Burkitt lymphoma is characterized by deregulation of MYC, but the contribution of other genetic mutations to the disease is largely unknown. Here, we describe the first completely sequenced genome from a Burkitt lymphoma tumor and germline DNA from the same affected individual. We further sequenced the exomes of 59 Burkitt lymphoma tumors and compared them to sequenced exomes from 94 diffuse large B-cell lymphoma (DLBCL) tumors. We identified 70 genes that were recurrently mutated in Burkitt lymphomas, including ID3, GNA13, RET, PIK3R1 and the SWI/SNF genes ARID1A and SMARCA4. Our data implicate a number of genes in cancer for the first time, including CCT6B, SALL3, FTCD and PC. ID3 mutations occurred in 34% of Burkitt lymphomas and not in DLBCLs. We show experimentally that ID3 mutations promote cell cycle progression and proliferation. Our work thus elucidates commonly occurring gene-coding mutations in Burkitt lymphoma and implicates ID3 as a new tumor suppressor gene.

Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma in adults. The disease exhibits a striking heterogeneity in gene expression profiles and clinical outcomes, but its genetic causes remain to be fully defined. Through whole genome and exome sequencing, we characterized the genetic diversity of DLBCL. In all, we sequenced 73 DLBCL primary tumors (34 with matched normal DNA). Separately, we sequenced the exomes of 21 DLBCL cell lines. We identified 322 DLBCL cancer genes that were recurrently mutated in primary DLBCLs. We identified recurrent mutations implicating a number of known and not previously identified genes and pathways in DLBCL including those related to chromatin modification (ARID1A and MEF2B), NF-κB (CARD11 and TNFAIP3), PI3 kinase (PIK3CD, PIK3R1, and MTOR), B-cell lineage (IRF8, POU2F2, and GNA13), and WNT signaling (WIF1). We also experimentally validated a mutation in PIK3CD, a gene not previously implicated in lymphomas. The patterns of mutation demonstrated a classic long tail distribution with substantial variation of mutated genes from patient to patient and also between published studies. Thus, our study reveals the tremendous genetic heterogeneity that underlies lymphomas and highlights the need for personalized medicine approaches to treating these patients.next-generation sequencing | cancer genetics | cancer heterogeneity D iffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma in adults (1). Although nearly half the patients can be cured with standard regimens, the majority of relapsed patients succumb. Thus, there is an urgent need to identify the genetic underpinnings of the disease and to identify novel treatment strategies. Gene expression profiling (2, 3) has uncovered distinct molecular signatures for DLBCL subtypes that have unique biology and prognoses. High-throughput sequencing has provided rich opportunities for the comprehensive identification of the genetic causes of cancer (4-6). Whereas exhaustive portraits of individual cancer genomes are emerging, the degree to which these genomes represent the disease is unclear.We generated a detailed analysis of a DLBCL genome by sequencing a primary human tumor and paired normal tissue (Dataset S1). We further characterized the genetic diversity of DLBCL by sequencing the exomes of 73 DLBCL primary tumors (34 with matched normal DNA) and 21 DLBCL cell lines for comparative purposes. This in-depth sequencing identified 322 DLBCL cancer genes that were recurrently mutated in DLBCLs. We also experimentally validated the effects of genetic alteration of PIK3CD, an oncogene that we identified in DLBCL. Our work provides one of the largest genetic portraits yet of human DLBCLs and offers insights into the molecular heterogeneity of the disease, especially in the context of other recently published studies in DLBCL (7, 8).

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• We identified novel recurrently mutated genes, including WHSC1, RB1, POT1, and SMARCA4, through exome sequencing of 56 cases of MCL.• Genetic mutations defining MCL and Burkitt lymphoma were associated with the epigenetically defined chromatin state of their respective B cells of origin.In this study, we define the genetic landscape of mantle cell lymphoma (MCL) through exome sequencing of 56 cases of MCL. We identified recurrent mutations in ATM, CCND1, MLL2, and TP53. We further identified a number of novel genes recurrently mutated in patients with MCL including RB1, WHSC1, POT1, and SMARCA4. We noted that MCLs have a distinct mutational profile compared with lymphomas from other B-cell stages.The ENCODE project has defined the chromatin structure of many cell types. However, a similar characterization of primary human mature B cells has been lacking. We defined, for the first time, the chromatin structure of primary human naïve, germinal center, and memory B cells through chromatin immunoprecipitation and sequencing for H3K4me1, H3K4me3, H3Ac, H3K36me3, H3K27me3, and PolII. We found that somatic mutations that occur more frequently in either MCLs or Burkitt lymphomas were associated with open chromatin in their respective B cells of origin, naïve B cells, and germinal center B cells. Our work thus elucidates the landscape of gene-coding mutations in MCL and the critical interplay between epigenetic alterations associated with B-cell differentiation and the acquisition of

