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BackgroundThe pandemic due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has tremendous consequences for our societies. Knowledge of the seroprevalence of SARS-CoV-2 is needed to accurately monitor the spread of the epidemic and also to calculate the infection fatality rate (IFR). These measures may help the authorities to make informed decisions and adjust the current societal interventions. Blood donors comprise approximately 4.7% of the similarly aged population of Denmark and blood is donated in all areas of the country. The objective of this study was to perform real-time seroprevalence surveying among blood donors as a tool to estimate previous SARS-CoV-2 infections and the population based IFR.MethodsAll Danish blood donors aged 17-69 years giving blood April 6 to 17 were tested for SARS-CoV-2 immunoglobulin M and G antibodies using a commercial lateral flow test. Antibody status was compared between areas and an estimate of the IFR was calculated. The seroprevalence was adjusted for assay sensitivity and specificity taking the uncertainties of the test validation into account when reporting the 95% confidence intervals (CI).ResultsThe first 9,496 blood donors were tested and a combined adjusted seroprevalence of 1.7% (CI: 0.9-2.3) was calculated. The seroprevalence differed across areas. Using available data on fatalities and population numbers a combined IFR in patients younger than 70 is estimated at 82 per 100,000 (CI: 59-154) infections.ConclusionsThe IFR was estimated to be slightly lower than previously reported from other countries not using seroprevalence data. The IFR, including only individuals with no comorbidity, is likely several fold lower than the current estimate. This may have implications for risk mitigation. We have initiated real-time nationwide anti-SARS-CoV-2 seroprevalence surveying of blood donations as a tool in monitoring the epidemic.

Summary Background Health-care workers are thought to be highly exposed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We aimed to investigate the prevalence of antibodies against SARS-CoV-2 in health-care workers and the proportion of seroconverted health-care workers with previous symptoms of COVID-19. Methods In this observational cohort study, screening was offered to health-care workers in the Capital Region of Denmark, including medical, nursing, and other students who were associated with hospitals in the region. Screening included point-of-care tests for IgM and IgG antibodies against SARS-CoV-2. Test results and participant characteristics were recorded. Results were compared with findings in blood donors in the Capital Region in the study period. Findings Between April 15 and April 23, 2020, we screened 29 295 health-care workers, of whom 28 792 (98·28%) provided their test results. We identified 1163 (4·04% [95% CI 3·82–4·27]) seropositive health-care workers. Seroprevalence was higher in health-care workers than in blood donors (142 [3·04%] of 4672; risk ratio [RR] 1·33 [95% CI 1·12–1·58]; p

Objectives: The prevalence of active, chronic, and former hepatitis E virus (HEV) infections was investigated in a cohort of immunocompromised patients. The association with transfusion transmitted HEV was evaluated, and the HEV seroprevalence was compared with that in healthy blood donors. Study design and methods: Serum samples from 4023 immunocompromised patients at Rigshospitalet, Denmark were retrospectively tested for HEV RNA and anti-HEV IgG. HEV RNA-positive patients were followed up by HEV testing, clinical symptoms, and transfusion history. Factors associated with anti-HEV were explored by multivariable logistic regression analysis. Samples from 1226 blood donors were retrospectively tested for anti-HEV IgG. Results: HEV RNA was detected in six patients (0.15%) with no indications of chronic HEV infection. HEV RNA prevalence rates among recipients of allogeneic haematopoietic stem cell transplantation (allo-HSCT) and solid organ transplantation (SOT) were 0.58% and 0.21%, respectively. Transfusion transmitted infections were refuted, and transfusion history was not associated with anti-HEV positivity. The difference in HEV seroprevalence between patients (22.0%) and blood donors (10.9%) decreased when adjusting for age and sex (odds ratio 1.20, 95% confidence interval 0.97–1.48). Conclusions: HEV viremia among allo-HSCT and SOT recipients suggests that clinicians should be aware of this diagnosis. The lack of association of blood transfusion with anti-HEV positivity supports food-borne transmission as the main transmission route of HEV common to both patients and blood donors. Keywords: Hepatitis E, Epidemiology, Immunocompromised, Transfusion transmission, HEV RNA, Anti-HEV IgG, Chronic HEV infection

Transmembrane protein 30A (TMEM30A) maintains the asymmetric distribution of phosphatidylserine, an integral component of the cell membrane and ‘eat-me’ signal recognized by macrophages. Integrative genomic and transcriptomic analysis of diffuse large B-cell lymphoma (DLBCL) from the British Columbia population-based registry uncovered recurrent biallelic TMEM30A loss-of-function mutations, which were associated with a favorable outcome and uniquely observed in DLBCL. Using TMEM30A-knockout systems, increased accumulation of chemotherapy drugs was observed in TMEM30A-knockout cell lines and TMEM30A-mutated primary cells, explaining the improved treatment outcome. Furthermore, we found increased tumor-associated macrophages and an enhanced effect of anti-CD47 blockade limiting tumor growth in TMEM30A-knockout models. By contrast, we show that TMEM30A loss-of-function increases B-cell signaling following antigen stimulation—a mechanism conferring selective advantage during B-cell lymphoma development. Our data highlight a multifaceted role for TMEM30A in B-cell lymphomagenesis, and characterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited. Integrative analysis in patients with diffuse large B-cell lymphoma uncovers that biallelic mutations on TMEM30A are associated with a favorable outcome and enhanced sensitivity to CD47 blockade.

