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Supplementary Methods, Supplementary Tables S1-S5, and Supplementary Figures S1-S15

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 β2-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.

1 - Serum and tumor cytokines according to MYD88 mutational status 2- Discrepancies in cell of origin distribution between gene expression and immunohistochemistry

Purpose: Mutations in MYD88 are found in different lymphoproliferative disorders associated with particular biologic characteristics and clinical impact. The aim of this study was to analyze the incidence of MYD88 mutations and its clinical impact in diffuse large B-cell lymphoma (DLBCL).Experimental Design: The incidence, clinicobiological features, and outcome of 213 patients (115 M/98 F; median age, 65 years) with DLBCL treated with immunochemotherapy in a single institution according to MYD88 mutational status as assessed by an allele-specific PCR assay were analyzed. The cell of origin (COO) was determined in 129 cases by gene expression.Results: MYD88 mutations were found in 47 cases (22%), including L265P in 39 and S219C and M232F in 4 cases, respectively. Patients with MYD88 L265P were older, presenting frequent extranodal involvement, and mostly corresponded to activated B-cell like (ABC) subtype, whereas no preference in COO was observed in patients with other MYD88 mutations. Five-year overall survival (OS) for MYD88 wild-type, MYD88 L265P, and other variants was 62%, 52%, and 75%, respectively (P = 0.05). International Prognostic Index (IPI) (HR, 2.71; P < 0.001) and MYD88 L265P (HR, 1.786; P = 0.023) were independent variables predicting OS in the multivariate analysis. However, MYD88 L265P lost its independent value when COO was included in the model.Conclusions: Our findings indicate that MYD88 L265P mutations, but no other variants, identify a subgroup of DLBCL mainly of ABC origin, with extranodal involvement and poor outcome. Clin Cancer Res; 22(11); 2755–64. ©2016 AACR.

Supplementary tables S1 to S6

Suppplementary Methods and Figures

Supplementary Table 2 from Genetic Variants in Apoptosis and Immunoregulation-Related Genes Are Associated with Risk of Chronic Lymphocytic Leukemia

The genetic mechanisms associated with splenic marginal zone lymphoma transformation (SMZL-T) are not well defined. We studied 41 SMZL patients that eventually underwent large B-cell lymphoma transformation. Tumor material was obtained only at diagnosis (9 patients), at diagnosis and transformation (18 patients), and only at transformation (14 patients). Samples were categorized in two groups: i) at diagnosis (SMZL, n=27 samples), and ii) 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 which acquired different genetic alterations in virtually all evaluable cases (12/13, 92%). Using whole genome sequencing from diagnostic and transformation samples in one 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 at transformation predicted for a shorter survival from transformation (P=0.001, P=0.042, and P=0.007, 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.

Supplementary Figure 1 from Genetic Variants in Apoptosis and Immunoregulation-Related Genes Are Associated with Risk of Chronic Lymphocytic Leukemia

Supplementary Table 1 from Genetic Variants in Apoptosis and Immunoregulation-Related Genes Are Associated with Risk of Chronic Lymphocytic Leukemia

Supplementary Figure Legend from Genetic Variants in Apoptosis and Immunoregulation-Related Genes Are Associated with Risk of Chronic Lymphocytic Leukemia

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.

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 FDCS 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.

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.

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.

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.

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 beta 2-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 center dot 33 (TERT), 12q13 (CDK2), 12q14 center dot 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.

