The Hierarchy of Somatic Mutations in Follicular Lymphoma

1. The Hierarchy of Somatic Mutations in Follicular Lymphoma Michael R. Green, Andrew Gentles, Ramesh Nair, Jonathan Irish, Ron Levy, Ash Alizadeh.

2. Follicular Lymphoma (FL) Follicular lymphoma histology black stain = T Cells (CD3) FL flow cytometry 3X B Cells (follicular structures) LymphomaB cell receptor Ig light chain (k) T Cells (infiltrating tumor) Tumor-infiltrating T cells (CD3) 10X LymphomaB cell receptor Ig light chain (k) BCL2 CD20

3. Follicular Lymphoma (FL) • Clonally rearranged immunoglobulin • Characterized by t(14;18)(q32;q21) translocation • Incurable using conventional therapy • Good candidate for molecularly-targeted therapies • Frequent mutation of MLL2 histone methyltransferase • Recurrent mutation of CREBBP histone acetyltransferase • Adapted from WHO 2008 • Solal-Celigny et al. Blood 2004;104:1258 Genetic “constants”

4. Histone Modification by MLL2 and CREBBP CREBBP/EP300 inactivating mutation MLL2/3 inactivating mutation HAT MLL ING H3 H4 K27 K4

5. Targeted Therapy: Hitting the Achilles Heel of Cancer 7 July 2012 8 July 2012 9 July 2012

6. The Theory of “Personalized Oncology” MacConaill and Garraway J. Clin. Oncol. 2010;28:5219 Roychowdhury et al. Sci. Transl. Med. 2011;3:111

7. The Reality of “Personalized Oncology” Peter C. Nowell (1976) Science.194(4260):23-8. Mutation 1 Mutation 2 Mutation 3 Catalogue of Mutations Mutation 1 Mutation 2 DRUG Mutation 3 RELAPSE

8. The Reality of “Personalized Oncology” Mutation 1 Mutation 2 Mutation 3 Catalogue of Mutations RELAPSE Mutation 1 Mutation 2 DRUG Mutation 3

9. Premise, Aim and Approach • Premise: Early genetic events are likely to be clonally dominant and represent good targets for mutation-directed therapy • Aim: To identify the hierarchy of genetic events in FL • Approach: Identify clonally dominant mutations • Consistently represented between intratumoral subpopulations • Maintained from diagnosis to relapse

10. Experimental approach FACS CD20int CD20hi T-cells DNA Extraction IgHV cloning/sequencing t(14;18) qPCR Tumor Purity Measurement Whole Exome Sequencing Sanger Validation Genetic “Constants”

11. Exome Sequencing Methodology • Constructed libraries from 3ug of DNA • Captured exome with withNimblegenSeqCap (v2) • Indexed with Illumina barcodes • 4-plexed samples on a single HiSeq 2000 lane (2x101bp)

12. Mutation Calling • Called somatic nucleotide variants (SNVs) with stringent implementation GATK: • GATK score of ≥250 in B-cells • GATK score of <50 in T-cells • Filtered silent mutations and those in dbSNP/1000genomes • Only considered cSNVs with: • ≥20X coverage in both T-cells and B-cells • <5% variant allele frequency (VAF) in T-cells • ≥5% VAF in B-cells  96% validation rate

13. Exome Sequencing and Mutation Detection • In 10 tumors from 8 cases, identified 877 coding SNVs in 572 unique genes • 95% of genes mutated in only 1/8 cases

14. Majority of Mutations in FL are Subclonal

15. Assessing sub-population skew Interrogated minor allele frequencies of 232 germ-line coding SNPs/patient (1856 total) Some noise around VAFs of heterozygous SNPs • By definition, variation in germline SNPs are false-positives* • Set thresholds to obtain confident calls At 16% VAF deviation, 85 false-positives • 4.58% error At 33% VAF deviation, 18 false-positives • 0.97% error *Possibility of LOH  over-estimating error

16. Mutation Frequencies in Tumor Subpopulations

17. More mutations, more clonal divergence

18. Illustrative Case of Diagnosis/Relapse Comparison CASE 128 • A 40 year old woman with enlarged lymph nodes and fevers found to have advanced follicular lymphoma • Diagnosis (1996) • Histology: FL grade 1 • Stage: 4B • Time to first treatment = 362 days • First treatment = CVP (1997) achieved Complete Remission (CR) • Second treatment = Id-vac (1998) • Relapse (1999) • Histology: FL grade 1 • Treatment: Fludarabine + Cyclophosphamide, CR • Second relapse in 2003, treated • Patient alive as of Feb 2013

19. Interrogation of FL Relapses: Case 128

20. Genetic Evolution of Case 128

21. Conclusions • The majority of mutations in FL are not recurrent and are subclonal. • MLL2 and TNFRSF14 • Skewed distribution in tumor cell subpopulations • Lost between diagnosis and relapse in LP-J128 • CREBBP • Equally represented in tumor cell subpopulations • Maintained between diagnosis and relapse Subclonal = Late event Clonally dominant = Early event

22. Conclusions

23. Acknowledgments Prof. Ron Levy Prof. Jonathan Irish Dr. June Myklebust Dr. ItaiKela Prof. Ash Alizadeh Shingo Kihira Dr. Chih Long Liu Prof. Sylvia Plevritis Dr. Andrew Gentles Dr. Ramesh Nair Prof. HanleeJi Dr. Eric Hopmans