The clinical significance of MYC and BCL2 genetic alterations in diffuse large B-cell lymphoma (DLBCL), apart from translocations, has not been comprehensively investigated using high-resolution genetic assays. In this study, we profiled MYC and BCL2 genetic alterations using next-generation sequencing and high-resolution SNP array in 347 de novo DLBCL cases treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) at the British Columbia Cancer Agency. Cell-of-origin (COO) subtype was determined by Lymph2Cx digital gene expression profiling. We showed that the incidence of MYC/BCL2 genetic alterations and their clinical significance were largely dependent on COO subtypes. It is noteworthy that the presence of BCL2 gain/amplification is significantly associated with poor outcome in activated B-cell-like and BCL2 translocation with poor outcome in germinal center B-cell subtypes, respectively. Both have prognostic significance independent of MYC/BCL2 dual expression and the International Prognostic Index (IPI). Furthermore, the combination of BCL2 genetic alterations with IPI identifies markedly worse prognostic groups within individual COO subtypes. Thus, high-resolution genomic assays identify extremely poor prognostic groups within each COO subtype on the basis of BCL2 genetic status in this large, uniformly R-CHOP-treated population-based cohort of DLBCL. These results suggest COO subtype-specific biomarkers based on BCL2 genetic alterations can be used to risk-stratify patients with DLBCL treated with immunochemotherapy.

The clinical significance of

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype worldwide, accounting for 40% of all non-Hodgkin lymphomas. DLBCL presents as an aggressive disease requiring immediate treatment. Although significant improvement in outcome has been achieved, ~40% of patients still experience treatment failure. Here, we characterized the recurrent genetic alterations and transcriptomic signatures in diagnostic biopsies from a population registry-based cohort of 347 patients with de novo DLBCL uniformly treated with R-CHOP. This analysis revealed bi-allelic loss of function mutations of TMEM30A that were associated with favorable treatment outcome. TMEM30A is a chaperone protein, involved in maintaining the asymmetric distribution of phosphatidylethanolamine and phosphatidylserine, an integral component of the plasma membrane and “eat-me” signal recognized by macrophages. Using TMEM30A knockout systems by CRISPR genome editing techniques, we have functionally characterized this loss-of-function mutation in representative human and mouse DLBCL cell line models. We have discovered that TMEM30A loss is associated with increased B-cell signaling following antigen stimulation, including a two-fold increase in the diffusion rate of B-cell receptor (BCR) clustering, using high resolution Single Particle Tracking (SPT) technology. In addition, we have measured three-fold increase in chemotherapeutic drug accumulation in both knockout cell lines and randomly selected patient biopsies with TMEM30A biallelic loss. This observation was validated in a xenograft mouse model, which presented improved survival and limited tumor growth following vincristine treatment in mice injected with TMEM30A null DLBCL cell lines compared with native cell lines. This phenotype explains the improved prognosis observed in DLBCL patients following R-CHOP treatment. Furthermore, we have observed over two fold higher numbers of tumor-associated macrophages in B-cell lymphoma syngeneic mouse models with Tmem30a loss-of-function, prior to any form of treatment, suggesting the existence of “hot” and primed tumors. Our data highlight a multi-faceted role for TMEM30A and plasma membrane physiology in B-cell lymphomagenesis, and characterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited. Characterization of these mechanisms will address a missing link in the cancer field as related insights in lymphoma will outline therapeutic approaches that can be extended to cancer therapy in general. Citation Format: Shannon Healy, Daisuke Ennishi, Ali Bashashati, Saeed Saberi, Christoffer Hother, Anja Mottok, Fong Chun Chan, Lauren Chong, Robert Kridel, Merrill Boyle, Barbara Meissner, Tomohiro Aoki, Katsuyoshi Takata, Bruce W. Woolcock, Elena Vigano, Libin Abraham, Michael Gold, Adele Telenius, Pedro Farinha, Graham Slack, Susana Ben-Neriah, Daniel Lai, Allen W. Zhang, Sohrab Salehi, Hennady P. Shulha, Derek S. Chiu, Sara Mostafavi, Alina S. Gerrie, Diego Villa, Laurie H. Sehn, Kerry J. J. Savage, Andrew J. J. Mungall, Andrew P. Weng, Marcel Bally, Ryan D. Morin, Gabriela V. Cohen Freue, Joseph M. Connors, Marco A. Marra, Sohrab P. Shah, Randy D. Gascoyne1, David W. Scott, Christian Steidl, Ulrich Steidl. TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3480.

The discovery that the Ten-Eleven Translocation (TET) hydroxylases cause DNA demethylation has fundamentally changed the notion of how DNA methylation is regulated. Clonal analysis of the hematopoetic stem cell compartment suggests that TET2 mutations can be early events in hematologic cancers and recent investigations have shown TET2 mutations in diffuse large B-cell lymphoma. However, the detection rates and the types of TET2 mutations vary, and the relation to global methylation patterns has not been investigated. Here, we show TET2 mutations in 12 of 100 diffuse large B-cell lymphomas with 7% carrying loss-of-function and 5% carrying missense mutations. Genome-wide methylation profiling using 450K Illumina arrays identified 315 differentially methylated genes between TET2 mutated and TET2 wild-type cases. TET2 mutations are primarily associated with hypermethylation within CpG islands (70%; P

Deletions of chromosome 5q are associated with poor outcomes in acute myeloid leukemia (AML) suggesting the presence of tumor suppressor(s) at the locus. However, definitive identification of putative tumor suppressor genes remains controversial. Here we show that a 106-nucleotide noncoding RNA vault RNA2-1 (vtRNA2-1), previously misannotated as miR886, could potentially play a role in the biology and prognosis of AML. vtRNA2-1 is transcribed by polymerase III and is monoallelically methylated in 75% of healthy individuals whereas the remaining 25% of the population have biallelic hypomethylation. AML patients without methylation of VTRNA2-1 have a considerably better outcome than those with monoallelic or biallelic methylation (n = 101, P = .001). We show that methylation is inversely correlated with vtRNA2-1 expression, and that 5-azanucleosides induce vtRNA2-1 and down-regulate the phosphorylated RNA-dependent protein kinase (pPKR), whose activity has been shown to be modulated by vtRNA2-1. Because pPKR promotes cell survival in AML, the data are consistent with vtRNA2-1 being a tumor suppressor in AML. This is the first study to show that vtRNA2-1 might play a significant role in AML, that it is either mono- or biallelically expressed in the blood cells of healthy individuals, and that its methylation state predicts outcome in AML.

