The genetic hallmark of Burkitt lymphoma (BL) is the t(8;14)(q24;q32) and its variants leading to activation of the MYC oncogene. It is a matter of debate whether true BL without MYC translocation exists. Here, we identified 59 lymphomas concordantly called BL by 2 gene expression classifiers among 753 B-cell lymphomas. Only 2 (3%) of these 59 molecular BL lacked a MYC translocation, which both shared a peculiar pattern of chromosome 11q aberration characterized by interstitial gains including 11q23.2-q23.3 and telomeric losses of 11q24.1-qter. We extended our analysis to 17 MYC-negative high-grade B-cell lymphomas with a similar 11q aberration and showed this aberration to be recurrently associated with morphologic and clinical features of BL. The minimal region of gain was defined by high-level amplifications in 11q23.3 and associated with overexpression of genes including PAFAH1B2 on a transcriptional and protein level. The recurrent region of loss contained a focal homozygous deletion in 11q24.2-q24.3 including the ETS1 gene, which was shown to be mutated in 4 of 16 investigated cases. These findings indicate the existence of a molecularly distinct subset of B-cell lymphomas reminiscent of BL, which is characterized by deregulation of genes in 11q.
Chromosomal translocations affecting the MYC oncogene are the biologic hallmark of Burkitt lymphomas but also occur in a subset of other mature B-cell lymphomas. If accompanied by a chromosomal break targeting the BCL2 and/or BCL6 oncogene these MYC translocation positive (MYC+) lymphomas are called double-hit lymphomas, otherwise the term single-hit lymphomas is applied. In order to characterize the biologic features of these MYC+ lymphomas other than Burkitt lymphoma we explored, after exclusion of molecular Burkitt lymphoma as defined by gene expression profiling, the molecular, pathological and clinical aspects of 80 MYC-translocation positive lymphomas (31 single-hit, 46 double-hit & 3 MYC+-lymphomas with unknown BCL6 status). Comparison of single-hit and double-hit lymphomas revealed no difference in MYC partner (IG/non-IG), genomic complexity, MYC expression or gene expression profile. Double-hit lymphomas showed more frequent GCB-like gene expression profile and higher IGH and MYC mutation frequencies. Gene expression profiling revealed 130 differentially expressed genes between BCL6+/MYC+ and BCL2+/MYC+ double-hit lymphomas. BCL2+/MYC+ double-hit lymphomas showed a more frequent GCB-like gene expression profile. Analysis of all lymphomas according to MYC partner (IG/non-IG) revealed no substantial differences. In this series of lymphomas, in which immunochemotherapy was administered in only a minority of cases, single-hit and double-hit lymphomas had a similar poor outcome in contrast with molecular Burkitt lymphoma and lymphomas without MYC break. Our data suggest that, after excluding molecular Burkitt lymphoma and pediatric cases, MYC+ lymphomas are biologically quite homogenous with single-hit and double-hit lymphomas as well as IG-MYC and non-IG-MYC+ lymphomas sharing various molecular characteristics.
The prognosis of germinal center-derived B-cell (GCB) lymphomas, including follicular lymphoma and diffuse large-B-cell lymphoma (DLBCL), strongly depends on age. Children have a more favorable outcome than adults. It is not known whether this is because of differences in host characteristics, treatment protocols, or tumor biology, including the presence of chromosomal alterations. By screening for novel IGH translocation partners in pediatric and adult lymphomas, we identified chromosomal translocations juxtaposing the IRF4 oncogene next to one of the immunoglobulin (IG) loci as a novel recurrent aberration in mature B-cell lymphoma. FISH revealed 20 of 427 lymphomas to carry an IG/IRF4-fusion. Those were predominantly GCB-type DLBCL or follicular lymphoma grade 3, shared strong expression of IRF4/MUM1 and BCL6, and lacked PRDM1/BLIMP1 expression and t(14;18)/BCL2 breaks. BCL6 aberrations were common. The gene expression profile of IG/IRF4-positive lymphomas differed from other subtypes of DLBCL. A classifier for IG/IRF4 positivity containing 27 genes allowed accurate prediction. IG/IRF4 positivity was associated with young age and a favorable outcome. Our results suggest IRF4 translocations to be primary alterations in a molecularly defined subset of GCB-derived lymphomas. The probability for this subtype of lymphoma significantly decreases with age, suggesting that diversity in tumor biology might contribute to the age-dependent differences in prognosis of lymphoma.
