[Show abstract][Hide abstract] ABSTRACT: The development of high-throughput sequencing technologies has advanced our understanding of cancer. However, characterizing somatic structural variants in tumor genomes is still challenging because current strategies depend on the initial alignment of reads to a reference genome. Here, we describe SMUFIN (somatic mutation finder), a single program that directly compares sequence reads from normal and tumor genomes to accurately identify and characterize a range of somatic sequence variation, from single-nucleotide variants (SNV) to large structural variants at base pair resolution. Performance tests on modeled tumor genomes showed average sensitivity of 92% and 74% for SNVs and structural variants, with specificities of 95% and 91%, respectively. Analyses of aggressive forms of solid and hematological tumors revealed that SMUFIN identifies breakpoints associated with chromothripsis and chromoplexy with high specificity. SMUFIN provides an integrated solution for the accurate, fast and comprehensive characterization of somatic sequence variation in cancer.
[Show abstract][Hide abstract] ABSTRACT: Prospective identification of patients with chronic lymphocytic leukemia (CLL) destined to progress would greatly facilitate their clinical management. Recently, whole-genome DNA methylation analyses identified three clinico-biological CLL subgroups with an epigenetic signature related to different normal B-cell counterparts. Here, we developed a clinically-applicable method to identify these subgroups and to study their clinical relevance. Using a support vector machine approach, we built a prediction model using five epigenetic biomarkers that was able to accurately classify CLL patients into the three subgroups, namely naive B cell-like, intermediate and memory B cell-like CLL. DNA methylation was quantified by highly reproducible bisulfite pyrosequencing (BPS) assays in two independent CLL series. In the initial series (n=211), the three subgroups showed differential levels of IGHV mutation (P<0.001) and VH usage (P<0.03), as well as different clinical features and outcome in terms of time-to-first-treatment (TTT) and overall survival (P<0.001). A multivariate Cox model showed that epigenetic classification was the strongest predictor of TTT (P<0.001) along with Binet stage (P<0.001). These findings were corroborated in a validation series (n=97). In this study, we developed a simple and robust method using epigenetic biomarkers to categorize CLLs into three subgroups with different clinico-biological features and outcome.Leukemia accepted article preview online, 25 August 2014 doi:10.1038/leu.2014.252.
[Show abstract][Hide abstract] ABSTRACT: Follicular lymphoma (FL) is one of the most common malignant lymphomas. The t(14;18)(q32;q21) is found in about 80% of cases and plays an important role in the lymphomagenesis. However, the molecular mechanisms involved in the development and transformation of this lymphoma are not fully understood. Gain-of function mutations of NOTCH1 or NOTCH2 have been recently reported in several B-cell lymphoid neoplasms, but the role of these mutations in FL is not known. In this study we have investigated the mutational status of these genes in 112 FL. NOTCH1 and NOTCH2 mutations were identified in five and two cases, respectively, (total 7/112, 6.3%). All mutations predicted for truncated protein in the PEST domain and were identical to those identified in other B cell lymphoid neoplasms. NOTCH mutated FL cases were characterized by lower frequency of t(14;18) (14% vs. 69%, P=0.01), higher incidence of splenic involvement (71% vs. 25%, P=0.02) and female predominance (100% vs 55%, P=0.04). A diffuse large B-cell lymphoma (DLBCL) component was more frequently identified in NOTCH mutated FL than wild-type cases (57% vs. 18%, P=0.03). These results indicate that NOTCH mutations are uncommon in FL but may occur in a subset of cases with distinctive characteristic clinicopathological features.
The Journal of Pathology 08/2014; · 7.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract We investigated the clinico-biological features, outcomes, and prognosis in 949 patients with chronic lymphocytic leukemia according to age. No biological differences (cytogenetics by fluorescent in situ hybridization, IGHV, ZAP-70, CD38, NOTCH1, SF3B1) were found across age groups. Elderly patients (>70 years; n=367) presented more frequently with advanced disease (Binet C/Rai III-IV: 10/12% vs. 5/5%; p<.001), were treated less frequently (23.8% vs. 41.9% at 3 years; p<.001) and in most cases received no highly effective regimens thereby achieving a lower overall response rate (49% with 14% complete response vs. 69%with 31% complete response; p<.001). They also had shorter overall survival (6.6 vs. 13.3 years; p<.001) and higher disease-unrelated mortality (34.9% vs. 6.9% at 10 years; p<.001). However, disease-attributable mortality was not significantly different between both groups. Combining Binet stage, ZAP-70, beta2-microglobulin and comorbidity two risk groups (low-risk: 0-1 parameters; high-risk: 2-4 parameters) with different overall survival (median: 6.8 vs. 11.4 years, p<.001) were identified. In patients requiring treatment, comorbidity at treatment (Cumulative Illness Rating Scale-T>4; hazard ratio 2.2, p<001) and response (Failure vs. Response: hazard ratio 1.60, p<.04) were the most important prognostic factors for overall survival. In conclusion, in our series elderly patients with chronic lymphocytic leukemia presented with no distinct biological features as compared to younger ones and had a poorer clinical outcome. This study highlights the importance of a comprehensive medical care, achieving response to therapy, and specific management strategies for elderly patients with chronic lymphocytic leukemia.
