB Y Dr Kalliopi Manola, Laboratory of Health Physics, Radiobiology & Cytogenetics, 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)

34 Discussion points (1) Number of viable frozen cells to be send for karyotyping (>10 X 10 6 ) 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)

35 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.

36 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)