Unraveling the genetics of transformed splenic marginal zone lymphoma.

The genetic mechanisms associated with splenic marginal zone lymphoma (SMZL) transformation are not well defined. We studied 41 patients with SMZL that eventually underwent large B-cell lymphoma transformation. Tumor material was obtained either only at diagnosis (9 patients), at diagnosis and transformation (18 patients), and only at transformation (14 patients). Samples were categorized in 2 groups: (1) at diagnosis (SMZL, n = 27 samples), and (2) at transformation (SMZL-T, n = 32 samples). Using copy number arrays and a next-generation sequencing custom panel, we identified that the main genomic alterations in SMZL-T involved TNFAIP3, KMT2D, TP53, ARID1A, KLF2, 1q gains, and losses of 9p21.3 (CDKN2A/B) and 7q31-q32. Compared with SMZL, SMZL-T had higher genomic complexity, and higher incidence of TNFAIP3 and TP53 alterations, 9p21.3 (CDKN2A/B) losses, and 6p gains. SMZL and SMZL-T clones arose by divergent evolution from a common altered precursor cell that acquired different genetic alterations in virtually all evaluable cases (92%, 12 of 13 cases). Using whole-genome sequencing of diagnostic and transformation samples in 1 patient, we observed that the SMZL-T sample carried more genomic aberrations than the diagnostic sample, identified a translocation t(14;19)(q32;q13) present in both samples, and detected a focal B2M deletion due to chromothripsis acquired at transformation. Survival analysis showed that KLF2 mutations, complex karyotype, and International Prognostic Index score at transformation were predictive of a shorter survival from transformation (P = .001; P = .042; and P = .007; respectively). In summary, SMZL-T are characterized by higher genomic complexity than SMZL, and characteristic genomic alterations that could represent key players in the transformation event.

Intravascular Large B-Cell Lymphoma Genomic Profile Is Characterized by Alterations in Genes Regulating NF-κB and Immune Checkpoints.

Intravascular large B-cell lymphoma (IVLBCL) is an uncommon lymphoma with an aggressive clinical course characterized by selective growth of tumor cells within the vessels. Its pathogenesis is still uncertain and there is little information on the underlying genomic alterations. In this study, we performed a clinicopathologic and next-generation sequencing analysis of 15 cases of IVLBCL using a custom panel for the detection of alterations in 68 recurrently mutated genes in B-cell lymphomagenesis. Six patients had evidence of hemophagocytic syndrome. Four patients presented concomitantly a solid malignancy. Tumor cells outside the vessels were observed in 7 cases, 2 with an overt diffuse large B-cell cell lymphoma. In 4 samples, tumor cells infiltrated lymphatic vessel in addition to blood capillaries. Programmed death-ligand 1 (PD-L1) was positive in tumor cells in 4 of 11 evaluable samples and in macrophages intermingled with tumor cells in 8. PD-L1 copy number gains were identified in a higher proportion of cases expressing PD-L1 than in negative tumors. The most frequently mutated gene was PIM1 (9/15, 60%), followed by MYD88L265P and CD79B (8/15, 53% each). In 6 cases, MYD88L265P and CD79B mutations were detected concomitantly. We also identified recurrent mutations in IRF4 , TMEM30A , BTG2 , and ETV6 loci (4/15, 27% each) and novel driver mutations in NOTCH2 , CCND3 , and GNA13 , and an IRF4 translocation in 1 case each. The mutational profile was similar in patients with and without evidence of hemophagocytic syndrome and in cases with or without dissemination of tumor cells outside the vessels. Our results confirm the relevance of mutations in B-cell receptor/nuclear factor-κB signaling and immune escape pathways in IVLBCL and identify novel driver alterations. The similar mutational profile in tumors with extravascular dissemination suggests that these cases may also be considered in the spectrum of IVLBCL.

MYC and TP53 Alterations but Not MAPK Pathway Mutations Are Common Oncogenic Mechanisms in Follicular Dendritic Cell Sarcomas.