This study aimed to identify microRNA species involved in the earliest metastatic event in cutaneous malignant melanoma (MM). Samples from 28 patients with MM [stage T2 (tumor), M0 (distant metastasis)] were grouped by the presence of micrometastasis in the sentinel lymph nodes (N0/N1). Melanoma cells were harvested from primary, cutaneous MM tumors by laser-capture microdissection, and microRNA expression profiles were obtained by the microarray technique. Results were validated by quantitative reverse transcription PCR. We found that miR-125b was downregulated in the primary cutaneous melanomas that produced early metastases (T2, N1, M0) compared with the sentinel lymph node-negative (T2, N0, M0) melanomas. MiR-125b has earlier been found to be downregulated in other tumor types and in atypic naevi compared with the common acquired naevi. In conclusion, miR-125b may be involved in an early progression of cutaneous MM.

The CD30-positive lymphoproliferations encompass a spectrum of disorders that share histological and phenotypic similarities but differ markedly in clinical behaviour. The basis for this diversity is not known, but it has been proposed that immune suppression by cytokines and/or regulatory T-cells (Tregs) may be implicated. In this study, skin biopsies from lymphomatoid papulosis (LyP) (n = 14), primary cutaneous anaplastic large cells lymphoma (C-ALCL) (n = 13) and systemic anaplastic large cells lymphoma (S-ALCL) with (n = 9) or without (n = 6) ALK expression were examined by immunohistology for FOXP3 expression in tumour cells and tumour infiltrating Tregs. Labelling of a majority of the neoplastic cells was seen in one case of C-ALCL. Another three cases (one LyP and two C-ALCL) displayed weak labelling of very occasional atypical T-cells. In the remaining 38 cases the atypical lymphoid infiltrate was FOXP3 negative. By contrast, all biopsies contained tumour infiltrating FOXP3-positive Tregs. Significant higher numbers were recorded in ALK negative S-ALCL and LyP than in C-ALCL and S-ALCL positive for ALK. In conclusion, it is shown that FOXP3 expression in cutaneous and systemic CD30-positive lymphoproliferations is generally confined to tumour infiltrating Tregs. These cells may have influence upon the clinical behaviour, possibly depending upon the net degree of Treg mediated immune suppression of tumour cells relative to tumour infiltrating, cytotoxic effector cells, thereby implicating the more favourable outcome of LyP compared to C-ALCL.

A cancer develops when a cell acquires specific growth advantages through the stepwise accumulation of heritable changes in gene function. Basically, this process is directed by changes in two different classes of genes: Tumor suppressor genes that inhibit cell growth and survival and oncogenes that promote cell growth and survival. Since several alterations are usually required for a cancer to fully develop, the malignant phenotype is determined by the compound status of tumor suppressor genes and oncogenes. Cancer genes may be changed by several mechanisms, which potentially alter the protein encoding nucleotide template, change the copy number of genes, or lead to increased gene transcription. Epigenetic alterations, which, by definition, comprise mitotically and meiotically heritable changes in gene expression that are not caused by changes in the primary DNA sequence, are increasingly being recognized for their roles in carcinogenesis. These epigenetic alterations may involve covalent modifications of amino acid residues in the histones around which the DNA is wrapped, and changes in the methylation status of cytosine bases (C) in the context of CpG dinucleotides within the DNA itself. Methylation of clusters of CpGs called "CpG-islands" in the promoters of genes has been associated with heritable gene silencing. The present review will focus on how disruption of the epigenome can contribute to cancer. In contrast to genetic alterations, gene silencing by epigenetic modifications is potentially reversible. Treatment by agents that inhibit cytosine methylation and histone deacetylation can initiate chromatin decondensation, demethylation and reestablishment of gene transcription. Accordingly, in the clinical setting, DNA methylation and histone modifications are very attractive targets for the development and implementation of new therapeutic approaches. Many clinical trials are ongoing, and epigenetic therapy has recently been approved by the United States Food and Drug Administration (US FDA) for use in the treatment of myelodysplastic syndrome (MDS) and primary cutaneous T-cell lymphoma (CTCL).