A role for microRNA (miRNA) has been recognized in nearly every biologic system examined thus far. A complete delineation of their role must be preceded by the identification of all miRNAs present in any system. We elucidated the complete small RNA transcriptome of normal and malignant B cells through deep sequencing of 31 normal and malignant human B-cell samples that comprise the spectrum of B-cell differentiation and common malignant phenotypes. We identified the expression of 333 known miRNAs, which is more than twice the number previously recognized in any tissue type. We further identified the expression of 286 candidate novel miRNAs in normal and malignant B cells. These miRNAs were validated at a high rate (92%) using quantitative polymerase chain reaction, and we demonstrated their application in the distinction of clinically relevant subgroups of lymphoma. We further demonstrated that a novel miRNA cluster, previously annotated as a hypothetical gene LOC100130622, contains 6 novel miRNAs that regulate the transforming growth factor-␤ pathway. Thus, our work suggests that more than a third of the miRNAs present in most cellular types are currently unknown and that these miRNAs may regulate important cellular functions. (Blood. 2010;116(23):e118-e127)

© F e r r a t a S t o r t i F o u n d a t i o nBCL2 translocations are more frequently found in the GCB subtype, whereas 18q21 locus amplification is more common in the ABC subtype of DLBCL. 3,8,10 The prognostic significance of BCL2 amplification or translocations in de novo DLBCL in the era of CHOP therapy alone, without rituximab, was controversial. [11][12][13][14][15][16][17][18][19][20] Some data on the prognostic significance of BCL2 aberrations in patients treated with R-CHOP have recently become available, with two studies reporting no influence of BCL2 gene rearrangements on the survival of DLBCL patients. 21,22 On the other hand, the concomitant presence of t(14;18) or variants and MYC rearrangements, referred to as double hit lymphomas, has consistently been associated with adverse outcome in DLBCL patients treated with R-CHOP. [23][24][25] Bcl-2 protein expression seems only partially related to BCL2 gene abnormalities as analyzed by fluorescence in situ hybridization (FISH), as Bcl-2 is expressed in a greater number of DLBCL cases than in those tumors carrying t(14;18)(q32;q21). [10][11][12] Indeed, in the absence of BCL2 translocations, amplification of 18q21 and/or activation of the nuclear factor κB (NF-κB) pathway can cause Bcl-2 protein overexpression. 26 The prognostic significance of Bcl-2 expression is also controversial, and comparison between different studies is hampered by the choice of different cut-offs of positive cells, and by the variability of treatments. In patients treated with R-CHOP, Bcl-2 protein did not correlate with outcome, 5,27 since the addition of rituximab seemed to improve survival of Bcl-2-positive patients, [28][29][30] apparently eliminating the gap between Bcl-2-positive and Bcl-2-negative patients found in the pre-rituximab era. This result does, however, appear to be contradicted in a very recent study in which Bcl-2 expression in GCB-DLBCL was associated with poorer outcome. 22 The goal of this study was to investigate the prognostic value of BCL2 gene aberrations and Bcl-2 expression in a large number of patients with de novo DLBCL, uniformly treated with R-CHOP, for whom MYC and GEP characterization was available. Design and Methods PatientsWe studied 327 cases of previously untreated de novo DLBCL, diagnosed between January 2002 and October 2009, and collected as part of the International DLBCL Rituxan-CHOP Consortium Program Study. These cases were analyzed for Bcl-2 protein expression, and BCL2 and MYC gene abnormalities, and gene expression profiling (GEP) was performed. All cases were reviewed by a group of hematopathologists (SMM, MAP, MBM, AT, and KHY), and the diagnoses were confirmed based on World Health Organization classification criteria. Patients with transformation from low grade lymphoma, those with composite follicular lymphoma, primary mediastinal large B-cell lymphoma, primary cutaneous and primary central nervous system DLBCL were excluded from the analysis due to the unique biological features of these types of lymphoma. All pat...