[Show abstract][Hide abstract] ABSTRACT: 15 on behalf of the European Research Initiative on CLL (ERIC) Through the European Research Initiative on chronic lymphocytic leukemia (CLL) (ERIC), we screened 3490 patients with CLL for mutations within the NOTCH1 (n ¼ 3334), SF3B1 (n ¼ 2322), TP53 (n ¼ 2309), MYD88 (n ¼ 1080) and BIRC3 (n ¼ 919) genes, mainly at diagnosis (75%) and before treatment (490%). BIRC3 mutations (2.5%) were associated with unmutated IGHV genes (U-CLL), del(11q) and trisomy 12, whereas MYD88 mutations (2.2%) were exclusively found among M-CLL. NOTCH1, SF3B1 and TP53 exhibited variable frequencies and were mostly enriched within clinically aggressive cases. Interestingly, as the timespan between diagnosis and mutational screening increased, so too did the incidence of SF3B1 mutations; no such increase was observed for NOTCH1 mutations. Regarding the clinical impact, NOTCH1 mutations, SF3B1 mutations and TP53 aberrations (deletion/mutation, TP53ab) correlated with shorter time-to-first-treatment (Po0.0001) in 889 treatment-naive Binet stage A cases. In multivariate analysis (n ¼ 774), SF3B1 mutations and TP53ab along with del(11q) and U-CLL, but not NOTCH1 mutations, retained independent significance. Importantly, TP53ab and SF3B1 mutations had an adverse impact even in U-CLL. In conclusion, we support the clinical relevance of novel recurrent mutations in CLL, highlighting the adverse impact of SF3B1 and TP53 mutations, even independent of IGHV mutational status, thus underscoring the need for urgent standardization/harmonization of the detection methods. Leukemia advance online publication, 11 July 2014; doi:10.1038/leu.2014.196 INTRODUCTION Recently, whole-genome or -exome sequencing studies have revealed a number of recurrently mutated genes in chronic lymphocytic leukemia (CLL). 1–4 Indeed, mutations in NOTCH1, SF3B1 and BIRC3 have been reported to occur in approximately 2–10% of CLL within a general practice (GP) setting with higher frequencies among patients with progressive or high-risk disease, and significantly correlated with poor outcome. 5–15 Consequently, it is reasonable to argue that integration of the newly discovered genetic lesions into a comprehensive prognostic model based on both chromosomal abnormalities and gene mutations would help to improve prognostication of patients. 10,13,16
[Show abstract][Hide abstract] ABSTRACT: Mutations in Toll-like receptor and MYD88 genes (TLR/MYD88) have been found in chronic lymphocytic leukemia (CLL) at low frequency. We analyzed the incidence, clinicobiological characteristics and outcome of patients with mutations in TLR/MYD88 in 587 CLL patients by either whole-exome or Sanger sequencing. Twenty-three patients (3.9%) had mutations, 19 in MYD88 (one with concurrent IRAK1 mutation), 2 TLR2 (one with concomitant TLR6 mutation), 1 IRAK1 and 1 TLR5. No mutations were found in IRAK2 and IRAK4. TLR/MYD88-mutated CLL cells overexpressed genes of the NFkB pathway. Patients with TLR/MYD88 mutations were significantly younger (83% ≤50 years) than unmutated, with this being the most frequent mutation in this subgroup of patients. Mutated TLR/MYD88 CLL had higher frequency of mutated IGHV and low expression of CD38 or ZAP-70. Overall survival (OS) was better in TLR/MYD88 mutated than unmutated in the whole series (10-year OS: 100% vs. 62%; p=0.002), and in the subset of patients ≤50 years (100% vs. 70%; p=0.02). In addition, relative OS of TLR/MYD88 mutated patients was similar to age- and gender-matched population. In summary, TLR/MYD88 mutations identify a population of young CLL patients with favorable outcome.