Follicular lymphoma is a clinically and genetically heterogeneous disease, but the prognostic value of somatic mutations has not been systematically assessed. We aimed to improve risk stratification of patients receiving first-line immunochemotherapy by integrating gene mutations into a prognostic model.We did DNA deep sequencing to retrospectively analyse the mutation status of 74 genes in 151 follicular lymphoma biopsy specimens that were obtained from patients within 1 year before beginning immunochemotherapy consisting of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). These patients were recruited between May 4, 2000, and Oct 20, 2010, as part of a phase 3 trial (GLSG2000). Eligible patients had symptomatic, advanced stage follicular lymphoma and were previously untreated. The primary endpoints were failure-free survival (defined as less than a partial remission at the end of induction, relapse, progression, or death) and overall survival calculated from date of treatment initiation. Median follow-up was 7·7 years (IQR 5·5-9·3). Mutations and clinical factors were incorporated into a risk model for failure-free survival using multivariable L1-penalised Cox regression. We validated the risk model in an independent population-based cohort of 107 patients with symptomatic follicular lymphoma considered ineligible for curative irradiation. Pretreatment biopsies were taken between Feb 24, 2004, and Nov 24, 2009, within 1 year before beginning first-line immunochemotherapy consisting of rituximab, cyclophosphamide, vincristine, and prednisone (R-CVP). Median follow-up was 6·7 years (IQR 5·7-7·6).We established a clinicogenetic risk model (termed m7-FLIPI) that included the mutation status of seven genes (EZH2, ARID1A, MEF2B, EP300, FOXO1, CREBBP, and CARD11), the Follicular Lymphoma International Prognostic Index (FLIPI), and Eastern Cooperative Oncology Group (ECOG) performance status. In the training cohort, m7-FLIPI defined a high-risk group (28%, 43/151) with 5-year failure-free survival of 38·29% (95% CI 25·31-57·95) versus 77·21% (95% CI 69·21-86·14) for the low-risk group (hazard ratio [HR] 4·14, 95% CI 2·47-6·93; p0·0001; bootstrap-corrected HR 2·02), and outperformed a prognostic model of only gene mutations (HR 3·76, 95% CI 2·10-6·74; p0·0001; bootstrap-corrected HR 1·57). The positive predictive value and negative predictive value for 5-year failure-free survival were 64% and 78%, respectively, with a C-index of 0·80 (95% CI 0·71-0·89). In the validation cohort, m7-FLIPI again defined a high-risk group (22%, 24/107) with 5-year failure-free survival of 25·00% (95% CI 12·50-49·99) versus 68·24% (58·84-79·15) in the low-risk group (HR 3·58, 95% CI 2·00-6·42; p0.0001). The positive predictive value for 5-year failure-free survival was 72% and 68% for negative predictive value, with a C-index of 0·79 (95% CI 0·69-0·89). In the validation cohort, risk stratification by m7-FLIPI outperformed FLIPI alone (HR 2·18, 95% CI 1·21-3·92), and FLIPI combined with ECOG performance status (HR 2·03, 95% CI 1·12-3·67).Integration of the mutational status of seven genes with clinical risk factors improves prognostication for patients with follicular lymphoma receiving first-line immunochemotherapy and is a promising approach to identify the subset at highest risk of treatment failure.Deutsche Krebshilfe, Terry Fox Research Institute.

MicroRNAs (miRNA) are a group of short noncoding RNAs that regulate gene expression at the posttranscriptional level. It has been shown that microRNAs are independent predictors of outcome in patients with diffuse large B-cell lymphoma (DLBCL) treated with the drug combination R-CHOP. Based on the measured growth inhibition of 60 human cancer cell lines (NCI60) in the presence of doxorubicine, cyclophosphamide, vincristine and etoposide as well as the baseline microRNA expression of the 60 cell lines, a microRNA based response predictor to CHOP was developed. The response predictor consisting of 20 microRNAs was blindly validated in a cohort of 116 de novo DLBCL patients treated with R-CHOP or R-CHOEP as first line treatment. The predicted sensitivity based on diagnostic FFPE samples matched the clinical response, with decreasing sensitivity in poor responders (P = 0.03). When the International Prognostic Index (IPI) was included in the prediction analysis, the separation between responders and non-responders improved (P = 0.001). Thirteen patients developed relapse, and five patients predicted sensitive to their second and third line treatment survived a median 1194 days, while eight patients predicted not sensitive to their second and third line treatment survived a median 187 days (90% CI: 485 days versus 227 days). Among the latter group it was predicted that four would have been sensitive to another second line treatment than the one they received. The predictions were almost the same when diagnostic biopsies were used as when relapse biopsies were used. These preliminary findings warrant testing in a larger cohort of relapse patients to confirm whether the miRNA based predictor can select the optimal second line treatment and increase survival.

OncomiRs miR-21 and miR-155 have been linked to lymphomagenesis, but information on their implication in diffuse large B-cell lymphoma (DLBCL) is limited. Here, we used locked nucleic acid-based in situ hybridization (ISH) detection techniques on formalin-fixed paraffin-embedded DLBCL tissue samples to identify miR-155 and miR-21 at the cellular level in 56 patients diagnosed with DLBCL, and compared them to miR array data. miR-155 was observed in tumor cells in 19/56 (33.9%) of the samples evaluated by ISH. miR-21 was localized to the stromal compartment in 41/56 (73.2%). A subset of these, 16/56 (28.6%), also showed labeling in tumor cells. When comparing ISH-scores and miR array data, miR-155 in tumor cells, identified by ISH, was associated with miR-155 expression in miR array data (P=0.030). Equally, miR-21 expression by miR array data were highly associated with miR-21 ISH-scores in the stromal cells (P=0.002), whereas no association between miR array data and ISH of miR-21 in tumor cells was observed (P=0.673). We found no association of miR-155 and miR-21 with overall survival or germinal center B-cell-like (GCB) versus non-GCB-like subtypes of DLBCL. In conclusion, miR-ISH added to the biological interpretation of miR expression in DLBCL compared with miR array data, but miR-155 and miR-21 ISH did not add prognostic information in this series.

