Pedro Jares

Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution. Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes. The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer.

Exome sequencing identifies recurrent mutations of the splicing factor SF3B1 gene in chronic lymphocytic leukemia

Here we perform whole-exome sequencing of samples from 105 individuals with chronic lymphocytic leukemia (CLL), the most frequent leukemia in adults in Western countries. We found 1,246 somatic mutations potentially affecting gene function and identified 78 genes with predicted functional alterations in more than one tumor sample. Among these genes, SF3B1, encoding a subunit of the spliceosomal U2 small nuclear ribonucleoprotein (snRNP), is somatically mutated in 9.7% of affected individuals. Further analysis in 279 individuals with CLL showed that SF3B1 mutations were associated with faster disease progression and poor overall survival. This work provides the first comprehensive catalog of somatic mutations in CLL with relevant clinical correlates and defines a large set of new genes that may drive the development of this common form of leukemia. The results reinforce the idea that targeting several well-known genetic pathways, including mRNA splicing, could be useful in the treatment of CLL and other malignancies.

Genetic and molecular pathogenesis of mantle cell lymphoma: perspectives for new targeted therapeutics.

Mantle cell lymphoma (MCL) is a well-defined lymphoid malignancy characterized by a rapid clinical evolution and poor response to current therapeutic protocols. The genetic and molecular mechanisms involved in its pathogenesis combine the dysregulation of cell proliferation and survival pathways with a high level of chromosome instability that seems related to the disruption of the DNA damage response pathway. Understanding these mechanisms and how they affect tumour behaviour is providing the rationale for the identification of reliable predictors of clinical evolution and the design of innovative therapeutic strategies that could open new avenues for the treatment of patients with MCL.

Epigenomic analysis detects widespread gene-body DNA hypomethylation in chronic lymphocytic leukemia.

We have extensively characterized the DNA methylomes of 139 patients with chronic lymphocytic leukemia (CLL) with mutated or unmutated IGHV and of several mature B-cell subpopulations through the use of whole-genome bisulfite sequencing and high-density microarrays. The two molecular subtypes of CLL have differing DNA methylomes that seem to represent epigenetic imprints from distinct normal B-cell subpopulations. DNA hypomethylation in the gene body, targeting mostly enhancer sites, was the most frequent difference between naive and memory B cells and between the two molecular subtypes of CLL and normal B cells. Although DNA methylation and gene expression were poorly correlated, we identified gene-body CpG dinucleotides whose methylation was positively or negatively associated with expression. We have also recognized a DNA methylation signature that distinguishes new clinico-biological subtypes of CLL. We propose an epigenomic scenario in which differential methylation in the gene body may have functional and clinical implications in leukemogenesis.

Genomic and Gene Expression Profiling Defines Indolent Forms of Mantle Cell Lymphoma

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.

KRAS mutation testing for predicting response to anti-EGFR therapy for colorectal carcinoma: proposal for an European quality assurance program.

Novel therapeutic agents targeting the epidermal growth factor receptor (EGFR) have improved outcomes for patients with colorectal carcinoma. However, these therapies are effective only in a subset of patients. Activating mutations in the KRAS gene are found in 30–40% of colorectal tumors and are associated with poor response to anti-EGFR therapies. Thus, KRAS mutation status can predict which patient may or may not benefit from anti-EGFR therapy. Although many diagnostic tools have been developed for KRAS mutation analysis, validated methods and standardized testing procedures are lacking. This poses a challenge for the optimal use of anti-EGFR therapies in the management of colorectal carcinoma. Here we review the molecular basis of EGFR-targeted therapies and the resistance to treatment conferred by KRAS mutations. We also present guideline recommendations and a proposal for a European quality assurance program to help ensure accuracy and proficiency in KRAS mutation testing across the European Union.

Non-coding recurrent mutations in chronic lymphocytic leukaemia.

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.

