Comparison of FISH, CpG-stimulation, chromosomal microarray and mate pair sequencing in 20 patients with CLL or lymphoma

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1 Comparison of FISH, CpG-stimulation, chromosomal microarray and mate pair sequencing in 20 patients with CLL or lymphoma Stephanie Smoley Cancer Genomics Consortium August 9 th, MFMER slide-1

2 Chronic Lymphocytic Leukemia (CLL)/ Small Lymphocytic Lymphoma (SLL) Disorder of the B-lymphocytes Most common form of adult leukemia/lymphoma in Western countries average age at diagnosis 71 years male:female ratio 2:1 Highly variable clinical course survival ranging 2-20 years 2016 MFMER slide-2

3 CLL staging Rai Stage Risk Characteristics Survival (years) 0 Low lymphocytosis >13 I Intermediate lymphocytosis plus lymphadenopathy 8 II Intermediate lymphocytosis plus hepatomegaly or splenomegaly w/ or w/o lymphadenopathy 5 III High lymphocytosis plus anemia w/ or w/o hepatomegaly, splenomegaly or lymphadenopathy 2 IV High lymphocytosis plus thrombocytopenia 1 Rai et al., Blood MFMER slide-3

4 Dohner Classification 17p- Nl/+12 11q- 13q- FISH abnormality 17p q Normal q- 133 Median Survival (months) Dohner et al., NEJM 2000 Shanafelt et al., J Clin Oncol MFMER slide-4

5 Why is prognostication so important? No cure has been found For asymptomatic patients watch and wait Many patients with CLL aren t healthy enough for treatment Targeted therapy 17p deletions Evolution 2016 MFMER slide-5

6 Cytogenetic testing for CLL FISH Limited targets ~30% are normal CpG stimulated karyotype Whole genome Low resolution Chromosomal microarray Whole genome High resolution Cannot detect rearrangements Mate Pair Sequencing Whole genome High resolution CNV and rearrangements 2016 MFMER slide-6

7 20 Test Patients RFR CLL or lymphoma Not all patients were confirmed to have CLL Chosen based on suitability for mate pair testing Highly enriched for IGH rearrangements 2016 MFMER slide-7

8 FISH vs. MPseq (n=20) 1* matched 1 case w/ FISH advantage: MPseq missed a low level 13q-, (*but identified the BCL2 partner as IGL) 4 cases w/ MPseq advantage: 2 reclassified 13q-x1 to 13q-x2 1 identified the IGH partner as BCL11A 1 identified a CCND1/IGH not visible by FISH testing FISH exceeds MPseq FISH & MPseq match MPseq exceeds FISH 2016 MFMER slide-8

9 CpG vs. MPseq (n=19) 1* match 1 case w/ CpG advantage MPseq missed low level add(18), (*but identified the IGH partner as BCL11A) 2 cases w/ MPseq advantage detected 13q- and identified the IGH partner as BCL11A add(14) characterized as BCL11A/IGH CpG exceeds MPseq CpG & MPseq match MPseq exceeds CpG 2016 MFMER slide-9

10 Example #1 CpG advantage Referrred for testing CLL FISH 11q- (77%) IGH separation (95.5%) 2016 MFMER slide-10

11 Example # ,XY,t(2;14)(p15;q32),del(11)(q22q23),add(18)(q21)[cp10] 2016 MFMER slide-11

12 Example #1 ATM IGH CpG ,XY,t(2;14)(p15;q32),del(11)(q22q23),add(18)(q21)[cp10] 2016 MFMER slide-12

13 Example #1 SERTAD2 SERTAD2 IGH locus IGH locus 2016 MFMER slide-13

14 Example # MFMER slide-14

15 Example #1 BCL11A/IGH 2016 MFMER slide-15

16 Example #2 MPseq advantage Referred for testing CLL CpG - 46,XY,t(5;6)(q13;q23)[10]/46,XY[10] FISH 13q-x1 (56%) 2016 MFMER slide-16

17 Example #2 CpG - 46,XY,t(5;6)(q13;q23)[10]/46,XY[10] FISH 13q-x1 (56%) 2016 MFMER slide-17

18 Example #2 CN=1.4 CN=0.8 CpG - 46,XY,t(5;6)(q13;q23)[10]/46,XY[10] FISH 13q-x1 (56%) 2016 MFMER slide-18

19 Example #2 Abbott Molecular D13S319 probe 2016 MFMER slide-19

20 Example #3 MPseq advantage Referred for testing CLL/SLL Flow cytometry indicative of follicular lymphoma or marginal zone lymphoma FISH for CCND1/IGH, CCND2, MYC and 7qwere all negative FISH showed ~90% 17p MFMER slide-20

21 Example #3 46,XX,+3,add(3)(p11)x2,t(5;20)(p13;q11.2), MFMER slide-21

22 Example #3 CpG - 46,XX,+3,add(3)(p11)x2,t(5;20)(p13;q11.2),-13 MPseq 46,XX,+3,add(3)(q10)x2,t(5;13)(q11.2;q22.1),t(11;14)(q13.3;q32.33) 2016 MFMER slide-22

23 Example #3 CpG - 46,XX,+3,add(3)(p11)x2,t(5;20)(p13;q11.2),-13 MPseq 46,XX,+3,add(3)(q10)x2,t(5;13)(q11.2;q22.1),t(11;14)(q13.3;q32.33) 2016 MFMER slide-23

24 Example #3 CpG - 46,XX,+3,add(3)(p11)x2,t(5;20)(p13;q11.2),-13 MPseq 46,XX,+3,add(3)(q10)x2,t(5;13)(q11.2;q22.1),t(11;14)(q13.3;q32.33) 2016 MFMER slide-24

25 Example #3 CCND1 CCND1 IGH locus IGH locus 2016 MFMER slide-25

26 Example #3 t(11;14)(q13.3;q32.33) Atlas of Genetics & Cytogen 2016 MFMER slide-26

27 chr11 chr14 t(11;14)(q13.3;q32.33) 2016 MFMER slide-27

28 chr11 chr14 Abbott CCND1 D-FISH probe Abbott IGH-XT D-FISH probe t(11;14)(q13.3;q32.33) 2016 MFMER slide-28

29 Abbott CCND1 D-FISH probe Insertional translocation Abbott IGH-XT D-FISH probe t(11;14)(q13.3;q32.33) 2016 MFMER slide-29

30 Conclusions: No single method provides all information FISH is more sensitive for follow-up and MRD MPseq is a useful diagnostic tool detecting more than any other method: Variant translocations (BCL11A, IGK, IGL) More accurately classifies heterozygous vs homozygous 13q deletions Novel changes 2016 MFMER slide-30

31 Acknowledgements: Genomics Lab: Umut Aypar, PhD Linda Baughn, PhD Patricia Greipp, DO Katherine Geiersbach, MD Nicole Hoppman, PhD Robert Jenkins, MD, PhD Hutton Kearney, PhD Rhett Ketterling, MD Beth Pitel, MS Jeannette Rustin William Sukov, MD Erik Thorland, PhD Daniel Van Dyke, PhD Bioinformatics: Eric Klee, PhD Saranya Sankaranarayan, MS Satwica Yerneni Biomarker Discovery: Athanasios (Saki) Gaitatzes, MS Sarah Johnson, MS James Smadbeck, PhD George Vasmatzis, PhD Clinical Genome Sequencing Lab: Joseph Blommel, MS Lisa Peterson 2016 MFMER slide-31

32 Questions? I think you should be more explicit here in step two 2016 MFMER slide-32