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, NR1H3

Introduction: Clonal evolution drives cancer development due to the emergence and/or selection of proliferatively advantageous subclones. Its understanding may facilitate the design of anticipation-based management strategies. Richter transformation (RT) is a paradigmatic tumor evolution in which chronic lymphocytic leukemia (CLL), an indolent neoplasia of mature B-cells, transforms into a high-grade lymphoma, usually diffuse large B-cell lymphoma (DLBCL), conferring a dismal prognosis. The evolutionary trajectories of RT and its driving (epi)genomic mechanisms remain largely unknown. Aims: To reconstruct the evolutionary history of RT and to reveal the molecular processes underlying this transformation. Methods: We characterized the whole genome (WGS), epigenome (DNA methylation, H3K27ac, ATAC-seq), and transcriptome (RNA-seq), combined with single-cell DNA and RNA sequencing analyses, of 19 CLL patients developing RT before (n=3) or after treatment with chemoimmunotherapy (n=6) and targeted therapies (BCR or BCL2 inhibitors, n=10). We analyzed 54 longitudinal samples covering up to 19 years of disease course. Results: Our WGS analyses uncovered that RT is characterized by a remarkable structural complexity. We also identified a novel treatment-independent RT-specific mutational process, which we named SBS-RT. The genetic driver landscape of RT is a compendium of alterations in genes involved in cell cycle, MYC, and NF-κB pathways, frequently targeted in single catastrophic events including chromothripsis and chromoplexy. The WGS-based phylogenic reconstruction and single-cell DNA/RNA-seq analyses identified a very early diversification of CLL leading to emergence of RT-cells carrying specific genetic drivers and transcriptomic profiles of RT already at CLL diagnosis. These small subclones were dormant for 6-19 years until rapid expansion associated with the clinical transformation. While the DNA methylome kept track of the cell of origin and proliferative history of RT cells, their chromatin configuration and transcriptional program converged into the overexpression of cell cycle regulators, Toll-like receptors, MYC, MTORC1, and OXPHOS related transcripts, as well as downregulation of BCR pathway. This phenotypic shift was related to de novo activation of key transcription factors. In vitro experiments confirmed that RT cells have a 4-fold higher oxygen consumption at routine respiration and electron transfer system capacity compared to CLL. The resistance of RT to BCR inhibition is consistent with its high OXPHOS and low BCR signaling, which mimics de novo DLBCL-OXPHOS insensitive to BCR inhibition. This OXPHOShigh-BCRlow transcriptional axis of RT can be exploited therapeutically. Conclusions: These findings demonstrate the early seeding of subclones driving advanced stages of cancer evolution and uncover therapeutic targets for the, once expanded, lethal Richter transformation. Citation Format: Ferran Nadeu, Romina Royo, Ramon Massoni-Badosa, Beatriz Garcia-Torre, Martí Duran-Ferrer, Kevin J. Dawson, Marta Kulis, Ander Diaz-Navarro, Neus Villamor, Juan L. Melero, Vicente Chapaprieta, Ana Dueso-Barroso, Julio Delgado, Riccardo Moia, Sara Ruiz-Gil, Domenica Marchese, Núria Verdaguer-Dot, Mónica Romo, Maria Rozman, Gerard Frigola, Alfredo Rivas-Delgado, Tycho Baumann, Miguel Alcoceba, Marcos González, Fina Climent, Pau Abrisqueta, Josep Castellví, Francesc Bosch, Marta Aymerich, Anna Enjuanes, Sílvia Ruiz-Gaspà, Armando López-Guillermo, Pedro Jares, Sílvia Beà, Dolors Colomer, Núria López-Bigas, Josep LlGelpí, David Torrents, Peter J. Campbell, Ivo Gut, Pablo M. Garcia-Roves, Davide Rossi, Gianluca Gaidano, Xose S. Puente, Holger Heyn, Francesco Maura, José I. Martín-Subero, Elías Campo. Early seeding of Richter transformation in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3795.

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 EBVpositive 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.

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., Immunoglobulin (Ig) rearrangement and translocation information are usually obtained by targeted sequencing of the respective loci. Here, the authors present the IgCaller algorithm, which extracts Ig heavy and light chain genetic properties from short-read whole-genome sequencing results to provide a feasible alternative to direct sequencing.

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 are 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 cut-off 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 cut-off of 30%. Centralized 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 present potential candidates, to be enhanced for risk-stratification in the future clinical trials.