[Show abstract][Hide abstract] ABSTRACT: A proportion of patients with chronic lymphocytic leukemia achieve a minimal residual disease negative status after therapy. We retrospectively evaluated the impact of minimal residual disease on the outcome of 255 consecutive patients receiving any frontline therapy in the context of a detailed prognostic evaluation, including IGHV, TP53, NOTCH1 and SF3B1 mutations. Median follow-up was 73 (range 2-202) months from disease evaluation. Median treatment-free survival for patients achieving a minimal residual disease negative complete response, minimal residual disease positive complete response, partial response and no response was 76, 40, 11 and 11 months, respectively (P < .001). Multivariate analysis revealed that three variables had a significant impact on treatment-free survival: minimal residual disease (P < .001), IGHV status (P < .001) and beta2-microglobulin (P = .012). Regarding overall survival, minimal residual disease positivity was predictive of an unfavorable outcome (P = .014), together with advanced age (P < .001), unmutated IGHV status (P = .001), TP53 mutations (P < .001) and elevated beta2-microglobulin (P = .003). In conclusion, for patients requiring frontline therapy, achievement of minimal residual disease negativity is associated with a significantly prolonged treatment-free and overall survival irrespective of other prognostic markers or treatment administered.
[Show abstract][Hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 10/2013; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PURPOSE: MicroRNAs (miRs) are post-transcriptional gene regulators that may be useful as diagnostic and/or prognostic biomarkers. We aim to study the expression profiles of a high number of miRs and their relationship with clinicopathological and biological relevant features in leukemic mantle cell lymphomas (MCL). EXPERIMENTAL DESIGN: Expression profiling of 664 miRs was investigated using a high-throughput quantitative real-time PCR platform in 30 leukemic MCL. Statistical and bioinformatic analysis were performed to define miRs associated with different clinicopathological parameters. Gene expression profiling was investigated by microarrays in 16 matching cases to study the potential genes and pathways targeted by selected miRs. The prognostic value of miR-34a was investigated in two independent series of 29 leukemic and 50 nodal MCL. RESULTS: Robust consensus clustering defined two main MCL subgroups with significant differences in the immunoglobulin (IGHV) mutational status, SOX11 expression, genomic complexity and nodal clinical presentation. Supervised analyses regarding IGHV and SOX11 categories identified 17 and 22 miRs differentially expressed, respectively. Enriched targets of these miRs corresponded to relevant pathways in MCL pathogenesis such as DNA stress response, CD40 signaling and chromatin modification. Additionally, we found seven miRs showing prognostic significance independently of IGHV status and SOX11 expression. Among them, miR-34a was also associated with poor prognosis in two independent series of leukemic and nodal MCL, and in cooperation with high expression of the MYC oncogene. CONCLUSIONS: We have identified miRs and target pathways related to clinical and biological variants of leukemic MCL, and validated miR-34a as a prognostic marker in MCL.
Clinical Cancer Research 05/2013; · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mantle cell lymphoma (MCL) is one of the most aggressive lymphoid neoplasms whose pathogenesis is not fully understood. The neural transcription factor SOX11 is overexpressed in most MCL but is not detected in other mature B-cell lymphomas or normal lymphoid cells. The specific expression of SOX11 in MCL suggests that it may be an important element in the development of this tumor but its potential function is not known. Here, we show that SOX11 promotes tumor growth in a MCL-xenotransplant mouse model. Using ChIP-chip analysis combined with gene expression profiling upon SOX11 knockdown, we identify target genes and transcriptional programs regulated by SOX11 including the block of mature B-cell differentiation, modulation of cell cycle, apoptosis and stem cell development. PAX5 emerges as one of the major SOX11 direct targets. SOX11 silencing downregulates PAX5, induces BLIMP1 expression and promotes the shift from a mature B-cell into the initial plasmacytic differentiation phenotype, in both primary tumor cells and in an in vitro model. Our results suggest that SOX11 contributes to tumor development by altering the terminal B-cell differentiation program of MCL and provide perspectives that may have clinical implications in the diagnosis and design of new therapeutic strategies.