Genomic studies have revealed surprising new insights into the genetics of human B-cell lymphomas. Of particular note the lysine-specific methyl transferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma (FL) and in diffuse large B-cell lymphoma (DLBCL). However, the biological consequences of KMT2D mutations in lymphoma are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and promotes lymphoma development in vivo. Integrative analyses of KMT2D function indicate specific and focal effects on H3K4 methylation and gene expression. These include B-cell growth and differentiation signals such as the CD40, JAK/STAT, and B-cell receptor pathways. Moreover, KMT2D targets include frequently mutated tumor suppressor genes such as TNFAIP3/A20, SOCS3, and TNFRSF14/HVEM. In this manner, KMT2D deficiency disrupts the physiological process of B-cell differentiation, facilitates malignant outgrowth, and can impede therapies acting on the CD40 and B-cell receptor pathways. Citation Format: Ana Ortega-Molina, Isaac W. Boss, Andres Canela, Heng Pan, Yanwen Jiang, Chunying Zhao, Man Jiang, Deqing Hu, Ji-Eun Lee, Hua-Tang Chen, Daisuke Ennishi, David W. Scott, Christoffer Hother, Shichong Liu, Xing-Jun Cao, Wayne Tam, Rita Shaknovich, Benjamin A. Garcia, Randy D. Gascoyne, Kai Ge, Ali Shilatifard, Olivier Elemento, Andre Nussenzweig, Ari M. Melnick, Hans-Guido Wendel. Functional characterization of the tumor suppressor lysine-specific methyltransferase KMT2D in lymphoma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr IA18.

Purpose To investigate changes in microRNA (miR) expression in ocular adnexal extranodal marginal zone lymphoma (EMZL) and diffuse large B-cell lymphoma (DLBCL), and to perform a pathway analysis of differentially expressed miRs. Methods Global miR expression profiling was performed using miRCURYTM LNA miR-arrays, v. 11.0, (Exiqon) on formalin-fixed paraffin-embedded tissue from 18 ocular adnexal EMZLs and 25 DLBCLs. The result of microarray was confirmed using quantitative real time (RT)-PCR. Predicted and validated targets of the significantly differentially expressed miRs were obtained from MiR-walk. Transcription factor (TF)-miR interactions were obtained from the TransmiR database. Results The microarray results revealed two up-regulated and 41 down-regulated miRs in the DLBCLs compared to the EMZLs. Further, the differentially expressed miRs separated EMZLs and DLBCLs into two distinct entities using a supervised clustering analysis. These findings were confirmed by quantitative RT-PCR. Five of the down-regulated miRs (let-7g, miR-26a, miR-29a, miR-29c and miR-221) were identified to be regulated negatively by MYC. A small set of miRs (let-7g, miR-199a-5, miR-26a, miR-27a* and miR-342-3p) that regulate NFKB1 were all down-regulated in the DLBCLs compared to the EMZL samples. Further, the NFKB1 regulated miRs (miR-29a/b/c and miR-125b) were down-regulated significantly in the DLBCLs compared with the EMZLs (log2 fold-changes: miR-29a/b/c < -1.9 and -1.1 for miR-125b). Conclusion We demonstrated that fundamental differences in miR expression exist between ocular adnexal EMZLs and DLBCLs, mainly due to differences in MYC and NF-kB regulatory pathways.

Background: MYC and BCL2 are critical driver genes for non-Hodgkin lymphoma including diffuse large B-cell lymphoma (DLBCL). However, the clinical impact of MYC and BCL2 genetic alterations, apart from translocations, has not been comprehensively investigated using high-resolution genetic assays, such as next generation sequencing and high-resolution SNP arrays. Moreover, correlations with cell of origin (COO) subtype, determined by gene expression profiling, have not been widely studied in a large homogeneously treated cohort. We determined the frequency and clinical impact of MYC and BCL2 genetic aberrations in DLBCL in a large population-based cohort uniformly treated with R-CHOP. Methods: We analyzed 347 newly diagnosed de novo DLBCL cases that were treated with R-CHOP in BC. Comprehensive clinical annotation was available through the BCCA Lymphoid Cancer Database. Deep targeted re-sequencing of the coding exons of MYC and BCL2 was performed using a Truseq Custom Amplicon assay (Illumina) on the Miseq platform. High-resolution copy number analyses were performed using Affymetrix SNP 6.0 arrays. Immunohistochemical staining and break-apart FISH assays for MYC and BCL2 were performed on tissue microarrays (n=332). Dual positivity for MYC (cut off; 40%) and BCL2 (cut off; 50%) proteins identified a dual protein expresser (DPE) phenotype. COO classification was achieved using the Lymph2Cx assay based on NanoString technology in 299 patients and the Hans algorithm in 32 cases with low tumor content ( Results: COO determination revealed 193 cases to be GCB subtype, 107 cases ABC/non-GCB and 30 were unclassifiable. Using next generation sequencing, 310 SNVs were detected in MYC (29/347; 8% of cases) and BCL2 (88/347; 25% of cases), with mean redundant coverage depths of 633-fold. All MYC and 98% of BCL2 mutations were misssense mutations. Analysis of copy number alterations using GISTIC 2.0 revealed significant focal gain/amplification (gain/amp) peaks affecting 8q24, including the MYC locus (68/341; 20% of cases) and 18q21, including the BCL2 locus (82/341; 24% of cases). MYC and BCL2 translocations were detected in 38/283 (13%) and 90/300 (30%) of tumors, respectively. MYC gain/amp, BCL2 mutation, BCL2 translocations and double MYC/BCL2 translocation (DHIT) were seen significantly more often in GCB-DLBCL (26% vs 10%, p=0.001; 35% vs 11%, p With a median follow up of 6.5 years for living patients, the presence of MYC translocation, BCL2 gain/amp and BCL2 mutation were associated with an inferior 5y-time to progression (TTP, 53% vs 30%, p=0.019; 60% vs 32%, p=0.009 and 52% vs 17%, p=0.026, respectively) in ABC subtype. In GCB subtype, BCL2 translocation, MYC gain/amp and MYC translocation were associated with an inferior 5y-TTP (85% vs 62%, p=0.001; 80% vs 63%, p=0.011; and 79% vs 61%, p=0.013, respectively). In a multivariate Cox model of TTP including IPI and DPE, BCL2 gain/amp remained prognostic (HR=2.4 [1.3-4.5], p=0.008) independent of IPI and DPE in ABC-DLBCL. In GCB-DLBCL, BCL2 translocation and/or MYC gain/amp showed strong prognostic value (HR=3.0 [1.4-6.4], p=0.006) independent of IPI and DPE. In the IPI high risk group (IPI=3-5), the presence of a BCL2 translocation and/or MYC gain/amp defined a remarkably poor outcome group in GCB-DLBCL (5y-TTP; 29%). Similar poor outcome was observed in ABC-DLBCL cases that harbored BCL2 gain/amp (5y-TTP; 22%). Conclusions: The DHIT genotype was only seen in GCB-DLBCL. High-resolution genomic assays identified extremely poor prognostic groups within each COO subtype on the basis of MYC and BCL2 genetic status in a large uniformly R-CHOP-treated population-based cohort of DLBCL. Figure 1. TTP according to MYC/BCL2 genetic alterations with IPI in ABC-DLBC (n=101) and GCB-DLBCL patients (n=166) treated with R-CHOP. Figure 1. TTP according to MYC/BCL2 genetic alterations with IPI in ABC-DLBC (n=101) and GCB-DLBCL patients (n=166) treated with R-CHOP. Disclosures Savage: Seattle Genetics: Honoraria, Speakers Bureau; BMS: Honoraria; Infinity: Honoraria; Roche: Other: Institutional research funding. Connors:Roche: Research Funding; Seattle Genetics: Research Funding. Scott:Celgene: Consultancy, Honoraria; NanoString: Patents & Royalties: Inventor on a patent that NanoString has licensed.