SOX11 expression is highly specific for mantle cell lymphoma and identifies the cyclin D1-negative subtype

Cyclin D1-negative mantle cell lymphoma is difficult to distinguish from other small B-cell lymphomas. This study shows that SOX11 mRNA and nuclear protein expression is a highly specific marker for both cyclin D1-positive and negative mantle cell lymphoma. See related perspective article on page 1488. Background Cyclin 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. Design and Methods The microarray database of 238 mature B-cell neoplasms was re-examined. SOX11 protein expression was investigated immunohistochemically in 12 cases of cyclin D1-negative mantle cell lymphoma, 54 cases of conventional mantle cell lymphoma, and 209 additional lymphoid neoplasms. Results SOX11 mRNA was highly expressed in conventional and cyclin D1-negative mantle cell lymphoma and in 33% of the cases of Burkitt’s lymphoma but not in any other mature lymphoid neoplasm. SOX11 nuclear protein was detected in 50 cases (93%) of conventional mantle cell lymphoma and also in the 12 cyclin D1-negative cases of mantle cell lymphoma, the six cases of lymphoblastic lymphomas, in two of eight cases of Burkitt’s lymphoma, and in two of three T-prolymphocytic leukemias but was negative in the remaining lymphoid neoplasms. Cyclin D2 and D3 mRNA levels were significantly higher in cyclin D1-negative mantle cell lymphoma than in conventional mantle cell lymphoma but the protein expression was not discriminative. The clinico-pathological features and outcomes of the patients with cyclin D1-negative mantle cell lymphoma identified by SOX11 expression were similar to those of patients with conventional mantle cell lymphoma. Conclusions SOX11 mRNA and nuclear protein expression is a highly specific marker for both cyclin D1-positive and negative mantle cell lymphoma.

Landscape of somatic mutations and clonal evolution in mantle cell lymphoma

Significance This is a comprehensive whole-genome/whole-exome analysis of mantle cell lymphoma (MCL). We sequenced 29 MCL cases and validated the findings by target sequencing of 172 additional tumors. We identified recurrent mutations in genes regulating chromatin modification and genes such as NOTCH2 that have a major impact on clinical outcome. Additionally, we demonstrated the subclonal heterogeneity of the tumors already at diagnosis and the modulation of the mutational architecture in the progression of the disease. The identification of new molecular mechanisms may open perspectives for the management of MCL patients. 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.

Gene expression profiling and not immunophenotypic algorithms predicts prognosis in patients with diffuse large B-cell lymphoma treated with immunochemotherapy

Diffuse large B-cell lymphomas (DLBCLs) can be divided into germinal-center B cell-like (GCB) and activated-B cell-like (ABC) subtypes by gene-expression profiling (GEP), with the latter showing a poorer outcome. Although this classification can be mimicked by different immunostaining algorithms, their reliability is the object of controversy. We constructed tissue microarrays with samples of 157 DLBCL patients homogeneously treated with immunochemotherapy to apply the following algorithms: Colomo (MUM1/IRF4, CD10, and BCL6 antigens), Hans (CD10, BCL6, and MUM1/IRF4), Muris (CD10 and MUM1/IRF4 plus BCL2), Choi (GCET1, MUM1/IRF4, CD10, FOXP1, and BCL6), and Tally (CD10, GCET1, MUM1/IRF4, FOXP1, and LMO2). GEP information was available in 62 cases. The proportion of misclassified cases by immunohistochemistry compared with GEP was higher when defining the GCB subset: 41%, 48%, 30%, 60%, and 40% for Colomo, Hans, Muris, Choi, and Tally, respectively. Whereas the GEP groups showed significantly different 5-year progression-free survival (76% vs 31% for GCB and activated DLBCL) and overall survival (80% vs 45%), none of the immunostaining algorithms was able to retain the prognostic impact of the groups (GCB vs non-GCB). In conclusion, stratification based on immunostaining algorithms should be used with caution in guiding therapy, even in clinical trials.