Introduction Outcomes for follicular lymphoma (FL) patients are generally good. Clinical and biological variables have been studied to identify patients at higher risk of early relapse, which markedly impacts survival. However, the genetic landscape of FL according to its clinical behavior (need of treatment and timing of relapse) is not well characterized, which was the aim of the present study. Patients and Methods We included 67 samples from 55 grade 1-3A FL patients from a single institution [55 samples at diagnosis (D), and 12 at relapse (R)]. Five groups were defined according to their clinical behavior: never required treatment after >5 years (y) of follow-up [never treated (NT), n=7]; treated and did not relapse after >9 y of follow-up, [non-relapsed (NR), n=19]; treated and relapsed beyond 24 months of frontline therapy [late relapse (LR), n=14]; treated and relapsed within 24 months of frontline treatment [early relapse (ER), n=11]; and primary refractory (PR, n=4). Of the 48 treated patients, 96% received R-CHOP. Patients developing histologic transformation were not included. DNA was extracted from FFPE tissue biopsies. Copy number alterations (CNA) were assessed in 50 D and 11 R samples [OncoScan CNV Assay (Thermofisher)], and single nucleotide variants (SNV) and insertions/deletions (indels) in 51 D and 10 R samples using a B-cell malignancy NGS panel examining 121 genes (SureSelectXT, Agilent Technologies). Genes and genomic regions were considered altered if they harbored SNV/indels and/or CNA. For comparisons and plotting, only the 52 D samples with both NGS and CNA data were considered. Non-parametric statistical tests were used. Results Median age was 56 y (range, 26−79), and 30 patients (55%) were female. Forty-two patients (76%) had stage III-IV disease, without significant differences among groups. Thirteen patients (25%) had a high-risk FLIPI score, a percentage that was higher in the LR and PR groups (50 and 67%, respectively, P=0.02). With a median follow-up of 12.9 y, 10-y overall survival estimates were 71, 100, 74, 82, and 0% for NT, NR, LR, ER, and PR patients, respectively. We detected CNA in all samples, with a median number of 7 (range, 1−27) for D samples, and of 6 (2−19) for R samples. We also identified SNV/indels in all samples, with a median number of 10 (1−23) for D samples, and of 9 (3−18) for R samples. The most commonly altered genes at diagnosis were KMT2D (82%), CREBBP (73%), SPEN (38%), TNFRSF14 (38%), ARID1A (33%), and BCL2 (33%) (Figure). There were no significant differences in the number of altered genes/regions among the five groups. Genes or regions with significantly different alteration frequency among groups were: CARD11 (57, 12, 0, 9, and 0% for NT, NR, LR, ER, and PR, respectively, P=0.014), CD70 (12% for NR, 50% for PR, and 0 for the remaining groups P=0.026), HIST1H1B (29% for NT, and 0% for the remaining groups, P=0.020), HVCN1 (43, 6, 0, 18, and 0% for NT, NR, LR, ER, and PR, respectively, P=0.048), KLHL6 (12% for NR, 75% for PR, and 0 for the remaining groups, P=0.002), PRKCB (0, 6, 0, 18, 50%, P=0.037), and 13q14.2-q14.3 loss (DLEU1/DLEU2) (24% for NR, 50% for PR, and 0 for the remaining groups, P=0.015). Two genes had a significantly lower alteration rate comparing the cases with a favorable (NT, NR, LR) and more aggressive (ER, PR) clinical behavior: CIITA (22 vs. 53%, P=0.043), and PRKCB (3 vs. 27%, P=0.044). Of the 51 patients for which the m7-FLIPI was calculated, six (12%) had a high-risk score. Of note, none of the high-risk patients belonged to the NT or NR groups. No significant differences were found in the number of altered genes/regions between D and R, or in the alteration frequency of each gene/region. We identified the presence of an ancestral common precursor cell with a divergent evolution in all paired cases. The median percentage of shared alterations between D and R was 50% (range 28−63%). Conclusions In this comprehensive genetic analysis of 55 FL patients, categorized into five groups according to their clinical behavior, alterations in the chromatin modifying genes KMT2D and CREBBP were the most frequent, and CIITA and PRKCB were more frequently altered in cases with a shorter duration of response. These data warrant further study of the mechanisms underlying these mutations, mutual exclusivity/co-occurrence, and relationships with B-cell biology and the tumor microenvironment. Disclosures Nadeu: Janssen: Honoraria. Giné:Gilead: Research Funding; Janssen: Research Funding; Roche: Research Funding. Armando:Janssen: Research Funding; Roche: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Celgene: Consultancy, Research Funding.

SummaryWe 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.

Purpose: Mutations in MYD88 are found in different lymphoproliferative disorders associated with particular biologic characteristics and clinical impact. The aim of this study was to analyze the incidence of MYD88 mutations and its clinical impact in diffuse large B-cell lymphoma (DLBCL). Experimental Design: The incidence, clinicobiological features, and outcome of 213 patients (115 M/98 F; median age, 65 years) with DLBCL treated with immunochemotherapy in a single institution according to MYD88 mutational status as assessed by an allele-specific PCR assay were analyzed. The cell of origin (COO) was determined in 129 cases by gene expression. Results: MYD88 mutations were found in 47 cases (22%), including L265P in 39 and S219C and M232F in 4 cases, respectively. Patients with MYD88 L265P were older, presenting frequent extranodal involvement, and mostly corresponded to activated B-cell like (ABC) subtype, whereas no preference in COO was observed in patients with other MYD88 mutations. Five-year overall survival (OS) for MYD88 wild-type, MYD88 L265P, and other variants was 62%, 52%, and 75%, respectively (P = 0.05). International Prognostic Index (IPI) (HR, 2.71; P < 0.001) and MYD88 L265P (HR, 1.786; P = 0.023) were independent variables predicting OS in the multivariate analysis. However, MYD88 L265P lost its independent value when COO was included in the model. Conclusions: Our findings indicate that MYD88 L265P mutations, but no other variants, identify a subgroup of DLBCL mainly of ABC origin, with extranodal involvement and poor outcome. Clin Cancer Res; 22(11); 2755–64. ©2016 AACR.