Diffuse large B-cell lymphoma (DLBCL) prognostication requires additional biologic markers. miRNAs may constitute markers for cancer diagnosis, outcome, or therapy response. In the present study, we analyzed the miRNA expression profile in a retrospective multicenter series of 258 DLBCL patients uniformly treated with chemoimmunotherapy. Findings were correlated with overall survival (OS) and progression-free survival (PFS). miRNA and gene-expression profiles were studied using microarrays in an initial set of 36 cases. A selection of miRNAs associated with either DLBCL molecular subtypes (GCB/ABC) or clinical outcome were studied by multiplex RT-PCR in a test group of 240 cases with available formalinfixed, paraffin-embedded (FFPE) diagnostic samples. The samples were divided into a training set (123 patients) and used to derive miRNA-based and combined (with IPI score) Cox regression models in an independent validation series (117 patients). Our model based on miRNA expression predicts OS and PFS and improves upon the predictions based on clinical variables. Combined models with IPI score identified a high-risk group of patients with a 2-year OS and a PFS probability of < 50%. In summary, a precise miRNA signature is associated with poor clinical outcome in chemoimmunotherapy-treated DLBCL patients. This information improves upon IPI-based predictions and identifies a subgroup of candidate patients for alternative therapeutic regimens. (Blood. 2011; 118(4):1034-1040) IntroductionDiffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma in adults, accounting for Ͼ 80% of aggressive lymphomas. 1 DLBCL is a heterogeneous group of tumors with different genetic abnormalities, clinical features, responses to treatment, and prognosis. 2 This heterogeneity hinders outcome prediction based on clinical and/or molecular parameters.Combination therapy that associates CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) with rituximab (R-CHOP) has become a standard treatment for DLBCL, leading to complete remission rates of 75%-80% and a 3-to 5-year PFS of 50%-60%. [3][4][5][6][7][8] Nevertheless, patients who fail to respond to first-line therapy or relapse continue to pose a challenge, and identification at diagnosis of poor-outcome cases is crucial for deciding between alternative treatment schemes.The International Prognostic Index (IPI) has been the primary clinical tool for predicting the outcome of patients with aggressive non-Hodgkin lymphoma. 9 Original IPI factors were redistributed in patients treated with R-CHOP to give a revised score (R-IPI) that distinguishes 3 prognostic categories, with 4-year survival rates ranging from 94%-55% for poor-risk patients. 7 Nevertheless, the R-IPI does not discriminate patients with Ͻ 50% probability of survival, which restricts its clinical value. 7 The biologic heterogeneity of DLBCL has been shown substantially to reflect the cell origin of these tumors from germinal center or activated B cells. These differences are significant ind...

Context.— The advent of molecular tools capable of subclassifying eosinophilia has changed the diagnostic and clinical approach to what was classically called hypereosinophilic syndrome. Objectives.— To review the etiologies of eosinophilia and to describe the current diagnostic approach to this abnormality. Data Sources.— Literature review. Conclusions.— Eosinophilia is a common, hematologic abnormality with diverse etiologies. The underlying causes can be broadly divided into reactive, clonal, and idiopathic. Classically, many cases of eosinophilia were grouped together into the umbrella category of hypereosinophilic syndrome, a clinical diagnosis of exclusion. In recent years, an improved mechanistic understanding of many eosinophilias has revolutionized the way these disorders are understood, diagnosed, and treated. As a result, specific diagnoses can now be assigned in many cases that were previously defined as hypereosinophilic syndrome. Most notably, chromosomal rearrangements, such as FIP1L1-PDGFRA fusions caused by internal deletions in chromosome 4, are now known to be associated with many chronic eosinophilic leukemias. When present, these specific molecular abnormalities predict response to directed therapies. Although an improved molecular understanding is revolutionizing the treatment of patients with rare causes of eosinophilia, it has also complicated the approach to evaluating and treating eosinophilia. Here, we review causes of eosinophilia and present a framework by which the practicing pathologist may approach this diagnostic dilemma. Finally, we consider recent cases as clinical examples of eosinophilia from a single institution, demonstrating the diversity of etiologies that must be considered.