[show abstract][hide abstract] ABSTRACT: DNA repair mechanisms are fundamental for B cell development, which relies on the somatic diversification of the immunoglobulin genes by V(D)J recombination, somatic hypermutation, and class switch recombination. Their failure is postulated to promote genomic instability and malignant transformation in B cells. By performing targeted sequencing of 73 key DNA repair genes in 29 B cell lymphoma samples, somatic and germline mutations were identified in various DNA repair pathways, mainly in diffuse large B cell lymphomas (DLBCLs). Mutations in mismatch repair genes (EXO1, MSH2, and MSH6) were associated with microsatellite instability, increased number of somatic insertions/deletions, and altered mutation signatures in tumors. Somatic mutations in nonhomologous end-joining (NHEJ) genes (DCLRE1C/ARTEMIS, PRKDC/DNA-PKcs, XRCC5/KU80, and XRCC6/KU70) were identified in four DLBCL tumors and cytogenetic analyses revealed that translocations involving the immunoglobulin-heavy chain locus occurred exclusively in NHEJ-mutated samples. The novel mutation targets, CHEK2 and PARP1, were further screened in expanded DLBCL cohorts, and somatic as well as novel and rare germline mutations were identified in 8 and 5% of analyzed tumors, respectively. By correlating defects in a subset of DNA damage response and repair genes with genomic instability events in tumors, we propose that these genes play a role in DLBCL lymphomagenesis.
Journal of Experimental Medicine 08/2013; · 13.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Non-Hodgkin lymphoma represents a diverse group of blood malignancies, of which follicular lymphoma (FL) is a common subtype. Previous genome-wide association studies (GWASs) have identified in the human leukocyte antigen (HLA) class II region multiple independent SNPs that are significantly associated with FL risk. To dissect these signals and determine whether coding variants in HLA genes are responsible for the associations, we conducted imputation, HLA typing, and sequencing in three independent populations for a total of 689 cases and 2,446 controls. We identified a hexa-allelic amino acid polymorphism at position 13 of the HLA-DR beta chain that showed the strongest association with FL within the major histocompatibility complex (MHC) region (multiallelic p = 2.3 × 10(-15)). Out of six possible amino acids that occurred at that position within the population, we classified two as high risk (Tyr and Phe), two as low risk (Ser and Arg), and two as moderate risk (His and Gly). There was a 4.2-fold difference in risk (95% confidence interval = 2.9-6.1) between subjects carrying two alleles encoding high-risk amino acids and those carrying two alleles encoding low-risk amino acids (p = 1.01 × 10(-14)). This coding variant might explain the complex SNP associations identified by GWASs and suggests a common HLA-DR antigen-driven mechanism for the pathogenesis of FL and rheumatoid arthritis.
The American Journal of Human Genetics 06/2013; · 11.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: Diffuse large B cell lymphoma (DLBCL) is a heterogeneous group of B cell lymphomas. MicroRNA expression provides a new and interesting tool for understanding the biology and clinical course of DLBCL. The present study presents microRNA-129-5p expression data from DLBCL patients treated with CHOP or R-CHOP. Patients with low microRNA-129-5p expression had a median survival of 23 months and a significantly shorter overall survival (P = 0.0042) compared to patients with high microRNA-129-5p expression, who had a median survival of 58 months. We also found that patients treated with R-CHOP only and displaying low microRNA-129-5p expression had a significantly shorter overall survival compared to patients with high microRNA-129-5p expression; all such patients were still alive at the time of last follow-up (P = 0.043). No significant difference was found among microRNA-129-5p expression in tumor tissue, the tissue surrounding the tumor, and normal controls. To our knowledge, this is the first report to show that the expression of microRNA-129-5p can affect the clinical outcome of DLBCL patients and that microRNA-129-5p may be involved in the biology of DLBCL development, although larger studies are necessary to confirm this. Further investigations may also help to elucidate the biological role of microRNA-129-5p in DLBCL.
International journal of hematology 03/2013; · 1.17 Impact Factor
[show abstract][hide abstract] ABSTRACT: Deregulation of microRNA (miRNA) expression has been documented in diffuse large B-cell lymphoma (DLBCL). However, the impact of miRNAs and their machinery in DLBCL is not fully determined. Here, we assessed the role of miRNA expression and their processing genes in DLBCL development. Using microarray and RT-qPCR approaches, we quantified global miRNAs and core components of miRNA-processing genes expression in 75 DLBCLs (56 de novo and 19 transformed) and 10 lymph nodes (LN). Differential miRNA signatures were identified between DLBCLs and LNs, or between the de novo and transformed DLBCLs. We also identified subsets of miRNAs associated with germinal center B-cell phenotype, BCL6 and IRF4 expression, and clinical staging. In addition, we showed a significant over-expression of TARBP2 in de novo DLBCLs as compared with LNs, and decreased expression of DROSHA, DICER, TARBP2 and PACT in transformed as compared with de novo cases. Interestingly, cases with high TARBP2 and DROSHA expression had a poorer chemotherapy response. We further showed that TARBP2 can regulate miRNA-processing efficiency in DLBCLs, and its expression inhibition decreases cell growth and increases apoptosis in DLBCL cell lines. Our findings provide new insights for the understanding of miRNAs and its machinery in DLBCL.
