Follicular lymphoma (FL) is an incurable malignancy1, with transformation to an aggressive subtype being a critical event during disease progression. Here we performed whole genome or exome sequencing on 10 FL-transformed FL pairs, followed by deep sequencing of 28 genes in an extension cohort and report the key events and evolutionary processes governing initiation and transformation. Tumor evolution occurred through either a ‘rich’ or ‘sparse’ ancestral common progenitor clone (CPC). We identified recurrent mutations in linker histones, JAK-STAT signaling, NF-κB signaling and B-cell development genes. Longitudinal analyses revealed chromatin regulators (CREBBP, EZH2 and MLL2) as early driver genes, whilst mutations in EBF1 and regulators of NF-κB signaling (MYD88 and TNFAIP3) were gained at transformation. Collectively, this study provides novel insights into the genetic basis of follicular lymphoma, the clonal dynamics of transformation and suggests that personalizing therapies to target key genetic alterations within the CPC represents an attractive therapeutic strategy.

Follicular lymphoma is an incurable B-cell malignancy1 characterized by the t(14;18) and mutations in one or more components of the epigenome2,3. Whilst frequent gene mutations in signaling pathways, including JAK-STAT, NOTCH and NF-κB, have also been defined2-7, the spectrum of these mutations typically overlap with the closely-related diffuse large B cell lymphoma (DLBCL)6-13. A combination of discovery exome and extended targeted sequencing revealed recurrent somatic mutations in RRAGC uniquely enriched in FL patients (17%). More than half of the mutations preferentially co-occurred with ATP6V1B2 and ATP6AP1 mutations, components of the vacuolar H+-adenosine triphosphate ATPase (v-ATPase) known to be necessary for amino acid-induced mTORC1 activation. The RagC mutants increased raptor binding whilst rendering mTORC1 signaling resistant to amino acid deprivation. Collectively, the activating nature of the RRAGC mutations, their existence within the dominant clone and stability during disease progression supports their potential as an excellent candidate to be therapeutically exploited.

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Key Points• EZH2 mutations occur in more than 25% of follicular lymphoma patients.• Mutations predominantly represent an early/clonal event in the pathogenesis of the disease.Gain of function mutations in the H3K27 methyltransferase EZH2 represent a promising therapeutic target in germinal center lymphomas. In this study, we assessed the frequency and distribution of EZH2 mutations in a large cohort of patients with follicular lymphoma (FL) (n 5 366) and performed a longitudinal analysis of mutation during the disease progression from FL to transformed FL (tFL) (n 5 33). Mutations were detected at 3 recurrent mutation hot spots (Y646, A682, and A692) in 27% of FL cases with variant allele frequencies (VAF) ranging from 2% to 61%. By comparing VAF of EZH2 with other mutation targets (CREBBP, MLL2, TNFRSF14, and MEF2B), we were able to distinguish patients harboring clonal EZH2 mutation from rarer cases with subclonal mutations. Overall, the high incidence of EZH2 mutations in FL and their stability during disease progression makes FL an appropriate disease to evaluate EZH2 targeted therapy. (Blood. 2013; 122(18):3165-3168) Introduction Next-generation sequencing (NGS) studies have shown frequent mutations in epigenetic regulators in almost all cases of follicular lymphoma (FL).1,2 These include EZH2, the catalytic subunit of PRC2, which catalyzes trimethylation of lysine 27 on histone H3 (H3K27me3), a repressive chromatin mark.3 Somatic gain-of-function mutations of EZH2 at codon Y646 (previously Y641) were identified in 7% to 22% of FLs and germinal center B-cell type diffuse large B-cell lymphomas leading to elevated H3K27 trimethylation [4][5][6][7][8] with mutations at codons A682 and A692 described in isolated cases of diffuse large B-cell lymphomas. 2,9-11 As highly selective EZH2 inhibitors have now been developed, [12][13][14] we set out to assess EZH2 mutation status, the effect of mutations on global gene expression, and the clonal representation of EZH2 mutations as the disease progresses. Study design Patient samplesGenomic DNA from 181 diagnostic FL patients with accompanying clinical and gene expression data 15 were obtained through the Lymphoma/Leukemia Molecular Profiling Project consortium. DNA from 185 additional FL patients (56 obtained at diagnosis and 129 at relapse) and 33 paired FL and transformed FL (tFL) samples were sourced from the tissue archive at the Barts Cancer The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.The publisher or recipient acknowledges right of the US government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.BLOOD, 31 OCTOBER 2013 x VOLUME 122, NUMBER 18 3165For personal use only. on May 9, 2018. by guest www.bloodjournal.org From Institute. The study was approved by the London Research Ethical Committee (05/Q0605/140) and was conducted in accordance with the...