This study was supported by the Instituto de Salud Carlos III (ISCIII) PMP15/00007; Ministerio de Economía y Competitividad (MINECO) SAF2015-64885-R; Generalitat de Catalunya AGAUR 2014-SGR-795; and Gilead Spain (GLD15/00288). ECa is an Academia Researcher of the 'Institució Catalana de Recerca i Estudis Avançats' (ICREA) of the Generalitat de Catalunya. FN is supported by a pre-doctoral fellowship of the MINECO (BES-2016-076372). We are indebted to the Genomics core facility of the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS). We are grateful to N Villahoz and MC Muro for their excellent work in the coordination of the CLL Spanish Consortium, and also thank C Capdevila, S Guijarro, L Pla and M Sánchez for their excellent technical assistance. We are also very grateful to all patients with CLL who have participated in this study., Nadeu, F., Clot, G., Delgado, J., Martín-García, D., Baumann, T., Salaverria, I., Beà, S., Pinyol, M., Jares, P., Navarro, A., Suárez-Cisneros, H., Aymerich, M., Rozman, M., Villamor, N., Colomer, D., González, M., Alcoceba, M., Terol, M.J., Navarro, B., Colado, E., Payer, Á., Puente, X.S., López-Otín, C., López-Guillermo, A., Enjuanes, A., Campo, E.

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.

Introduction: Previous studies have highlighted the potential of cell-free DNA (cfDNA) to assess the mutational profile and copy number alterations (CNA) in diffuse large B-cell lymphoma (DLBCL), usually performed in tissue biopsies, both at diagnosis and relapse. In addition, the quantitative levels of cfDNA might be a surrogate of the lymphoma tumor burden and could therefore predict response to therapy, progression-free survival (PFS) and overall survival (OS). The aim of this study was to analyze the mutational profile and CNA at diagnosis using cfDNA, to compare these results with those obtained from formalin-fixed paraffin-embedded (FFPE) biopsies, and to estimate the correlation of pre-treatment levels of cfDNA with FDG-PET/CT Total Metabolic Tumor Volume (TMTV) and their impact in the outcome of DLBCL patients. Methods: We included 79 patients (41M/38F , median age 63 years) diagnosed with DLBCL according to the WHO criteria in a single institution between 2016 and 2018. All patients received chemoimmunotherapy. After frontline treatment, 56 patients achieved a complete (CR) response, 4 partial response, 16 were refractory, including 7 early deaths, and in 3 cases the response was not yet evaluable. Samples were obtained at diagnosis before starting treatment. cfDNA extraction was performed from 2-4 mL of plasma collected in PAXgene Blood ccfDNA tubes (Qiagen) and size distribution of DNA fragments was analyzed using the Agilent 2100 Bioanalyzer. Tumor genomic DNA (gDNA) was isolated from FFPE diagnostic tissue biopsies. A panel of 115 genes was enriched using a hybridization capture-based protocol from 10-30 ng of cfDNA and 150 ng of gDNA (ThruPlex Tag-seq kit and SureSelectXT enrichment reagents, Agilent Technologies) and sequenced in a MiSeq instrument (Illumina). CNA were examined using CNVkit software toolkit. cfDNA levels were reported as haploid genome equivalents per mL of plasma and expressed as a base 10 logarithm (log hGE/mL). Quantitative analysis of TMTV was performed using the semiautomatic MIM software, with a fixed SUV>2.5 thresholding method for segmentation. Results: A median of 15.6 ng/mL (range: 4-754 ng/mL) of cfDNA was obtained. At least one mutation could be detected in 69/79 cases (87.3%). The median number of mutations per sample was 6 (range: 0-41 mutations). The genes most frequently mutated at diagnosis in plasma samples were KMT2D, TP53, TNFRSF14, MYD88, BCL2, SOCS1, CREBBP, MYC and EP300. In 45 cases, paired FFPE samples were available. Sensitivity of cfDNA to detect mutations in baseline FFPE samples was 69% (95%CI: 64.1-73.9). In 28 of the 45 cases, >70% of the mutations were observed both in the cfDNA and FFPE samples. In the remaining 17 cases, the number of mutations identified in cfDNA was lower than the one observed in the FFPE sample (Figure 1a). Of note, most of the cases in which mutations were not detected in the cfDNA corresponded to localized stages (11/17, 65%) and/or primary extranodal DLBCL (12/17, 71%). The CNA profile in cfDNA was similar to that reported in tissue, with recurrent deletions of TNSFRS14 (11%), TNFAIP3 (14%), CDKN2A (7%), or TP53 (7%), as well as gains of REL (7%) or BCL2 (5%), among others (Figure 1b). Median pre-treatment TMTV (N=48) was 292 cm3 (0-4,171 cm3). Higher TMTV predicted for a poorer PFS (HR 4.85; p=0.005). cfDNA concentration significantly correlated with TMTV (R=0.546, p3 log hGE/mL) had a significantly lower CR rate, PFS, and OS than those with low levels (CR rate, 47% vs. 92%, p Conclusions: cfDNA represents a valuable tool to easily assess the genomic landscape and tumor burden in DLBCL, as well as to predict response to therapy and outcome in these patients. Figure 1 Disclosures Gine: Janssen: Other: Travel expenses, Research Funding; Roche: Other: Travel expenses, Research Funding; Gilead: Other: Travel expenses, Research Funding. Lopez-Guillermo:Gilead: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Janssen: Research Funding.

