MINCR is a MYC-induced lncRNA able to modulate MYC's transcriptional network in Burkitt lymphoma cells.

Despite the established role of the transcription factor MYC in cancer, little is known about the impact of a new class of transcriptional regulators, the long noncoding RNAs (lncRNAs), on MYC ability to influence the cellular transcriptome. Here, we have intersected RNA-sequencing data from two MYC-inducible cell lines and a cohort of 91 B-cell lymphomas with or without genetic variants resulting in MYC overexpression. We identified 13 lncRNAs differentially expressed in IG-MYC-positive Burkitt lymphoma and regulated in the same direction by MYC in the model cell lines. Among them, we focused on a lncRNA that we named MYC-induced long noncoding RNA (MINCR), showing a strong correlation with MYC expression in MYC-positive lymphomas. To understand its cellular role, we performed RNAi and found that MINCR knockdown is associated with an impairment in cell cycle progression. Differential gene expression analysis after RNAi showed a significant enrichment of cell cycle genes among the genes down-regulated after MINCR knockdown. Interestingly, these genes are enriched in MYC binding sites in their promoters, suggesting that MINCR acts as a modulator of the MYC transcriptional program. Accordingly, MINCR knockdown was associated with a reduction in MYC binding to the promoters of selected cell cycle genes. Finally, we show that down-regulation of Aurora kinases A and B and chromatin licensing and DNA replication factor 1 may explain the reduction in cellular proliferation observed on MINCR knockdown. We, therefore, suggest that MINCR is a newly identified player in the MYC transcriptional network able to control the expression of cell cycle genes.

B-cell prolymphocytic leukemia: a specific subgroup of mantle cell lymphoma.

B-cell prolymphocytic leukemia (B-PLL) is a rare mature B-cell malignancy that may be hard to distinguish from mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). B-PLL cases with a t(11;14) were redefined as MCL in the World Health Organization 2008 classification. We evaluated 13 B-PLL patients [7 being t(11;14)-positive (B-PLL+) and 6 negative (B-PLL-)] and compared them with MCL and CLL patients. EuroFlow-based immunophenotyping showed significant overlap between B-PLL+ and B-PLL-, as well as between B-PLL and MCL, whereas CLL clustered separately. Immunogenotyping showed specific IGHV gene usage partly resembling MCL. Gene expression profiling showed no separation between B-PLL+ and B-PLL- but identified 3 subgroups. One B-PLL subgroup clustered close to CLL and another subgroup clustered with leukemic MCL; both were associated with prolonged survival. A third subgroup clustered close to nodal MCL and was associated with short survival. Gene expression profiles of both B-PLL+ and B-PLL- showed best resemblance with normal immunoglobulin M-only B-cells. Our data confirm that B-PLL+ is highly comparable to MCL, indicate that B-PLL- also may be considered as a specific subgroup of MCL, and suggest that B-PLL is part of a spectrum, ranging from CLL-like B-PLL, to leukemic MCL-like B-PLL, to nodal MCL-like B-PLL.

Alterations of microRNA and microRNA-regulated messenger RNA expression in germinal center B-cell lymphomas determined by integrative sequencing analysis.

MicroRNA are well-established players in post-transcriptional gene regulation. However, information on the effects of microRNA deregulation mainly relies on bioinformatic prediction of potential targets, whereas proof of the direct physical microRNA/target messenger RNA interaction is mostly lacking. Within the International Cancer Genome Consortium Project "Determining Molecular Mechanisms in Malignant Lymphoma by Sequencing", we performed miRnome sequencing from 16 Burkitt lymphomas, 19 diffuse large B-cell lymphomas, and 21 follicular lymphomas. Twenty-two miRNA separated Burkitt lymphomas from diffuse large B-cell lymphomas/follicular lymphomas, of which 13 have shown regulation by MYC. Moreover, we found expression of three hitherto unreported microRNA. Additionally, we detected recurrent mutations of hsa-miR-142 in diffuse large B-cell lymphomas and follicular lymphomas, and editing of the hsa-miR-376 cluster, providing evidence for microRNA editing in lymphomagenesis. To interrogate the direct physical interactions of microRNA with messenger RNA, we performed Argonaute-2 photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation experiments. MicroRNA directly targeted 208 messsenger RNA in the Burkitt lymphomas and 328 messenger RNA in the non-Burkitt lymphoma models. This integrative analysis discovered several regulatory pathways of relevance in lymphomagenesis including Ras, PI3K-Akt and MAPK signaling pathways, also recurrently deregulated in lymphomas by mutations. Our dataset reveals that messenger RNA deregulation through microRNA is a highly relevant mechanism in lymphomagenesis.