The micronucleus test (MNT) is a well-established assay in genotoxicity testing and human biomonitoring. The cytokinesis-block micronucleus test (CBMNT) is the preferred method for measuring MN in cultured human lymphocytes from human subjects exposed to genotoxins. It is, however, unclear to what extent mutagen exposure either leads to the formation of MN already in vivo or to the formation of MN ex vivo during cell culture as a consequence of persisting DNA damage. To address this question, we investigated peripheral blood of 22 patients who had received cytostatic therapies including drugs with clastogenic and aneugenic effects. We also performed the MNT with blood samples from 13 healthy controls without relevant mutagen exposure. The incidence of MN was studied 24, 48 and 72 h after the start of the culture in mononuclear lymphocytes in cultures without cytochalasin B and also at 72 h in binucleated lymphocytes in the standard CBMNT. The mean frequency of binuclear cells with MN in the CBMNT was clearly increased in blood samples from patients (29.3 versus 10.2 per 1000 in controls). In contrast, mononuclear lymphocytes analysed 24 or 48 h after start of the cultures only revealed a marginal increase in MN frequencies in comparison to controls. These results suggest that mutagen exposure in vivo mainly leads to the formation of MN during ex vivo proliferation of lymphocytes as a consequence of persistent damage. Characterization of MN in binuclear lymphocytes from patients by fluorescence in situ hybridization (FISH) with a pan-centromeric probe indicated that MN arose by clastogenic and aneugenic mechanisms. A high portion of MN was relatively large and exhibited several centromere signals. If the results of this study with patients exposed to cytostatic drugs also apply to other kinds of mutagen exposure, increased MN frequencies in the CBMNT can only be expected for exposures leading to persistent damage in peripheral lymphocytes and MN formation during ex vivo lymphocyte culture.
Lymphomas are assumed to originate at different stages of lymphocyte development through chromosomal aberrations. Thus, different lymphomas resemble lymphocytes at distinct differentiation stages and show characteristic morphologic, genetic, and transcriptional features. Here, we have performed a microarray-based DNA methylation profiling of 83 mature aggressive B-cell non-Hodgkin lymphomas (maB-NHLs) characterized for their morphologic, genetic, and transcriptional features, including molecular Burkitt lymphomas and diffuse large B-cell lymphomas. Hierarchic clustering indicated that methylation patterns in maB-NHLs were not strictly associated with morphologic, genetic, or transcriptional features. By supervised analyses, we identified 56 genes de novo methylated in all lymphoma subtypes studied and 22 methylated in a lymphoma subtype-specific manner. Remarkably, the group of genes de novo methylated in all lymphoma subtypes was significantly enriched for polycomb targets in embryonic stem cells. De novo methylated genes in all maB-NHLs studied were expressed at low levels in lymphomas and normal hematopoietic tissues but not in nonhematopoietic tissues. These findings, especially the enrichment for polycomb targets in stem cells, indicate that maB-NHLs with different morphologic, genetic, and transcriptional background share a similar stem cell-like epigenetic pattern. This suggests that maB-NHLs originate from cells with stem cell features or that stemness was acquired during lymphomagenesis by epigenetic remodeling.