CONTEXT.­: Despite their stromal origin, follicular dendritic cells (FDCs) share many functions with hematopoietic system cells. FDC neoplasms are currently classified by the World Health Organization along with those of a histiocytic nature. However, the molecular alterations driving oncogenesis in FDC sarcomas (FDCSs) are beginning to be unveiled and do not seem to concur with those described in histiocytic neoplasms, namely MAPK pathway activation. OBJECTIVE.­: To identify molecular alterations driving tumorigenesis in FDCS. DESIGN.­: We investigated the role of MYC and TP53 in FDC-derived tumor oncogenesis and assessed comprehensively the status of the MAPK pathway in 16 FDCSs, 6 inflammatory pseudotumor (IPT)-like FDCSs, and 8 IPTs. RESULTS.­: MYC structural alterations (both amplifications and rearrangements) were identified in 5 of 14 FDCSs (35.7%), all associated with MYC overexpression. TP53 mutations were identified in 4 of 14 FDCSs (28.6%), all of which displayed intense and diffuse p53 expression. None of these alterations were identified in any IPT-like FDCSs or in IPT cases. No MAPK pathway gene alterations were identified in any of the cases studied. CONCLUSIONS.­: The presence of MYC and TP53 alterations and the lack of association with Epstein-Barr virus segregate classical FDCS from IPT-like FDCS, pointing at different oncogenic mechanisms in both entities. Our results suggest a possible oncogenic role of MYC and TP53 alterations in FDCS. The absence of MAPK pathway alterations confirms the lack of a significant role of this pathway in the oncogenesis of FDC-derived neoplasms.

Detection of early seeding of Richter transformation in chronic lymphocytic leukemia.

Richter transformation (RT) is a paradigmatic evolution of chronic lymphocytic leukemia (CLL) into a very aggressive large B cell lymphoma conferring a dismal prognosis. The mechanisms driving RT remain largely unknown. We characterized the whole genome, epigenome and transcriptome, combined with single-cell DNA/RNA-sequencing analyses and functional experiments, of 19 cases of CLL developing RT. Studying 54 longitudinal samples covering up to 19 years of disease course, we uncovered minute subclones carrying genomic, immunogenetic and transcriptomic features of RT cells already at CLL diagnosis, which were dormant for up to 19 years before transformation. We also identified new driver alterations, discovered a new mutational signature (SBS-RT), recognized an oxidative phosphorylation (OXPHOS)high-B cell receptor (BCR)low-signaling transcriptional axis in RT and showed that OXPHOS inhibition reduces the proliferation of RT cells. These findings demonstrate the early seeding of subclones driving advanced stages of cancer evolution and uncover potential therapeutic targets for RT.

NR1H3 (LXRα) is associated with pro-inflammatory macrophages, predicts survival and suggests potential therapeutic rationales in diffuse large b-cell lymphoma.

The role of macrophages (Mo) and their prognostic impact in diffuse large B-cell lymphomas (DLBCL) remain controversial. By regulating the lipid metabolism, Liver-X-Receptors (LXRs) control Mo polarization/inflammatory response, and their pharmacological modulation is under clinical investigation to treat human cancers, including lymphomas. Herein, we surveyed the role of LXRs in DLBCL for prognostic purposes. Comparing bulk tumors with purified malignant and normal B-cells, we found an intriguing association of NR1H3, encoding for the LXR-α isoform, with the tumor microenvironment (TME). CIBERSORTx-based purification on large DLBCL datasets revealed a high expression of the receptor transcript in M1-like pro-inflammatory Mo. By determining an expression cut-off of NR1H3, we used digital measurement to validate its prognostic capacity on two large independent on-trial and real-world cohorts. Independently of classical prognosticators, NR1H3high patients displayed longer survival compared with NR1H3low cases and a high-resolution Mo GEP dissection suggested a remarkable transcriptional divergence between subgroups. Overall, our findings indicate NR1H3 as a Mo-related biomarker identifying patients at higher risk and prompt future preclinical studies investigating its mouldability for therapeutic purposes.

Cell-Free DNA for Genomic Analysis in Primary Mediastinal Large B-Cell Lymphoma.