Introduction: Genomic studies of chronic lymphocytic leukemia (CLL) have uncovered >80 potential driver mutations. The vast majority of these mutations affect coding regions, and just two potential drivers have been identified in non-coding elements. Aim: To describe the biological and clinical impact of a recurrent A>C mutation at the third base of the small nuclear RNA U1, the non-coding component of the spliceosome involved in the recognition of the 5' splice site (5'SS). Methods: Whole-genome sequencing (WGS) and RNA-seq from 318 CLL patients were used to identify and characterize a highly recurrent A>C point mutation occurring at position 3 of the U1 snRNA gene (g.3A>C mutation). The U1 wild-type and mutant forms were introduced into three CLL cell lines (JVM3, HG3, MEC1) to validate in vitro the predicted effect of this alteration. We screened two independent cohorts including a total of 1,314 CLL patients for the presence of the mutation using the rhAmp SNP genotyping assay, and integrated the U1 mutational status with well-known driver alterations, IGHV and epigenetic subgroups, and clinical parameters. Results: The U1 mutation was found in 8/78 (10.3%) CLL cases analyzed by WGS. Given its role in 5'SS recognition by base-pairing, we reasoned that this mutation was likely to alter the splicing and expression patterns of CLL. We were able to confirm widespread specific alterations in the transcriptome by comparing RNA-seq data between wild-type and g.3A>C mutated samples. Applying this knowledge to an algorithm aimed to infer the U1 mutational status from expression data, we were able to identify 4 mutated cases among 240 additional cases that had RNA-seq but no WGS. In total, 12/318 (3.8%) CLL patients analyzed by WGS and/or RNA-seq harbored this mutation. This g.3A>C U1 mutation changes the preferential A-U base-pairing between U1 and 5'SS to C-G base-pairing, creating novel splice junctions and altering the splicing pattern of 3,193 introns in 1,519 genes. In addition to altered splicing, 869 genes were differentially expressed between mutated and wild-type cases. We identified specific cancer genes (e.g. MSI2, POLD1, or CD44) and pathways (B-cell receptor signaling, promotion of apoptosis, telomere maintenance, among others) altered by the U1 mutation. To confirm a causal link between this mutation and splicing changes, we introduced exogenous U1 genes with or without the mutation into three cell lines. Subsequent RNA-seq of these cell lines recapitulated the altered splicing and expression patterns observed in CLL patients. We next screened for the presence of the U1 mutation 1,057 patients (cohort 1) using the rhAmp assay and it was found in 30 (2.8%) cases. The distribution of the mutation was similar in Binet stages and CLL vs monoclonal B-cell lymphocytosis. However, the U1 mutation was almost always found in IGHV unmutated CLL (29/30, p=9.0e-11) and within the naïve-like CLL epigenetic subgroup (p=3.7e-7). None of the U1 mutated cases had mutations in the SF3B1 splicing factor. Considering only pre-treatment CLL samples, U1 mutation was associated with a shorter time to first treatment independently of the Binet stage, IGHV mutational status, epigenetic subgroups, and mutations in the well-known CLL drivers SF3B1, NOTCH1, ATMor TP53. In cohort 2 (n=257), this mutation was found in 13 (5.1%) patients, confirming its enrichment in IGHV unmutated cases, naïve-like epigenetic subgroup, and splicing modulation. Despite the relatively small number of pre-treatment samples carrying the U1 mutation (7/178) and short follow-up of the patients (median 2.6 years), the effect of this mutation on time to first treatment in cohort 2 was compatible with the one observed in cohort 1. Finally, we screened for the U1 mutation a cohort of diffuse large B-cell lymphoma (n=108), mantle cell lymphoma (n=101), follicular lymphoma (n=87), splenic marginal zone lymphoma (n=12), acute myeloid leukemia (n=52), and myelodysplastic syndrome (n=67). The mutation was not present in any of the samples analyzed. Conclusions: Here we have reported that the third base of the small nuclear RNA U1 is recurrently mutated in CLL, proved its effect in splicing and gene expression, and shown that this mutation is independently associated with faster disease progression. The g.3A>C U1 mutation represents a novel non-coding driver alteration in CLL with potential clinical and therapeutic implications. Disclosures Ramirez Payer: GILEAD SCIENCES: Research Funding. Terol:Astra Zeneca: Consultancy; Gilead: Research Funding; Abbvie: Consultancy; Janssen: Consultancy, Research Funding; Roche: Consultancy. Lopez-Guillermo:Celgene: Consultancy, Research Funding; Janssen: Research Funding; Roche: Consultancy, Research Funding; Gilead: Consultancy, Research Funding.

