Chronic lymphocytic leukaemia (CLL) is a frequent disease in which the genetic alterations determining the clinicobiological behaviour are not fully understood. Here we describe a comprehensive evaluation of the genomic landscape of 452 CLL cases and 54 patients with monoclonal B-lymphocytosis, a precursor disorder. We extend the number of CLL driver alterations, including changes in ZNF292, ZMYM3, ARID1A and PTPN11. We also identify novel recurrent mutations in non-coding regions, including the 3' region of NOTCH1, which cause aberrant splicing events, increase NOTCH1 activity and result in a more aggressive disease. In addition, mutations in an enhancer located on chromosome 9p13 result in reduced expression of the B-cell-specific transcription factor PAX5. The accumulative number of driver alterations (0 to ≥4) discriminated between patients with differences in clinical behaviour. This study provides an integrated portrait of the CLL genomic landscape, identifies new recurrent driver mutations of the disease, and suggests clinical interventions that may improve the management of this neoplasia.

Mantle cell lymphoma (MCL) is an aggressive tumor, but a subset of patients may follow an indolent clinical course. To understand the mechanisms underlying this biological heterogeneity, we performed whole-genome and/or whole-exome sequencing on 29 MCL cases and their respective matched normal DNA, as well as 6 MCL cell lines. Recurrently mutated genes were investigated by targeted sequencing in an independent cohort of 172 MCL patients. We identified 25 significantly mutated genes, including known drivers such as ataxia-telangectasia mutated (ATM), cyclin D1 (CCND1), and the tumor suppressor TP53; mutated genes encoding the anti-apoptotic protein BIRC3 and Toll-like receptor 2 (TLR2); and the chromatin modifiers WHSC1, MLL2, and MEF2B. We also found NOTCH2 mutations as an alternative phenomenon to NOTCH1 mutations in aggressive tumors with a dismal prognosis. Analysis of two simultaneous or subsequent MCL samples by whole-genome/whole-exome (n = 8) or targeted (n = 19) sequencing revealed subclonal heterogeneity at diagnosis in samples from different topographic sites and modulation of the initial mutational profile at the progression of the disease. Some mutations were predominantly clonal or subclonal, indicating an early or late event in tumor evolution, respectively. Our study identifies molecular mechanisms contributing to MCL pathogenesis and offers potential targets for therapeutic intervention.next-generation sequencing | cancer genetics | cancer heterogeneity M antle cell lymphoma (MCL) is a mature B-cell neoplasm characterized by the t(11;14)(q13;q32) translocation leading to the overexpression of cyclin D1 (1). CCND1 is a weak oncogene that requires the cooperation of other oncogenic events to transform lymphoid cells (2). Molecular studies have identified alterations in components of the cell-cycle regulation, DNA damage response, and cell survival pathways (3, 4), but the profile of mutated genes contributing to the pathogenesis of MCL and cooperating with CCND1 is not well defined (1). Most MCL cases have a rapid evolution and an aggressive behavior with an unfavorable outcome with current therapies (5). Paradoxically, a subset of patients follows an indolent clinical evolution with stable disease even in the absence of chemotherapy (6, 7). This favorable behavior has been associated with IGHV-mutated (8, 9) and lack of expression of SOX11 (10, 11), a transcription factor highly specific of MCL that contributes to the aggressive behavior of this tumor (12). However, the molecular mechanisms responsible for this clinical heterogeneity are not well understood.To gain insight into the molecular pathogenesis of MCL we performed whole-genome sequencing (WGS) and whole-exome sequencing (WES) of 29 MCL and further investigated mutated genes in an expanded series of patients. We also analyzed the subclonal heterogeneity of the tumors and their modulation during the evolution of the disease. Results Landscape of Mutations in MCL.We performed WGS and WES of 4 and 29 MCL, respectively. These patients were re...

Mantle cell lymphoma (MCL) is typically a very aggressive disease with poor outcomes, but some cases display an indolent behavior that might not necessitate treatment at diagnosis. To define molecular criteria that might permit recognition of such cases, we compared the clinicopathologic features, gene expression, and genomic profile of patients who had indolent or conventional disease (iMCL or cMCL). Patients with iMCL displayed nonnodal leukemic disease with predominantly hypermutated IGVH and noncomplex karyotypes. iMCL and cMCL shared a common gene expression profile that differed from other leukemic lymphoid neoplasms. However, we identified a signature of 13 genes that was highly expressed in cMCL but underexpressed in iMCL. SOX11 was notable in this signature and we confirmed a restriction of SOX11 protein expression to cMCL. To validate the potential use of SOX11 as a biomarker for cMCL, we evaluated SOX11 protein expression in an independent series of 112 cases of MCL. Fifteen patients with SOX11-negative tumors exhibited more frequent nonnodal presentation and better survival compared with 97 patients with SOX11-positive MCL (5-year overall survival of 78% versus 36%, respectively; P = 0.001). In conclusion, we defined nonnodal presentation, predominantly hypermutated IGVH, lack of genomic complexity, and absence of SOX11 expression as qualities of a specific subtype of iMCL with excellent outcomes that might be managed more conservatively than cMCL.