High-throughput sequencing of cell-free DNA (cfDNA) has emerged as a promising noninvasive approach in lymphomas, being particularly useful when a biopsy specimen is not available for molecular analysis, as it frequently occurs in primary mediastinal large B-cell lymphoma (PMBL). We used cfDNA for genomic characterization in 20 PMBL patients by means of a custom NGS panel for gene mutations and low-pass whole-genome sequencing (WGS) for copy number analysis (CNA) in a real-life setting. Appropriate cfDNA to perform the analyses was obtained in 18/20 cases. The sensitivity of cfDNA to detect the mutations present in paired FFPE samples was 69% (95% CI: 60-78%). The mutational landscape found in cfDNA samples was highly consistent with that of the tissue, with the most frequently mutated genes being B2M (61%), SOCS1 (61%), GNA13 (44%), STAT6 (44%), NFKBIA (39%), ITPKB (33%), and NFKBIE (33%). Overall, we observed a 75% concordance to detect CNA gains/losses between DNA microarray and low-pass WGS. The sensitivity of low-pass WGS was remarkably higher for clonal CNA (18/20, 90%) compared to subclonal alterations identified by DNA microarray. No significant associations between cfDNA amount and tumor burden or outcome were found. cfDNA is an excellent alternative source for the accurate genetic characterization of PMBL cases.

Revised International Prognostic Index and genetic alterations are associated with early failure to R-CHOP in patients with diffuse large B-cell lymphoma.

Relapsed or refractory diffuse large B-cell lymphoma (DLBCL) cases have a poor outcome. Here we analysed clinico-biological features in 373 DLBCL patients homogeneously treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP), in order to identify variables associated with early failure to treatment (EF), defined as primary refractoriness or relapse within 12 months from diagnosis. In addition to clinical features, mutational status of 106 genes was studied by targeted next-generation sequencing in 111 cases, copy number alterations in 87, and gene expression profile (GEP) in 39. Ninety-seven cases (26%) were identified as EF and showed significantly shorter overall survival (OS). Patients with B symptoms, advanced stage, high levels of serum lactate dehydrogenase (LDH) or ß2-microglobulin, low lymphocyte/monocyte ratio and higher Revised International Prognostic Index (R-IPI) scores, as well as those with BCL2 rearrangements more frequently showed EF, with R-IPI being the most important in logistic regression. Mutations in NOTCH2, gains in 5p15·33 (TERT), 12q13 (CDK2), 12q14·1 (CDK4) and 12q15 (MDM2) showed predictive importance for EF independently from R-IPI. GEP studies showed that EF cases were significantly enriched in sets related to cell cycle regulation and inflammatory response, while cases in response showed over-representation of gene sets related to extra-cellular matrix and tumour microenvironment.

MAPK and JAK-STAT pathways dysregulation in plasmablastic lymphoma.

Plasmablastic lymphoma (PBL) is an aggressive B-cell lymphoma with an immunoblastic/large-cell morphology and terminal B-cell differentiation. The differential diagnosis from Burkitt lymphoma, plasma cell myeloma and some variants of diffuse large B-cell lymphoma may be challenging because of the overlapping morphological, genetic and immunophenotypic features. Furthermore, the genomic landscape in PBL is not well known. To characterize the genetic and molecular heterogeneity of these tumors, we investigated 34 cases of PBL using an integrated approach, including fluorescence in situ hybridization, targeted sequencing of 94 B-cell lymphoma-related genes, and copy-number arrays. PBL were characterized by high genetic complexity including MYC translocations (87%), gains of 1q21.1-q44, trisomy 7, 8q23.2- q24.21, 11p13-p11.2, 11q14.2-q25, 12p and 19p13.3-p13.13, losses of 1p33, 1p31.1-p22.3, 13q and 17p13.3-p11.2, and recurrent mutations of STAT3 (37%), NRAS and TP53 (33%), MYC and EP300 (19%) and CARD11, SOCS1 and TET2 (11%). Pathway enrichment analysis suggested a cooperative action between MYC alterations and MAPK (49%) and JAK-STAT (40%) signaling pathways. Of note, Epstein-Barr virus (EBV)-negative PBL cases had higher mutational and copy-number load and more frequent TP53, CARD11 and MYC mutations, whereas EBV-positive PBL tended to have more mutations affecting the JAK-STAT pathway. In conclusion, these findings further unravel the distinctive molecular heterogeneity of PBL identifying novel molecular targets and the different genetic profile of these tumors in relation to EBV infection.

A Cyclin D1-Dependent Transcriptional Program Predicts Clinical Outcome in Mantle Cell Lymphoma.