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.

Chronic lymphocytic leukemia (CLL) cells are characterized by several chromosomal lesions. Some of these aberrations imply chromosome breaks as a result of unrepaired double strand breaks (DSBs) in the DNA. The ATM (ataxia telangiectasia-mutated) protein is the principal integrator of cellular responses to DSBs. ATM deletion is also an adverse prognostic factor in CLL. Taking this into account, we evaluated if genetic and/or epigenetic variation in the ATM gene may modulate the individual susceptibility to develop CLL. Our case-control association study was performed in a large Spanish population of 1,503 individuals, including 742 patients with CLL and 761 controls. We identified one haplotype within the ATM gene that confers an increased risk of CLL development (OR = 1.33; 95% CI: 1.10–1.60). Two polymorphisms of this ATM haplotype eliminated one CpG site each in Introns 15 and 61, causing changes in DNA methylation pattern. These data provide the first evidence for the existence of a putative “hepitype” in the ATM gene associated with CLL risk.© 2011 Wiley-Liss, Inc.

BACKGROUND Pediatric aggressive large B-cell lymphomas (LBCL) share morphological and phenotypic features with adult types but seem to have better prognosis. Additionally, a specific subtype carrying IRF4 translocations (LBCL-IRF4) has been recently identified in this age group. In adults, the cell-of-origin (COO) distinction of diffuse large B-cell lymphoma (DLBCL) based on gene expression signatures (germinal center B-cell like, GCB and activated B-cell like, ABC) and more recently clusters of genetic alterations have identified molecularly distinct DLBCL subsets that may benefit from novel therapeutic targets. The integration of pediatric population in these clinically relevant molecular subgroups and its clinical importance is not well known. The aim of this study was to characterize the molecular heterogeneity of LBCL in pediatric and young adult patients and evaluate their potential clinical impact. DESIGN Sixty-one LBCL diagnosed in patients ≤25 years-old (median age 14 years old, male/female 38/23) were included in the study. Molecular analyses included fluorescence in situ hybridization for MYC, BCL2, IRF4 and BCL6, copy number (CN) analysis (Oncoscan, Affymetrix), COO Lymph2Cx assay (NanoString) and targeted next generation sequencing of 96 B-cell lymphoma driver genes (SureSelect XT, Agilent Technologies). CNA and mutational profiles were compared to those previously published in adult DLBCL. Correlation of molecular features and event free survival (EFS) was performed using Kaplan-Meier curves. RESULTS Histologically, 33 were DLBCL, 20 LBCL-IRF4 and 8 high grade B-cell lymphomas, not otherwise specified (HGBCL). Nodal disease was present in 57%, mainly in the cervical region. COO distribution was: 68% GCB, 18% ABC and 13% unclassified. Most of LBCL-IRF4 cases were GCB-COO (73%). The IRF4 translocation was demonstrated in 16 out of 19 cases diagnosed as LBCL-IRF4 and an IGH rearrangement was seen in the other 3. Five cases carried MYC-breaks (3 DLBCL and 2 HGBCL) and two cases carried BCL6-breaks (1DLBCL and 1HGBCL). BCL2 rearrangements were absent. CN analysis detected alterations in 46/51 cases with recurrent gains (>15%) of 1q, 2p16, 11q, trisomies 7 and 12, and recurrent losses (>10%) of 1p36, 6q21-q22, 15q24, 17p13 and 19p13. Recurrent homozygous deletions were observed at 19p13/CD70 (6 cases), 9p13/CDKN2A (3 cases) and 13q14/RB1 (2 cases). Alteration patterns suggestive of chromothripsis were found in 10% (5/51) of the cases. No ABC-DLBCL related alterations such as 3p21-p14, 6q21-q25, 9p21.3 and 17p13 losses were seen. Targeted