Background: Diffuse large B cell lymphoma (DLBCL) is divided into two distinct molecular subtypes, germinal center B cell (GCB) subtype and activated B cell (ABC) subtype. Genetic landscape studies of DLBCL have revealed several GCB-DLBCL specific mutations, including CREBBP, GNA13, EZH2, TNFRSF14, BCL2 and MEF2B. Functional studies have recently shown that the inactivation of Gα13 signaling pathway genes, including GNA13, together with BCL2 over-expression, allows GC B-cells to escape the germinal center niche and widely disseminate. Although these findings revealed a critical role of genetic alterations of Gα13 signaling pathway in GC-driven mouse models of lymphomagenesis, clinical correlation is lacking. Here we analyzed the clinical impact of genetic alterations of Gα13 signaling pathway in a large population-based DLBCL cohort. Methods: We analyzed 347 newly diagnosed de novo DLBCL cases that were uniformly treated with R-CHOP at the BC Cancer Agency. Comprehensive clinical annotation was available through the BCCA Lymphoid Cancer Database. Deep targeted re-sequencing of the coding exons of GNA13, P2RY8, ARHGEF1, S1PR2 and RHOA was performed using a Truseq Custom Amplicon assay (Illumina) and/or Fluidigm Access Array chips. High-resolution copy number analyses were performed using Affymetrix SNP 6.0 arrays. Immunohistochemical staining and break-apart FISH assays for MYC and BCL2 were performed on tissue microarrays (n=332). Cell-of-origin classification was available in 331 cases, according to gene expression profiling by the Lymph2Cx assay using the NanoString platform (Scott, Blood 2014; 123) in 299 patients and the Hans algorithm (Hans, Blood 2004; 103) in 32 cases with low tumor content ( Results: Using next generation sequencing, 225 SNVs and 5 Indels were detected in GNA13 (16%), P2RY8 (18%), ARHGEF1 (6%), S1PR2 (3%) and RHOA (6%). SNP 6.0 microarrays revealed heterozygous deletions in GNA13 (2%), ARHGEF1 (1%), S1PR2 (4%) and RHOA (8%), but homozygous deletion was not found in any of these five loci. GNA13, P2RY8 and ARHGEF1 mutations were significantly more frequent in the GCB subtype than ABC subtype (26% vs. 6%; p In the cases with mutations of any of the five Gα13 signaling pathway genes, BCL2 over-expression (cut off; 50%) and translocation was associated with increasing stage (p=.018 and p=.005, respectively), but not in wt cases (p=.53 and p=.63, respectively). Specifically, in the cases with GNA13 and P2RY8 mutations individually, BCL2 over-expression was associated with advanced stage (stage III/IV, p=.018 and p=.037, respectively), but not in wild type (wt) cases. Importantly, BCL2 over-expression in the cases harboring Gα13 pathway mutations was not significantly associated with other poor risk features, including any other IPI factors or bone marrow involvement, indicating that genetic alterations in Gα13 signaling pathway accompanied by BCL2 over-expression might promote lymphoma dissemination into lymph nodes but not extranodal sites. With a median follow up of 6.5 years for living patients, there was no prognostic impact of harboring any isolated Gα13 pathway mutation in GCB-DLBCL patients. However, in cases with any Gα13 pathway mutations, BCL2 over-expression was significantly associated with an inferior 5y-time to progression (TTP; 90% vs 62%, p=.003) and disease-specific survival (DSS; 90% vs 71%, p=.042), but not in wt cases (Fig 1). In a Cox model of TTP including the IPI, BCL2 over-expression remained prognostic in the cases harboring any Gα13 pathway mutations (HR=4.13 [1.42-12.01], p=.009), but not in wt cases (HR=1.70 [0.62-4.68], p=.31). In cases with any Gα13 pathway alterations including copy number loss, BCL2 over-expression was also significantly associated with an inferior TTP (HR=3.64 [1.39-9.57], p=.009) independent of IPI, but not in the cases without genetic alterations (HR=1.75 [0.57-5.34], p=.33). Conclusions: Genetic alterations in Gα13 signaling pathway genes cooperate with BCL2 over-expression to promotes lymphoma dissemination to nodal sites and is associated with the poor outcome in GCB-DLBCL Figure 1. TTP and DSS according to BCL2 over-expression with/without Gα13 signaling pathway mutations in GCB-DLBCL patients (n=185) treated with R-CHOP Figure 1. TTP and DSS according to BCL2 over-expression with/without Gα13 signaling pathway mutations in GCB-DLBCL patients (n=185) treated with R-CHOP Disclosures Savage: Seattle Genetics: Honoraria, Speakers Bureau; BMS: Honoraria; Infinity: Honoraria; Roche: Other: Institutional research funding. Connors:Roche: Research Funding; Seattle Genetics: Research Funding. Scott:Celgene: Consultancy, Honoraria; NanoString: Patents & Royalties: Inventor on a patent that NanoString has licensed.