Follicular lymphoma (FL) is a morphologically and genetically well-characterized B-cell non-Hodgkin lymphoma that can show predominantly follicular, combined follicular and diffuse, or predominantly diffuse growth patterns. Although approximately 85% of FLs harbor the translocation t(14;18)(q32;q21) and consistently display a follicular growth pattern, predominantly diffuse FLs are less well characterized on the phenotypical, molecular, and clinical level. We studied 35 predominantly diffuse FL by immunohistochemistry, classical chromosome banding analysis, fluorescence in situ hybridization (FISH), and gene expression profiling. A total of 28 of 29 analyzable cases lacked t(14;18), and 27 of 29 cases revealed a unifying chromosomal aberration, a deletion in 1p36. Morphologically, 12 FLs were grade 1 and 23 were grade 2, and the immunophenotype with frequent expression of CD10, BCL6, and CD23 was in line with a germinal center B-cell phenotype. The gene expression profiles of 4 predominantly diffuse FLs fell into the spectrum of typical FL, with a unique enrichment of specific gene signatures. Remarkably, patients with diffuse FL frequently presented with low clinical stage and large but localized inguinal tumors. These results suggest that predominantly diffuse FL represent a distinct subtype of t(14;18)-negative nodal FL with a unifying genetic alteration (deletion of 1p36) and characteristic clinical features.
Follicular lymphoma (FL) is characterized by a large number of chromosomal aberrations. However, their exact genomic extension and involved target genes remain to be determined. For this purpose, we used array-based intermediate-high resolution genomic profiling in combination with Affymetrix gene expression analysis. Tumor specimens from 128 FL patients were analyzed for the presence of genomic aberrations and the results were correlated to clinical data sets and mRNA expression levels. In 114 (89%) of the 128 analyzed cases, a total of 688 genomic aberrations (384 gains/amplifications and 304 losses) were detected. Frequent genomic aberrations were: -1p36 (18%), +2p15 (24%), -3q (14%), -6q (25%), +7p (19%), +7q (23%), +8q (14%), -9p (16%), -11q (15%), +12q (20%), -13q (11%), -17p (16%), +18p (18%), and +18q (28%). Critical segments of these imbalances were delineated to genomic fragments with a minimum size down to 0.2 Mb. By comparison of these with mRNA gene expression data, putative candidate genes were identified. Moreover, we found that deletions affecting the tumor suppressor gene CDKN2A/B on 9p21 were detected in nontransformed FL grade I-II. For this aberration as well as for -6q25 and -6q26, an association with inferior survival was observed.
Translocations affecting chromosome subband 6p25.3 containing the IRF4 gene have been recently described as characteristic alterations in a molecularly distinct subset of germinal center B-cell-derived lymphomas. Secondary changes have yet only been described in few of these lymphomas. Here, we performed array-comparative genomic hybridization and molecular inversion probe microarray analyses on DNA from 12 formalin-fixed paraffin-embedded and two fresh-frozen IRF4 translocation-positive lymphomas, which together with the previously published data on nine cases allowed the extension of copy number analyses to a total of 23 of these lymphomas. All except one case carried chromosomal imbalances, most frequently gains in Xq28, 11q22.3-qter, and 7q32.1-qter and losses in 6q13-16.1, 15q14-22.31, and 17p. No recurrent copy-neutral losses of heterozygosity were observed. TP53 point mutations were detected in three of six cases with loss of 17p. Overall this study unravels a recurrent pattern of secondary genetic alterations in IRF4 translocation-positive lymphomas.
Diffuse large B-cell lymphoma is the most frequent type of B-cell lymphoma in adult patients but also occurs in children. Patients are currently assigned to therapy regimens based on arbitrarily chosen age limits only (eg, 18 or 60 years) and not biologically justified limits. A total of 364 diffuse large B-cell lymphomas and related mature aggressive B-cell lymphomas other than Burkitt lymphoma from all age groups were analyzed by comprehensive molecular profiling. The probability of several biologic features previously reported to be associated with poor prognosis in diffuse large B-cell lymphoma, such as ABC subtype, BCL2 expression, or cytogenetic complexity, increases with age at diagnosis. Similarly, various genetic features, such as IRF4 translocations, gains in 1q21, 18q21, 7p22, and 7q21, as well as changes in 3q27, including gains and translocations affecting the BCL6 locus, are significantly associated with patient age, but no cut-offs between age groups could be defined. If age was incorporated in multivariate analyses, genetic complexity lost its prognostic significance, whereas the prognostic impact of ABC subtype and age were additive. Our data indicate that aging is a major determinant of lymphoma biology. They challenge current concepts regarding both prognostic biomarkers and treatment stratification based on strict age cut-offs.
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