sequencing detected a total of 434 variants in 44 of 47 cases (mean 9.2 mutations/case). A pipeline for selection of driver mutations revealed a total of 270 mutations (62%) with potential functional effect. Recurrent mutations found in >15% of the cases affected IRF4, SOCS1, PIM1, CARD11, ACTB and CCND3 genes. In comparison to adult DLBCL, pediatric and young adult cases had significantly lower incidence of MYD88 (5 cases), CREBBP,TP53 (3 cases each) and TNFRSF14 (2 cases) mutations which are strongly associated with the definition of established mutational clusters in adult DLBCL. In our cohort, the morphological subtypes displayed different molecular profiles. IRF4 variants (some cases with >7 variants) and mutations in NF-kB pathway (CARD11, CD79B and MYD88-non L265P) were found specifically in the LBCL-IRF4 subgroup, whereas mutations in GCB related genes such as SOCS1 and EZH2 were particularly seen in cases with DLBCL diagnosis. All 49 patients with available follow-up received chemotherapy as first line treatment (41% containing Rituximab). Variables significantly associated with poor EFS in univariate analysis were age >18 years, ABC-COO, Stage IV, high genetic complexity (chromothripsis and/or >10 CN alterations), 2p16/REL gain/amplification, 9p21.3/CDKN2A homozygous deletions, MYC rearrangements and mutations in MYC, TP53 and DDX3X genes. CONCLUSION Despite pediatric/young-adult LBCL having overlapping features with adult disease our findings suggest that LBCL in young age have specific molecular mechanisms and highlight potential key drivers of these lymphomas in this age group. Disclosures No relevant conflicts of interest to declare.

To identify new risk variants for chronic lymphocytic leukemia (CLL), we conducted a genome-wide association study of 299,983 tagging SNPs, with validation in four additional series totaling 2,503 cases and 5,789 controls. We identified four new risk loci for CLL at 2q37.3 (rs757978, FARP2; odds ratio (OR) = 1.39; P = 2.11 x 10(-9)), 8q24.21 (rs2456449; OR = 1.26; P = 7.84 x 10(-10)), 15q21.3 (rs7169431; OR = 1.36; P = 4.74 x 10(-7)) and 16q24.1 (rs305061; OR = 1.22; P = 3.60 x 10(-7)). We also found evidence for risk loci at 15q25.2 (rs783540, CPEB1; OR = 1.18; P = 3.67 x 10(-6)) and 18q21.1 (rs1036935; OR = 1.22; P = 2.28 x 10(-6)). These data provide further evidence for genetic susceptibility to this B-cell hematological malignancy.

Alendronate is a well-established treatment for osteoporosis and suppresses bone resorption by a direct effect on osteoclasts and their precursors. The effect of alendronate on osteoclasts is produced, at least in part, by the receptor activator of nuclear factor kappaB ligand (RANKL) and the osteoprotegerin (OPG) synthesized by the osteoblasts. This study analyzes the effect of alendronate in cell viability, alkaline phosphatase (ALP) activity and RANKL and OPG expression in primary human osteoblasts (hOB). Alendronate at concentrations lower than 10⁻⁵ M did not have a toxic effect on hOB in vitro and did not modify the ALP activity at least for 72 h. Alendronate did not change OPG expression in basal, 10% fetal bovine serum (FBS), and vitamin D-treated cultures. Similar results were observed at the protein level. Unexpectedly, alendronate at 10⁻⁷ and 10⁻⁵ M concentrations increased the RANKL expression with the presence of vitamin D in differentiated hOB, and this induction of RANKL mRNA levels by alendronate was dose-dependent. However, this effect was not observed in basal and 10% FBS culture conditions. Thus, we conclude that alendronate does not affect the ALP activity and OPG gene expression in differentiated hOB, but may increase RANKL gene expression induced by vitamin D.