Smad4 is important in the TGF-β pathway and required for transcriptional activation and inhibition of cell growth after TGF-β1 stimulation. We demonstrate that miR-130a is differentially expressed during granulopoiesis and targets Smad4 mRNA. The transcript for Smad4 is present throughout neutrophil maturation, but Smad4 protein is undetectable in the most immature cells, where miR-130a is highly expressed. Two miR-130a binding sites were identified in the 3′-untranslated region of the Smad4 mRNA. Overexpression of miR-130a in HEK293, A549, and 32Dcl3 cells repressed synthesis of Smad4 protein without affecting Smad4 mRNA level. Repression of Smad4 synthesis in a granulocytic cell line by miR-130a reduced its sensitivity to TGF-β1–induced growth inhibition. This effect was reversed by inhibiting the activity of miR-130a with an antisense probe or by expressing a Smad4 mRNA lacking miR-130a binding sites. High endogenous miR-130a and Smad4 mRNA levels and low expression of Smad4 protein were found in the t(8;21)(q22;q22) acute myelogenous leukemia–derived cell line Kasumi-1. When miR-130a was inhibited by an antisense RNA, the amount of Smad4 protein increased in Kasumi-1 cells and rendered it susceptible for TGF-β1–mediated cell growth inhibition. Our data indicate that miR-130a is involved in cell cycle regulation of granulocytic cells through engagement of Smad4 in the TGF-β pathway.

BACKGROUND: The hypomethylating agent 5-Azacytidine (5-Aza-CR) is the first drug to prolong overall survival in patients with myelodysplastic syndrome (MDS). Surprisingly, the deoxyribonucleoside analog 5-Aza-2'deoxycytidine (5-Aza-CdR) did not have a similar effect on survival in a large clinical trial. Both drugs are thought to exert their effects after incorporation into DNA by covalent binding of DNA methyltransferase (DNMT). While 5-Aza-CdR is incorporated into only DNA, 5-Aza-CR is also incorporated into RNA. Here, we have analyzed whether this difference in nucleic acid incorporation may influence the capacities of these drugs to regulate the expression of mRNA and microRNAs (miRNA), which may potentially affect the activities of the drugs in patients. METHODOLOGY/PRINCIPAL FINDINGS: A hematopoietic (HL-60; acute myeloid leukemia) and a solid (T24; transitional cell carcinoma) cancer cell line were treated with equitoxic doses of 5-Aza-CR and 5-Aza-CdR for 24 hrs, and the immediate (day 2) and lasting (day 8) effects on RNA expression examined. There was considerable overlap between the RNAs heritably upregulated by both drugs on day 8 but more RNAs were stably induced by the deoxy analog. Both drugs strongly induced expression of cancer testis antigens. On day 2 more RNAs were downregulated by 5-Aza-CR, particularly at higher doses. A remarkable downregulation of miRNAs and a significant upregulation of tRNA synthetases and other genes involved in amino acid metabolism was observed in T24 cells. CONCLUSIONS/SIGNIFICANCE: Overall, this suggests that significant differences exist in the immediate action of the two drugs, however the dominant pattern of the lasting, and possible heritable changes, is overlapping.

The CD30-positive lymphoproliferations encompass a spectrum of disorders that share histological and phenotypic similarities but differ markedly in clinical behaviour. The basis for this diversity is not known, but it has been proposed that immune suppression by cytokines and/or regulatory T-cells (Tregs) may be implicated. In this study, skin biopsies from lymphomatoid papulosis (LyP) (n = 14), primary cutaneous anaplastic large cells lymphoma (C-ALCL) (n = 13) and systemic anaplastic large cells lymphoma (S-ALCL) with (n = 9) or without (n = 6) ALK expression were examined by immunohistology for FOXP3 expression in tumour cells and tumour infiltrating Tregs. Labelling of a majority of the neoplastic cells was seen in one case of C-ALCL. Another three cases (one LyP and two C-ALCL) displayed weak labelling of very occasional atypical T-cells. In the remaining 38 cases the atypical lymphoid infiltrate was FOXP3 negative. By contrast, all biopsies contained tumour infiltrating FOXP3-positive Tregs. Significant higher numbers were recorded in ALK negative S-ALCL and LyP than in C-ALCL and S-ALCL positive for ALK. In conclusion, it is shown that FOXP3 expression in cutaneous and systemic CD30-positive lymphoproliferations is generally confined to tumour infiltrating Tregs. These cells may have influence upon the clinical behaviour, possibly depending upon the net degree of Treg mediated immune suppression of tumour cells relative to tumour infiltrating, cytotoxic effector cells, thereby implicating the more favourable outcome of LyP compared to C-ALCL.