Blood Cancer Journal 01/2013; 3:e152. · 1.40 Impact Factor
[show abstract][hide abstract] ABSTRACT: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous group of B-cell lymphomas. A new and important tool for understanding the biology and clinical course of DLBCL is microRNA expression. This study presents microRNA-200c expression data from 61 DLBCL patients treated with CHOP or R-CHOP. Patients with high microRNA-200c expression had a median survival of 20.3 months and a significantly shorter overall survival (P=0.019) compared to patients with low microRNA-200c expression, who had a median survival of 35.8 months. We also found that patients treated with R-CHOP only and displaying high microRNA-200c expression had a significantly shorter overall survival compared to patients with low microRNA-200c expression, where all patients were still alive at the time of the last follow-up (P=0.0036). Lastly, we found that patients with high microRNA-200c expression had a significantly shorter time from initial diagnosis to the first relapse compared to patients with low microRNA-200c expression (P=0.0001). To our knowledge, this is the first study showing that the expression of microRNA-200c affects the clinical outcome of DLBCL patients, and that microRNA-200c is involved in the biology of DLBCL development, although larger studies are necessary to confirm this. Further investigations may also help to elucidate the biological role of microRNA-200c in DLBCL.
[show abstract][hide abstract] ABSTRACT: Genetic heterogeneity is common in tumors, explicable by the development of subclones with distinct genetic and epigenetic alterations. We describe an in vitro model for cancer heterogeneity, comprising the diffuse large B-cell lymphoma cell line U-2932 which expresses two sets of cell surface markers representing twin populations flow-sorted by CD20 vs CD38 expression. U-2932 populations were traced to subclones of the original tumor with clone-specific immunoglobulin IgV(H)4-39 hypermutation patterns. BCL6 was overexpressed in one subpopulation (R1), MYC in the other (R2), both clones overexpressed BCL2. According to the combined results of immunoglobulin hypermutation and cytogenetic analysis, R1 and R2 derive from a mother clone with genomic BCL2 amplification, which acquired secondary rearrangements leading to the overexpression of BCL6 (R1) or MYC (R2). Some 200 genes were differentially expressed in R1/R2 microarrays including transcriptional targets of the aberrantly expressed oncogenes. Other genes were regulated by epigenetic means as shown by DNA methylation analysis. Ectopic expression of BCL6 in R2 variously modulated new candidate target genes, confirming dual silencing and activating functions. In summary, stable retention of genetically distinct subclones in U-2932 models tumor heterogeneity in vitro permitting functional analysis of oncogenes against a syngenic background.Leukemia advance online publication, 8 January 2013; doi:10.1038/leu.2012.358.
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: Cernunnos is involved in the nonhomologous end-joining (NHEJ) process during DNA double-strand break (DSB) repair. Here, we
studied immunoglobulin (Ig) class switch recombination (CSR), a physiological process which relies on proper repair of the
DSBs, in B cells from Cernunnos-deficient patients. The pattern of in vivo generated CSR junctions is altered in these cells,
with unusually long microhomologies and a lack of direct end-joining. The CSR junctions from Cernunnos-deficient patients
largely resemble those from patients lacking DNA ligase IV, Artemis, or ATM, suggesting that these factors are involved in
the same end-joining pathway during CSR. By screening 269 mature B cell lymphoma biopsies, we also identified a somatic missense
Cernunnos mutation in a diffuse large B cell lymphoma sample. This mutation has a dominant-negative effect on joining of a subset of
DNA ends in an in vitro NHEJ assay. Translocations involving both Ig heavy chain loci and clonal-like, dynamic IgA switching
activities were observed in this tumor. Collectively, our results suggest a link between defects in the Cernunnos-dependent
NHEJ pathway and aberrant CSR or switch translocations during the development of B cell malignancies.
Journal of Experimental Medicine 02/2012; 209(2):291-305. · 13.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: The p16(INK4a) tumor suppressor gene can be inactivated by a variety of events including promoter hypermethylation. In diffuse large B-cell lymphoma (DLBCL), p16(INK4a) methylation has been associated with advanced disease stage and higher IPI. The prognostic impact of p16(INK4a) methylation in DLBCL remains unclear; however, it has been suggested to correlate with inferior outcome. To further investigate the clinical impact of p16(INK4a) methylation in DLBCL, promoter methylation of this gene was assessed quantitatively by pyrosequencing. Forty-two of 113 (37%) DLBCL patients with methylation level above 5% were categorized as methylated and subsequently divided into low, intermediate and high methylation categories. Overall, no association was shown between the extent of p16(INK4a) methylation and patients' clinical characteristics, except disease stage (P=0.049). Moreover, we could not reveal any impact of p16(INK4a) methylation on lymphoma-specific survival. Although >25% of p16(INK4a) methylation correlated with a better progression-free survival (P=0.048) in patients <65 years old, the significance of this finding, if any, needs to be further investigated. In conclusion, our finding questions the role of p16(INK4a) promoter methylation as a negative prognostic factor in DLBCL.
