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

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

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 Genetic “constants” Adapted from WHO 2008 Solal-Celigny et al. Blood 2004;104:1258

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

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

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

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

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

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

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

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

Mutation Calling GATK score of ≥250 in B-cells 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

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 Previously, populations frequencies were only way of assessing

Majority of Mutations in FL are Subclonal

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

Mutation Frequencies in Tumor Subpopulations

More mutations, more clonal divergence

Illustrative Case of Diagnosis/Relapse Comparison 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) Treatment: Fludarabine + Cyclophosphamide, CR Second relapse in 2003, treated Patient alive as of Feb 2013

Interrogation of FL Relapses: Case 128 Previously, populations frequencies were only way of assessing

Genetic Evolution of Case 128

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 Previously, populations frequencies were only way of assessing Clonally dominant = Early event

Conclusions

Acknowledgments Prof. Ron Levy Prof. Ash Alizadeh Prof. Jonathan Irish Dr. June Myklebust Dr. Itai Kela Prof. Ash Alizadeh Shingo Kihira Dr. Chih Long Liu Prof. Sylvia Plevritis Dr. Andrew Gentles Dr. Ramesh Nair Prof. Hanlee Ji Dr. Eric Hopmans