The influence of osteoprotegerin and RANKL as regulators of osteoclastogenesis and bone remodeling in liver disease and in the development of osteoporosis in primary biliary cirrhosis (PBC) is uncertain. Therefore, 68 women with PBC and 20 healthy females were studied by assessing circulating osteoprotegerin and RANKL. Bone mineral density and markers of bone turnover were measured as well. Osteoprotegerin-mRNA expression was also assessed in liver tissue from 16 patients and 5 controls. Osteoprotegerin was higher in PBC than in controls (5.4 +/- 0.2 vs. 2.9 +/- 0.2 pM/l, P0.0001), whilst RANKL was lower in patients than in controls (0.39 +/- 0.06 vs. 1.40 +/- 0.16 pM/l, P0.0001). Osteoprotegerin was more elevated in patients with more advanced disease, as defined by bilirubin above 1.2 mg/dl (6.6 +/- 0.6 vs. 5.2 +/- 0.2 pM/l, P = 0.02) or by Mayo over 4 (5.9 +/- 0.3 vs. 4.8 +/- 0.2 pM/l, P = 0.02). Osteoprotegerin and RANKL were unrelated with osteoporosis, and no associations were found with markers of bone remodeling, except for RANKL, which was particularly decreased in patients with low osteocalcin. This marker of bone formation was also higher in patients with elevated circulating osteoprotegerin. Liver osteoprotegerin gene expression was similar in patients and controls, and no correlation was found between liver osteoprotegerin-mRNA and patients' respective circulating levels. In conclusion, osteoprotegerin and RANKL are abnormal in patients with PBC, regardless of osteoporosis. The elevated circulating osteoprotegerin is associated with the severity of disease, but not with gene expression in the liver.

El tabaco es un conocido factor de riesgo de osteoporosis, pero los mecanismos por los que se produce esta asociacion siguen siendo objeto de debate. Estudios previos han evaluado las variaciones de osteoprotegerina plasmatica, una citoquina que regula la osteoclastogenesis y la resorcion osea, en fumadores, con resultados dispares. En este estudio se analizaron los niveles de osteoprotegerina plasmatica en un grupo de 49 voluntarios jovenes sanos, pero no existieron diferencias entre fumadores y no fumadores. Tampoco se encontraron diferencias tras ajustar por las diferencias basales existentes en estrona en varones y en edad e ingesta de calcio en mujeres. En conclusion, fumar no parece influir en los niveles plasmaticos de osteoprotegerina en voluntarios jovenes sanos. PALABRAS CLAVE: osteoprotegerina, tabaco, jovenes. While tobacco smoke is a known risk factor for osteoporosis, the mechanisms that produce this association must still elucidated. Several previous studies have evaluated the variations in serum levels of osteoprotegerin, a cytokine that regulates osteoclastogenesis and bone resorption in smokers, however, the results have been inconclusive. This study, which makes an analysis of serum levels of osteoprotegerin in a group of 49 healthy young volunteers, did not find any influence of smoking on this parameter. There were also no differences following adjustment for baseline differences in estrone in men and for age and calcium intake in women. In conclusion, tobacco smoking does not seem to influence osteoprotegerin serum levels in healthy young volunteers.

Splenic diffuse red pulp small B-cell lymphoma (SDRPL) is considered an indolent neoplasm and its pathogenesis is not well known. We investigated the molecular characteristics of 19 SDRPL patients, 5 of them with progressive disease. IGHV genes were mutated in 9/13 (69%). Cytogenetic and molecular studies identified complex karyotypes in 2 cases, and IGH rearrangements in 3, with PAX5 and potentially TCL1 as partners in each one of them. Copy number arrays showed aberrations in 69% of the tumors, including recurrent losses of 10q23, 14q31-q32, and 17p13 in 3, and 9p21 in 2 cases. Deletion of 7q31.3-q32.3 was present in only 1 case and no trisomies 3 or 18 were detected. NOTCH1 and MAP2K1 were mutated in 2 cases each, whereas BRAF, TP53, and SF3B1 were mutated each in single cases. No mutations were found in NOTCH2 or MYD88. Four of the 5 patients with aggressive disease had mutations in NOTCH1 (2 cases), TP53 (1 case), and MAP2K1 (1 case). The progression-free survival of patients with mutated genes was significantly shorter than in the unmutated (P=0.011). These findings show that SDRPL share some mutated genes but not chromosomal alterations, with other splenic lymphomas, that may confer a more aggressive behavior.

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.