PURPOSE: Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation leading to cyclin D1 overexpression. Cyclin D1 is a major cell-cycle regulator and also regulates transcription, but the impact of cyclin D1-mediated transcriptional dysregulation on MCL pathogenesis remains poorly understood. The aim of this study was to define a cyclin D1-dependent gene expression program and analyze its prognostic value. EXPERIMENTAL DESIGN: We integrated genome-wide expression analysis of cyclin D1-silenced and overexpressing cells with cyclin D1 chromatin-binding profiles to identify a cyclin D1-dependent transcriptional program in MCL cells. We analyzed this gene program in two MCL series of peripheral blood samples (n = 53) and lymphoid tissues (n = 106) to determine its biological and clinical relevance. We then obtained a simplified signature of this program and evaluated a third series of peripheral blood MCL samples (n = 81) by NanoString gene expression profiling to validate our findings. RESULTS: We identified a cyclin D1-dependent transcriptional program composed of 295 genes that were mainly involved in cell-cycle control. The cyclin D1-dependent gene program was overexpressed in MCL tumors directly proportional to cyclin D1 levels. High expression of this program conferred an adverse prognosis with significant shorter overall survival of the patients. These observations were validated in an independent cohort of patients using a simplified 37-gene cyclin D1 signature. The cyclin D1-dependent transcriptional program was also present in multiple myeloma and breast tumors with cyclin D1 overexpression. CONCLUSIONS: We identified a cyclin D1-dependent transcriptional program that is overexpressed in MCL and predicts clinical outcome.

Comparative analysis of targeted next-generation sequencing panels for the detection of gene mutations in chronic lymphocytic leukemia: an ERIC multi-center study.

Next-generation sequencing (NGS) has transitioned from research to clinical routine, yet the comparability of different technologies for mutation profiling remains an open question. We performed a European multicenter (n=6) evaluation of three amplicon-based NGS assays targeting 11 genes recurrently mutated in chronic lymphocytic leukemia. Each assay was assessed by two centers using 48 pre-characterized chronic lymphocytic leukemia samples; libraries were sequenced on the Illumina MiSeq instrument and bioinformatics analyses were centralized. Across all centers the median percentage of target reads ≥100x ranged from 94.2- 99.8%. In order to rule out assay-specific technical variability, we first assessed variant calling at the individual assay level i.e., pairwise analysis of variants detected amongst partner centers. After filtering for variants present in the paired normal sample and removal of PCR/sequencing artefacts, the panels achieved 96.2% (Multiplicom), 97.7% (TruSeq) and 90% (HaloPlex) concordance at a variant allele frequency (VAF) >0.5%. Reproducibility was assessed by looking at the inter-laboratory variation in detecting mutations and 107 of 115 (93% concordance) mutations were detected by all six centers, while the remaining eight variants (7%) were undetected by a single center. Notably, 6 of 8 of these variants concerned minor subclonal mutations (VAF <5%). We sought to investigate low-frequency mutations further by using a high-sensitivity assay containing unique molecular identifiers, which confirmed the presence of several minor subclonal mutations. Thus, while amplicon-based approaches can be adopted for somatic mutation detection with VAF >5%, after rigorous validation, the use of unique molecular identifiers may be necessary to reach a higher sensitivity and ensure consistent and accurate detection of low-frequency variants.

Mutational Landscape and Tumor Burden Assessed by Cell-free DNA in Diffuse Large B-Cell Lymphoma in a Population-Based Study.

