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.

Key Points• Richter syndrome has genomic complexity intermediate between chronic lymphocytic leukemia and diffuse large B-cell lymphoma.• Inactivation of TP53 and of CDKN2A is a main mechanism in the transformation to Richter syndrome.Richter syndrome (RS) occurs in up to 15% of patients with chronic lymphocytic leukemia (CLL). Although RS, usually represented by the histologic transformation to a diffuse large B-cell lymphoma (DLBCL), is associated with a very poor outcome, especially when clonally related to the preexisting CLL, the mechanisms leading to RS have not been clarified. To better understand the pathogenesis of RS, we analyzed a series of cases including 59 RS, 28 CLL phase of RS, 315 CLL, and 127 de novo DLBCL. RS demonstrated a genomic complexity intermediate between CLL and DLBCL. Cell-cycle deregulation via inactivation of TP53 and of CDKN2A was a main mechanism in the histologic transformation from CLL phase, being present in approximately one half of the cases, and affected the outcome of the RS patients. A second major subgroup was characterized by the presence of trisomy 12 and comprised one third of the cases. Although RS shared some of the lesions seen in de novo DLBCL, its genomic profile was clearly separate. The CLL phase preceding RS had not a generalized increase in genomic complexity compared with untransformed CLL, but it presented clear differences in the frequency of specific genetic lesions. (Blood. 2013;122(15):2673-2682

Summary MicroRNAs (miRNA, miR) are negative regulators of gene expression that play an important role in diverse biological processes such as development, cell growth, apoptosis and haematopoiesis, suggesting their association with cancer. Here we analysed the expression signatures of 157 miRNAs in 58 diffuse large B‐cell lymphoma (DLBCL), 46 follicular lymphoma (FL) and seven non‐neoplastic lymph nodes (LN). Comparison of the possible combinations of DLBCL‐, FL‐ and LN resulted in specific DLBCL‐ and FL‐signatures, which include miRNAs with previously published function in haematopoiesis (MIRN150 and MIRN155) or tumour development (MIRN210, MIRN10A, MIRN17‐5P and MIRN145). As compared to LN, some miRNAs are differentially regulated in both lymphoma types (MIRN155, MIRN210, MIRN106A, MIRN149 and MIRN139). Conversely, some miRNAs show lymphoma‐specific aberrant expression, such as MIRN9/9*, MIRN301, MIRN338 and MIRN213 in FL and MIRN150, MIRN17‐5P, MIRN145, MIRN328 and others in DLBCL. A classification tree was computed using four miRNAs (MIRN330, MIRN17‐5P, MIRN106a and MIRN210) to correctly identify 98% of all 111 cases that were analysed in this study. Finally, eight miRNAs were found to correlate with event‐free and overall survival in DLBCL including known tumour suppressors (MIRN21, MIRN127 and MIRN34a) and oncogenes (MIRN195 and MIRNLET7G).

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...

© F e r r a t a S t o r t i F o u n d a t i o nchemistry (IHC) has been used as a surrogate marker for TP53 gene mutations in hematologic and other malignancies, including large B-cell lymphoma. [22][23][24] Recently, it was demonstrated that p53 nuclear expression correlated with hemizygous TP53 mutation and outcomes in relapsed myeloma patients treated with lenalidomide. 25 AML following MDS has a dismal prognosis, which makes prediction essential, especially in patients who potentially could be cured by stem cell transplantation. In order to develop a clinically useful prognostic tool, we measured p53 protein expression by IHC in a cohort of 85 patients with lower-risk del(5q) MDS from the MDS-004 clinical trial, and compared outcomes and responses to lenalidomide treatment in patients with respect to strong nuclear p53 expression. We demonstrate the independent prognostic value of p53 IHC in this population of patients, and show that strong nuclear staining reflects underlying TP53 mutations. Our findings underscore the importance of including molecular markers such as TP53 mutations in risk-assessment for del(5q) MDS patients. Methods PatientsFormalin-fixed paraffin-embedded BM trephines from patients enrolled in the phase III, randomized, double-blind, placebo-controlled MDS-004 trial were retrieved. 5 The MDS-004 trial assessed the efficacy and safety of lenalidomide in 205 red blood cell transfusion-dependent patients with low-or intermediate-1-risk del(5q) MDS. The inclusion criteria and treatment schedule were as previously described; 5 a bone marrow biopsy was recommended, but not mandatory. The present study was conducted under the ethical consent for the MDS-004 trial. The original ethical permit did not include any type of sequencing; therefore, TP53 deep-sequencing analysis was only possible in a subset of patients who were still alive and provided consent to an ethical permit obtained after the MDS-004 trial had been completed. In Sweden, gene-sequencing analysis was performed under a separate national ethical permit, which was used for the pyrosequencing of laser-microdissected BM cells. Bone marrow morphology and immunohistochemistryOverall, 131 BM trephines from 85 of the 205 patients (41%; IHC study cohort) obtained at baseline and follow-up were assessed in a blinded fashion. Serial BM biopsies were available for 25% (21 of 85) of the patients. BM cellularity and fibrosis were assessed according to European consensus guidelines. 26 The percentage and intensity of p53 staining was assessed based on a total manual count of 1000 BM hematopoietic cells (lymphocytes/lymphoid aggregates excluded) and graded as: 0 (negative); 1+ (weakly positive); 2+ (moderately positive); and 3+ (strongly positive). The entire BM trephine was also assessed using a Modified Quick Score; 27 a score of ≥3 was used to define p53-positive staining as previously described. 28 In addition, all samples were assessed blindly for the percentage of p53-DO1 strongly positive (3+) cells by three hematopathologists fro...