Leukemia research 10/2010; 35(4):438-43. · 2.36 Impact Factor
[show abstract][hide abstract] ABSTRACT: Transcription factors (TFs) are critical for B-cell differentiation, affecting gene expression both by repression and transcriptional activation. Still, this information is not used for classification of B-cell lymphomas (BCLs). Traditionally, BCLs are diagnosed based on a phenotypic resemblance to normal B-cells; assessed by immunohistochemistry or flow cytometry, by using a handful of phenotypic markers. In the last decade, diagnostic and prognostic evaluation has been facilitated by global gene expression profiling (GEP), providing a new powerful means for the classification, prediction of survival, and response to treatment of lymphomas. However, most GEP studies have typically been performed on whole tissue samples, containing varying degrees of tumor cell content, which results in uncertainties in data analysis. In this study, global GEP analyses were performed on highly purified, flow-cytometry sorted tumor-cells from eight subgroups of BCLs. This enabled identification of TFs that can be uniquely associated to the tumor cells of chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), hairy cell leukemia (HCL), and mantle cell lymphoma (MCL). The identified transcription factors influence both the global and specific gene expression of the BCLs and have possible implications for diagnosis and treatment.
American Journal of Hematology 06/2010; 85(6):418-25. · 4.00 Impact Factor
[show abstract][hide abstract] ABSTRACT: Follicular lymphoma (FL) frequently transforms into the more aggressive diffuse large B cell lymphoma (DLBCL-tr), but no protein biomarkers have been identified for predictive or early diagnosis. Gene expression analyses have identified genes changing on transformation but have failed to be reproducible in different studies, reflecting the heterogeneity within the tumor tissue and between tumor samples. Gene expression analyses on Affymetrix Human Genome U133 Plus 2.0 arrays were performed, using flow cytometry sorted tumor cells derived from FL and transformed DLBCL. To identify molecular targets associated with the transformation, subsequent immunohistochemistry (IHC) analyses of the corresponding proteins were performed. Using highly purified cells, this study identified 163 genes, which were significantly deregulated during the transformation in a majority of cases. Among the upregulated transcripts, 13 genes were selected for validation using IHC, based on the availability of commercial antibodies, and galectin-3 and NEK2 proteins specifically identify DLBCL-tr, when compared with FL. We demonstrate that by purifying tumor cells through cell sorting, thereby reducing the heterogeneity due to infiltrating cells, it was possible to identify distinct differences between tumor entities rather than variations due to cellular composition. Galectin-3 and NEK2 both identified a subgroup of DLBCL-tr, and the function of these protein markers also suggests a biological role in the transformation process.
American Journal of Hematology 09/2009; 84(12):803-8. · 4.00 Impact Factor
[show abstract][hide abstract] ABSTRACT: Presence of TP53 mutations has been associated with poor prognosis in diffuse large B-cell lymphoma (DLBCL), although this has remained controversial. The TP53 codon 72 polymorphism has shown negative impact on cancer survival, but this has not been analyzed in DLBCL. Furthermore, the MDM2 SNP309 has been associated with earlier age of onset in DLBCL. Here, we investigated the clinical impact of TP53 mutations, MDM2 SNP309 and TP53 codon 72 polymorphisms on survival in DLBCL of germinal center (GC) and non-GC subtypes. Thirteen of the 102 (12.7%) patients displayed TP53 mutations. Overall, TP53 mutations had a significant effect on lymphoma-specific survival (LSS, P=0.009) and progression-free survival (PFS, P=0.028). In particular, inferior survival was observed in TP53-mutated DLBCLs of GC subtype (LSS, P=0.002 and PFS, P=0.006). Neither MDM2 SNP309 nor the TP53 codon 72 polymorphism had an impact on age of onset or survival. Altogether, our data suggests that TP53 mutations are associated with poor outcome in GC-DLBCL patients.
Leukemia Research 09/2008; 33(1):60-6. · 2.76 Impact Factor
[show abstract][hide abstract] ABSTRACT: Antibody microarrays enable extensive protein expression profiling, and provide a valuable complement to DNA microarray-based gene expression profiling. In this study, we used DotScan antibody microarrays that contain antibodies against 82 different cell surface antigens, to determine phenotypic protein expression profiles for human B cell sub-populations. We then demonstrated that the B cell protein profile can be used to delineate the relationship between normal B cells and malignant counterparts. Principle component analysis showed that the lymphomas did not cluster with the normal memory B cells or germinal centre B cells, but they did cluster with germinal centre founder cells and naïve B cells.
Cancer Letters 07/2008; 265(1):98-106. · 4.26 Impact Factor