PURPOSE: We analyzed the utility of cell-free DNA (cfDNA) in a prospective population-based cohort to determine the mutational profile, assess tumor burden, and estimate its impact in response rate and outcome in patients with diffuse large B-cell lymphoma (DLBCL). EXPERIMENTAL DESIGN: A total of 100 patients were diagnosed with DLBCL during the study period. Mutational status of 112 genes was studied in cfDNA by targeted next-generation sequencing. Paired formalin-fixed, paraffin-embedded samples and volumetric PET/CT were assessed when available. RESULTS: Appropriate cfDNA to perform the analyses was obtained in 79 of 100 cases. At least one mutation could be detected in 69 of 79 cases (87%). The sensitivity of cfDNA to detect the mutations was 68% (95% confidence interval, 56.2-78.7). The mutational landscape found in cfDNA samples was highly consistent with that shown in the tissue and allowed genetic classification in 43% of the cases. A higher amount of circulating tumor DNA (ctDNA) significantly correlated with clinical parameters related to tumor burden (elevated lactate dehydrogenase and ß2-microglobulin serum levels, advanced stage, and high-risk International Prognostic Index) and total metabolic tumor volume assessed by PET/CT. In patients treated with curative intent, high ctDNA levels (>2.5 log hGE/mL) were associated with lower complete response (65% vs. 96%; P < 0.004), shorter progression-free survival (65% vs. 85%; P = 0.038), and overall survival (73% vs. 100%; P = 0.007) at 2 years, although it did not maintain prognostic value in multivariate analyses. CONCLUSIONS: In a population-based prospective DLBCL series, cfDNA resulted as an alternative source to estimate tumor burden and to determine the tumor mutational profile and genetic classification, which have prognostic implications and may contribute to a future tailored treatment.

PI3Kδ inhibition reshapes follicular lymphoma-immune microenvironment cross talk and unleashes the activity of venetoclax.

Despite idelalisib approval in relapsed follicular lymphoma (FL), a complete characterization of the immunomodulatory consequences of phosphatidylinositol 3-kinase δ (PI3Kδ) inhibition, biomarkers of response, and potential combinatorial therapies in FL remain to be established. Using ex vivo cocultures of FL patient biopsies and follicular dendritic cells (FDCs) to mimic the germinal center (n = 42), we uncovered that PI3Kδ inhibition interferes with FDC-induced genes related to angiogenesis, extracellular matrix formation, and transendothelial migration in a subset of FL samples, defining an 18-gene signature fingerprint of idelalisib sensitivity. A common hallmark of idelalisib found in all FL cases was its interference with the CD40/CD40L pathway and induced proliferation, together with the downregulation of proteins crucial for B-T-cell synapses, leading to an inefficient cross talk between FL cells and the supportive T-follicular helper cells (TFH). Moreover, idelalisib downmodulates the chemokine CCL22, hampering the recruitment of TFH and immunosupressive T-regulatory cells to the FL niche, leading to a less supportive and tolerogenic immune microenvironment. Finally, using BH3 profiling, we uncovered that FL-FDC and FL-macrophage cocultures augment tumor addiction to BCL-XL and MCL-1 or BFL-1, respectively, limiting the cytotoxic activity of the BCL-2 inhibitor venetoclax. Idelalisib restored FL dependence on BCL-2 and venetoclax activity. In summary, idelalisib exhibits a patient-dependent activity toward angiogenesis and lymphoma dissemination. In all FL cases, idelalisib exerts a general reshaping of the FL immune microenvironment and restores dependence on BCL-2, predisposing FL to cell death, providing a mechanistic rationale for investigating the combination of PI3Kδ inhibitors and venetoclax in clinical trials.

IgCaller for reconstructing immunoglobulin gene rearrangements and oncogenic translocations from whole-genome sequencing in lymphoid neoplasms.

Immunoglobulin (Ig) gene rearrangements and oncogenic translocations are routinely assessed during the characterization of B cell neoplasms and stratification of patients with distinct clinical and biological features, with the assessment done using Sanger sequencing, targeted next-generation sequencing, or fluorescence in situ hybridization (FISH). Currently, a complete Ig characterization cannot be extracted from whole-genome sequencing (WGS) data due to the inherent complexity of the Ig loci. Here, we introduce IgCaller, an algorithm designed to fully characterize Ig gene rearrangements and oncogenic translocations from short-read WGS data. Using a cohort of 404 patients comprising different subtypes of B cell neoplasms, we demonstrate that IgCaller identifies both heavy and light chain rearrangements to provide additional information on their functionality, somatic mutational status, class switch recombination, and oncogenic Ig translocations. Our data thus support IgCaller to be a reliable alternative to Sanger sequencing and FISH for studying the genetic properties of the Ig loci.

Reproducibility of histologic prognostic parameters for mantle cell lymphoma: cytology, Ki67, p53 and SOX11.