Quality assessment program for EuroFlow protocols: summary results of four-year (2010-2013) quality assurance rounds.

Flow cytometric immunophenotyping has become essential for accurate diagnosis, classification, and disease monitoring in hemato-oncology. The EuroFlow Consortium has established a fully standardized "all-in-one" pipeline consisting of standardized instrument settings, reagent panels, and sample preparation protocols and software for data analysis and disease classification. For its reproducible implementation, parallel development of a quality assurance (QA) program was required. Here, we report on the results of four consecutive annual rounds of the novel external QA EuroFlow program. The novel QA scheme aimed at monitoring the whole flow cytometric analysis process (cytometer setting, sample preparation, acquisition and analysis) by reading the median fluorescence intensities (MedFI) of defined lymphocytes' subsets. Each QA participant applied the predefined reagents' panel on blood cells of local healthy donors. A uniform gating strategy was applied to define lymphocyte subsets and to read MedFI values per marker. The MedFI values were compared with reference data and deviations from reference values were quantified using performance score metrics. In four annual QA rounds, we analyzed 123 blood samples from local healthy donors on 14 different instruments in 11 laboratories from nine European countries. The immunophenotype of defined cellular subsets appeared sufficiently standardized to permit unified (software) data analysis. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%, average MedFI in each QA round ranged from 86 to 125% from overall median. Calculation of performance scores was instrumental to pinpoint standardization failures and their causes. Overall, the new EuroFlow QA system for the first time allowed to quantify the technical variation that is introduced in the measurement of fluorescence intensities in a multicentric setting over an extended period of time. EuroFlow QA is a proficiency test specific for laboratories that use standardized EuroFlow protocols. It may be used to complement, but not replace, established proficiency tests. © 2014 International Society for Advancement of Cytometry.

Mutational mechanisms shaping the coding and noncoding genome of germinal center derived B-cell lymphomas.

B cells have the unique property to somatically alter their immunoglobulin (IG) genes by V(D)J recombination, somatic hypermutation (SHM) and class-switch recombination (CSR). Aberrant targeting of these mechanisms is implicated in lymphomagenesis, but the mutational processes are poorly understood. By performing whole genome and transcriptome sequencing of 181 germinal center derived B-cell lymphomas (gcBCL) we identified distinct mutational signatures linked to SHM and CSR. We show that not only SHM, but presumably also CSR causes off-target mutations in non-IG genes. Kataegis clusters with high mutational density mainly affected early replicating regions and were enriched for SHM- and CSR-mediated off-target mutations. Moreover, they often co-occurred in loci physically interacting in the nucleus, suggesting that mutation hotspots promote increased mutation targeting of spatially co-localized loci (termed hypermutation by proxy). Only around 1% of somatic small variants were in protein coding sequences, but in about half of the driver genes, a contribution of B-cell specific mutational processes to their mutations was found. The B-cell-specific mutational processes contribute to both lymphoma initiation and intratumoral heterogeneity. Overall, we demonstrate that mutational processes involved in the development of gcBCL are more complex than previously appreciated, and that B cell-specific mutational processes contribute via diverse mechanisms to lymphomagenesis.

DNA methylome analysis in Burkitt and follicular lymphomas identifies differentially methylated regions linked to somatic mutation and transcriptional control.

Although Burkitt lymphomas and follicular lymphomas both have features of germinal center B cells, they are biologically and clinically quite distinct. Here we performed whole-genome bisulfite, genome and transcriptome sequencing in 13 IG-MYC translocation-positive Burkitt lymphoma, nine BCL2 translocation-positive follicular lymphoma and four normal germinal center B cell samples. Comparison of Burkitt and follicular lymphoma samples showed differential methylation of intragenic regions that strongly correlated with expression of associated genes, for example, genes active in germinal center dark-zone and light-zone B cells. Integrative pathway analyses of regions differentially methylated in Burkitt and follicular lymphomas implicated DNA methylation as cooperating with somatic mutation of sphingosine phosphate signaling, as well as the TCF3-ID3 and SWI/SNF complexes, in a large fraction of Burkitt lymphomas. Taken together, our results demonstrate a tight connection between somatic mutation, DNA methylation and transcriptional control in key B cell pathways deregulated differentially in Burkitt lymphoma and other germinal center B cell lymphomas.