[Show abstract][Hide abstract] ABSTRACT: Cyclin D1-negative mantle cell lymphomas (MCL) are not well characterized, in part due to the difficulties in their recognition. SOX11 has been recently identified as a reliable biomarker of MCL, also expressed in the cyclin D1-negative variant. We investigated 40 lymphomas with MCL morphology and immunophenotype, negative for cyclin D1 expression/t(11;14)(q13;q32) but SOX11-positive. These tumors presented clinically with generalized lymphadenopathy, advanced stage, and had a poor outcome (5-year overall survival 48%). Chromosomal rearrangements of the CCND2 locus were detected in 55% of the cases, with an IG gene as partner in 18/22 cases, in particular with light chains (10 IGK@, 5 IGL@). No mutations in the phosphorylation motifs of CCND1, CCND2 and CCND3 were detected. The global genomic profile and the high complexity of the 32 cyclin D1-negative SOX11-positive MCL analyzed by copy number arrays were similar to the conventional cyclin D1/SOX11-positive MCL. 17p deletions and high Ki67 expression conferred a significantly worse outcome to the patients. This comprehensive characterization of a large series of cyclin D1-negative MCL indicates that these tumors are clinically and biologically similar to the conventional cyclin D1-positive MCL and provides a basis for the proper identification and clinical management of these patients.
[Show abstract][Hide abstract] ABSTRACT: PURPOSE: We evaluated the antitumoral properties of the multikinase inhibitor sorafenib in mantle cell lymphoma (MCL), an aggressive B-lymphoma for which current therapies have shown limited efficacy. EXPERIMENTAL DESIGN: Sensitivity to sorafenib was analyzed in MCL cell lines and primary samples in the context of BCR stimulation and microenvironment simulation. Sorafenib signaling was characterized by quantitative PCR, western blot, immunofluorescence and protein immunoprecipitation. Migration analysis included flow cytometry counting, actin polymerization assays and siRNA-mediated knockdown of FAK. In vivo antitumor effect of sorafenib and bortezomib was analyzed in MCL xenograft mouse model. RESULTS: Sorafenib rapidly dephosphorylates the BCR-associated kinases, SYK and LYN, as well as FAK, a SRC target involved in focal adhesion. In this line, sorafenib displays strong synergy with the SYK inhibitor, R406. Sorafenib also blocks Mcl-1 and cyclin D1 translation, which promotes an unbalance between pro- and anti-apoptotic proteins and facilitates Bax release from cyclin D1, leading to the induction of mitochondrial apoptosis and caspase-dependent and independent mechanisms. Moreover, sorafenib inhibits MCL cell migration and CXCL12-induced actin polymerization. FAK knockdown partially prevents this inhibitory effect, indicating that FAK is a relevant target of sorafenib. Furthermore, sorafenib enhances the antitumoral effect of bortezomib in a MCL xenograft mouse model, as well as overcomes stroma-mediated bortezomib-resistance in MCL cells. CONCLUSIONS: We demonstrate for the first time that sorafenib interferes BCR signaling, protein translation and modulates the microenvironment prosurvival signals in MCL, suggesting that sorafenib, alone or in combination with bortezomib, may represent a promising approach to treat MCL patients.
Clinical Cancer Research 12/2012; · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: NOTCH1 has been found recurrently mutated in a subset of patients with chronic lymphocytic leukemia (CLL). To analyze biological features and clinical impact of NOTCH1 mutations in CLL, we sequenced this gene in 565 patients. NOTCH1 mutations, found in 63 patients (11%), were associated with unmutated IGHV, high expression of CD38 and ZAP-70, trisomy 12, advanced stage and elevated lactate dehydrogenase. Sequential analysis in 200 patients demonstrated acquisition of mutation in one case (0.5%) and disappearance after treatment in two. Binet A and B patients with NOTCH1-mutated had a shorter time to treatment. NOTCH1-mutated patients were more frequently refractory to therapy and showed shorter progression-free and overall survival after complete remission. Overall survival was shorter in NOTCH1-mutated patients, although not independently from IGHV. NOTCH1 mutation increased the risk of transformation to diffuse large B-cell lymphoma independently from IGHV, with this being validated in resampling tests of replicability. In summary, NOTCH1 mutational status, that was rarely acquired during the course of the disease, identify a genetic subgroup with high risk of transformation and poor outcome. This recently identified genetic subgroup of CLL patients deserves prospective studies to define their best management.Leukemia advance online publication, 8 January 2013; doi:10.1038/leu.2012.357.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 12/2012; · 10.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.