Mantle cell lymphoma (MCL) shows a clinical aggressiveness that varies from patient to patient. Despite major advances in outcomes with current immunochemotherapy, the future development of therapies requires risk stratification to tailor therapy intensity. Within the group of reference pathologists for the ongoing trials of the European MCL Network, we performed a round robin test on a tissue microarray to evaluate the reproducibility in assessing the biomarkers of outcome in MCL. Cytological subtype, Ki67-index and expression of p53 and SOX11 were evaluated on 20 diagnostic tumour samples by eight participating labs independently. We demonstrate that the assessment of the proliferation index by counting the Ki67 positive cells as well as assessment of SOX11 and p53 expression status is reproducible between labs. For the most established prognostic biomarker, Ki67, the intra-class correlation coefficient was very good when assessed as a continuous parameter (0.87). The agreement was lower when the values were analysed in a dichotomized way applying the commonly used cutoff of 30% (kappa = 0.65, complete concordance of all labs in 13/20 (65%)). Cases with discrepant results between labs in the dichotomized analysis showed mean values close to the cutoff of 30%. Centralised scoring and digital image analysis revealed results in line with the scores from individual labs. All cases in our cohort were additionally assessed for gene expression signatures and of TP53 gene alterations. Given the good reproducibility when guidelines of assessment are applied, the biomarker studied in this inter-laboratory test presents potential candidates to be enhanced for risk-stratification in the future clinical trials.

Distinct molecular profile of IRF4-rearranged large B-cell lymphoma.

Pediatric large B-cell lymphomas (LBCLs) share morphological and phenotypic features with adult types but have better prognosis. The higher frequency of some subtypes such as LBCL with IRF4 rearrangement (LBCL-IRF4) in children suggests that some age-related biological differences may exist. To characterize the genetic and molecular heterogeneity of these tumors, we studied 31 diffuse LBCLs (DLBCLs), not otherwise specified (NOS); 20 LBCL-IRF4 cases; and 12 cases of high-grade B-cell lymphoma (HGBCL), NOS in patients ≤25 years using an integrated approach, including targeted gene sequencing, copy-number arrays, and gene expression profiling. Each subgroup displayed different molecular profiles. LBCL-IRF4 had frequent mutations in IRF4 and NF-κB pathway genes (CARD11, CD79B, and MYD88), losses of 17p13 and gains of chromosome 7, 11q12.3-q25, whereas DLBCL, NOS was predominantly of germinal center B-cell (GCB) subtype and carried gene mutations similar to the adult counterpart (eg, SOCS1 and KMT2D), gains of 2p16/REL, and losses of 19p13/CD70. A subset of HGBCL, NOS displayed recurrent alterations of Burkitt lymphoma-related genes such as MYC, ID3, and DDX3X and homozygous deletions of 9p21/CDKN2A, whereas other cases were genetically closer to GCB DLBCL. Factors related to unfavorable outcome were age >18 years; activated B-cell (ABC) DLBCL profile, HGBCL, NOS, high genetic complexity, 1q21-q44 gains, 2p16/REL gains/amplifications, 19p13/CD70 homozygous deletions, and TP53 and MYC mutations. In conclusion, these findings further unravel the molecular heterogeneity of pediatric and young adult LBCL, improve the classification of this group of tumors, and provide new parameters for risk stratification.

Cyclin D1 overexpression induces global transcriptional downregulation in lymphoid neoplasms.

Cyclin D1 is an oncogene frequently overexpressed in human cancers that has a dual function as cell cycle and transcriptional regulator, although the latter is widely unexplored. Here, we investigated the transcriptional role of cyclin D1 in lymphoid tumor cells with cyclin D1 oncogenic overexpression. Cyclin D1 showed widespread binding to the promoters of most actively transcribed genes, and the promoter occupancy positively correlated with the transcriptional output of targeted genes. Despite this association, the overexpression of cyclin D1 in lymphoid cells led to a global transcriptional downmodulation that was proportional to cyclin D1 levels. This cyclin D1-dependent global transcriptional downregulation was associated with a reduced nascent transcription and an accumulation of promoter-proximal paused RNA polymerase II (Pol II) that colocalized with cyclin D1. Concordantly, cyclin D1 overexpression promoted an increase in the Poll II pausing index. This transcriptional impairment seems to be mediated by the interaction of cyclin D1 with the transcription machinery. In addition, cyclin D1 overexpression sensitized cells to transcription inhibitors, revealing a synthetic lethality interaction that was also observed in primary mantle cell lymphoma cases. This finding of global transcriptional dysregulation expands the known functions of oncogenic cyclin D1 and suggests the therapeutic potential of targeting the transcriptional machinery in cyclin D1-overexpressing tumors.