Key Points• Clonal and subclonal mutations of NOTCH1 and TP53, clonal mutations of SF3B1, and ATM mutations in CLL have an impact on clinical outcome.• Clonal evolution in longitudinal samples occurs before and after treatment and may have an unfavorable impact on overall survival.Genomic studies have revealed the complex clonal heterogeneity of chronic lymphocytic leukemia (CLL). The acquisition and selection of genomic aberrations may be critical to understanding the progression of this disease. In this study, we have extensively characterized the mutational status of TP53, SF3B1, BIRC3, NOTCH1, and ATM in 406 untreated CLL cases by ultra-deep next-generation sequencing, which detected subclonal mutations down to 0.3% allele frequency. Clonal dynamics were examined in longitudinal samples of 48 CLL patients. We identified a high proportion of subclonal mutations, isolated or associated with clonal aberrations. TP53 mutations were present in 10.6% of patients (6.4% clonal, 4.2% subclonal), ATM mutations in 11.1% (7.8% clonal, 1.3% subclonal, 2% germ line mutations considered pathogenic), SF3B1 mutations in 12.6% (7.4% clonal, 5.2% subclonal), NOTCH1 mutations in 21.8% (14.2% clonal, 7.6% subclonal), and BIRC3 mutations in 4.2% (2% clonal, 2.2% subclonal). ATM mutations, clonal SF3B1, and both clonal and subclonal NOTCH1 mutations predicted for shorter time to first treatment irrespective of the immunoglobulin heavy-chain variable-region gene (IGHV) mutational status. Clonal and subclonal TP53 and clonal NOTCH1 mutations predicted for shorter overall survival together with the IGHV mutational status. Clonal evolution in longitudinal samples mainly occurred in cases with mutations in the initial samples and was observed not only after chemotherapy but also in untreated patients. These findings suggest that the characterization of the subclonal architecture and its dynamics in the evolution of the disease may be relevant for the management of CLL patients. (Blood. 2016;127(17):2122-2130

BackgroundCyclin D1-negative mantle cell lymphoma is difficult to distinguish from other small B-cell lymphomas. The clinical and pathological characteristics of patients with this form of lymphoma have not been well defined. Overexpression of the transcription factor SOX11 has been observed in conventional mantle cell lymphoma. The aim of this study was to determine whether this gene is expressed in cyclin D1-negative mantle cell lymphoma and whether its detection may be useful to identify these tumors.

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Chronic lymphocytic leukemia (CLL) has heterogeneous clinical and biological behavior. Whole-genome and -exome sequencing has contributed to the characterization of the mutational spectrum of the disease, but the underlying transcriptional profile is still poorly understood. We have performed deep RNA sequencing in different subpopulations of normal B-lymphocytes and CLL cells from a cohort of 98 patients, and characterized the CLL transcriptional landscape with unprecedented resolution. We detected thousands of transcriptional elements differentially expressed between the CLL and normal B cells, including protein-coding genes, noncoding RNAs, and pseudogenes. Transposable elements are globally derepressed in CLL cells. In addition, two thousand genes-most of which are not differentially expressed-exhibit CLL-specific splicing patterns. Genes involved in metabolic pathways showed higher expression in CLL, while genes related to spliceosome, proteasome, and ribosome were among the most down-regulated in CLL. Clustering of the CLL samples according to RNA-seq derived gene expression levels unveiled two robust molecular subgroups, C1 and C2. C1/C2 subgroups and the mutational status of the immunoglobulin heavy variable (IGHV ) region were the only independent variables in predicting time to treatment in a multivariate analysis with main clinico-biological features. This subdivision was validated in an independent cohort of patients monitored through DNA microarrays. Further analysis shows that B-cell receptor (BCR) activation in the microenvironment of the lymph node may be at the origin of the C1/C2 differences.

Mantle cell lymphoma (MCL) is a heterogeneous disease with most patients following an aggressive clinical course while others have an indolent behavior. We performed an integrative and multidisciplinary analysis of 177 MCL to determine whether the immunogenetic features of the clonotypic B cell receptors may identify different subsets of tumors. ‘Truly unmutated’ (100% identity) IGHV genes were found in 24% cases, 40% were ‘minimally/borderline mutated’ (99.9-97%), 19% ‘significantly mutated’ (96.9-95%) and 17% ‘hypermutated’ (<95%). Tumors with high (≥97%) or low (<97%) mutational load used different IGHV genes and their gene expression profiles were also different for several gene pathways. A gene set enrichment analysis showed that MCL with high and low IGHV mutations were enriched in memory and naïve B-cell signatures, respectively. Furthermore, the highly mutated tumors displayed less genomic complexity, were preferentially SOX11 negative, and showed more frequently non-nodal disease. The best cut-off of germline identity of IGHV genes to predict survival was 3%. Patients with high and low mutational load had significant different outcome with 5-year overall survival of 59% and 40%, respectively (P=0.004). Nodal presentation and SOX11 expression also predicted for poor overall survival. In a multivariate analysis, IGHV gene status and SOX11 expression were independent risk factors. In conclusion, these observations suggest the idea that MCL with mutated IGHV, SOX11 negativity, and non-nodal presentation correspond to a subtype of the disease with more indolent behavior.