The reference epigenome and regulatory chromatin landscape of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a frequent hematological neoplasm in which underlying epigenetic alterations are only partially understood. Here, we analyze the reference epigenome of seven primary CLLs and the regulatory chromatin landscape of 107 primary cases in the context of normal B cell differentiation. We identify that the CLL chromatin landscape is largely influenced by distinct dynamics during normal B cell maturation. Beyond this, we define extensive catalogues of regulatory elements de novo reprogrammed in CLL as a whole and in its major clinico-biological subtypes classified by IGHV somatic hypermutation levels. We uncover that IGHV-unmutated CLLs harbor more active and open chromatin than IGHV-mutated cases. Furthermore, we show that de novo active regions in CLL are enriched for NFAT, FOX and TCF/LEF transcription factor family binding sites. Although most genetic alterations are not associated with consistent epigenetic profiles, CLLs with MYD88 mutations and trisomy 12 show distinct chromatin configurations. Furthermore, we observe that non-coding mutations in IGHV-mutated CLLs are enriched in H3K27ac-associated regulatory elements outside accessible chromatin. Overall, this study provides an integrative portrait of the CLL epigenome, identifies extensive networks of altered regulatory elements and sheds light on the relationship between the genetic and epigenetic architecture of the disease.

Decoding the DNA Methylome of Mantle Cell Lymphoma in the Light of the Entire B Cell Lineage.

We analyzed the in silico purified DNA methylation signatures of 82 mantle cell lymphomas (MCL) in comparison with cell subpopulations spanning the entire B cell lineage. We identified two MCL subgroups, respectively carrying epigenetic imprints of germinal-center-inexperienced and germinal-center-experienced B cells, and we found that DNA methylation profiles during lymphomagenesis are largely influenced by the methylation dynamics in normal B cells. An integrative epigenomic approach revealed 10,504 differentially methylated regions in regulatory elements marked by H3K27ac in MCL primary cases, including a distant enhancer showing de novo looping to the MCL oncogene SOX11. Finally, we observed that the magnitude of DNA methylation changes per case is highly variable and serves as an independent prognostic factor for MCL outcome.

Clinical impact of clonal and subclonal TP53, SF3B1, BIRC3, NOTCH1, and ATM mutations in chronic lymphocytic leukemia.

Genomic studies have revealed the complex clonal heterogeneity of chronic lymphocytic leukemia (CLL). The acquisition and selection of genomic aberrations may be critical to understanding the progression of this disease. In this study, we have extensively characterized the mutational status of TP53, SF3B1, BIRC3, NOTCH1, and ATM in 406 untreated CLL cases by ultra-deep next-generation sequencing, which detected subclonal mutations down to 0.3% allele frequency. Clonal dynamics were examined in longitudinal samples of 48 CLL patients. We identified a high proportion of subclonal mutations, isolated or associated with clonal aberrations. TP53 mutations were present in 10.6% of patients (6.4% clonal, 4.2% subclonal), ATM mutations in 11.1% (7.8% clonal, 1.3% subclonal, 2% germ line mutations considered pathogenic), SF3B1 mutations in 12.6% (7.4% clonal, 5.2% subclonal), NOTCH1 mutations in 21.8% (14.2% clonal, 7.6% subclonal), and BIRC3 mutations in 4.2% (2% clonal, 2.2% subclonal). ATM mutations, clonal SF3B1, and both clonal and subclonal NOTCH1 mutations predicted for shorter time to first treatment irrespective of the immunoglobulin heavy-chain variable-region gene (IGHV) mutational status. Clonal and subclonal TP53 and clonal NOTCH1 mutations predicted for shorter overall survival together with the IGHV mutational status. Clonal evolution in longitudinal samples mainly occurred in cases with mutations in the initial samples and was observed not only after chemotherapy but also in untreated patients. These findings suggest that the characterization of the subclonal architecture and its dynamics in the evolution of the disease may be relevant for the management of CLL patients.