Udgivelsesdato: 2008-May Allelic loss at chromosome 9q31-34 is a frequent event in many lymphoproliferative malignancies. Here, we examined DBC1 at 9q33.1 as a potential target in lymphomagenesis. DBC1 is a putative tumor suppressor that has been shown to be involved in the regulation of cell growth and programmed cell death. The methylation status of the DBC1 promoter CpG island was examined by methylation-specific PCR, bisulfite sequencing, and methylation-specific melting curve analysis. DBC1 was hypermethylated in 5 of 5 B-cell-derived lymphoma cell lines, 41 of 42 diffuse large B-cell lymphomas, 24 of 24 follicular lymphomas, 5 of 5 mantle cell lymphomas, 4 of 4 small lymphocytic lymphomas, 1 of 2 lymphoplasmacytoid lymphomas, and in 12 of 12 acute lymphoblastic leukemias, but was unmethylated in 1 case of splenic marginal zone lymphoma, in 12 of 12 multiple myelomas, in 24 of 24 reactive lymph nodes, and in 12 of 12 samples of blood lymphocytes from random donors. DBC1 hypermethylation was associated with transcriptional silencing in lymphoma cell lines, and reexpression of this gene could be induced by treatment with the demethylating agent, 5-aza-2'-deoxycytidine. Our data suggest that hypermethylation of the DBC1 promoter region is a frequent event during the development of lymphoproliferative malignancies, and that DBC1 hypermethylation may serve as a marker for these cancers.

MicroRNAs (miRNAs) are a recently discovered group of small RNA molecules involved in the regulation of gene expression. Analogously to mRNAs, the non-protein-encoding pri-miRNAs are synthesized by RNA polymerase II and post-transcriptionally modified by addition of a 5'-cap and a 3'-poly (A) tail. Subsequently, the pri-miRNA undergoes a number of processing steps in the nucleus and cytoplasm, and ends up as a mature approximately 22 nt miRNA, which can exert its function by binding to the 3'-untranslated region of a subset of mRNAs. Binding of the miRNA to the mRNA results in a reduced translation rate and/or increased degradation of the mRNA. In this way a large number of cellular pathways, such as cellular proliferation, differentiation, and apoptosis, are regulated by mi-RNAs. As corruption of these pathways is the hallmark of many cancers, dysregulation of miRNA biogenesis or expression levels may lead to tumorigenesis. The mechanisms that alter the expression of miRNAs are similar to those that change the expression levels of mRNAs of tumor suppressor- and oncogenes, i.e. gross genomic aberrations, epigenetic changes, and minor mutations affecting the expression level, processing, or target-interaction potential of the miRNA. Furthermore, expression profiling of miRNAs has been found to be useful for classification of different tumor types. Taken together, miRNAs can be classified as onco-miRs or tumor suppressor-miRs, and may turn out to be potential targets for cancer therapy.

Introduction: Primary mediastinal large B cell lymphoma (PMBCL) is a distinct subtype of aggressive B cell lymphoma that arises from thymic medullary B cells and characteristically presents as a mass in the anterior mediastinum. A proportion of PMBCL patients suffer from refractory or relapsing disease, and subsequent salvage therapies are frequently ineffective. Development of targeted therapies is impeded by the lack of knowledge about the mutational landscape in the lymphoma genomes and mutation-associated phenotypes. We recently reported somatic mutations in the transcriptome of 7 PMBCL patients and 3 cell lines by next-generation sequencing and found a hotspot mutation in exon 8 of IL4R (Gunawardana et al, Nat Genet, 2014). Interleukin (IL)-4 is a type II cytokine that regulates immune responses through binding to a receptor complex consisting of the IL4 receptor alpha (IL4Rα) chain and IL13Rα or the common gamma chain (γc). Activation of IL4R by IL-4 and/or IL-13 initiates intracellular signal transduction mediated by the phosphorylation of JAnus Kinase-Signal Transducer and Activation of Transcription (JAK-STAT) pathway. We hypothesize that constitutively active JAK-STAT observed in PMBCL is in part due to gain-of-function IL4R mutations signaling through this oncogenic pathway. Methods and samples: A sequencing cohort consisting of fresh-frozen pretreatment lymph node biopsies from 62 PMBCL cases were selected from the BCCA tissue archive. DNA from PMBCL tumors and 3 PMBCL-derived cell lines were extracted for IL4R exonic PCR amplification and Sanger sequencing or high-throughput sequencing on an Illumina MiSeq instrument. WT and mutant (I242N) IL4R cDNA were cloned into mammalian expression vectors and expressed in the lymphoma cell line DEV and in engineered HEK293 cells expressing STAT6. Supernatant from cultured cells were used to measure the activity of STAT6-dependent expression of secreted embryonic alkaline phosphatase (SEAP). Extracted RNA and protein from transfected cells were used for qRT-PCR and Western blotting, respectively. Results: Somatic IL4R mutations were found in 18 of 65 (28%) cases confirming a hotspot mutation (I242N) in exon 8 in 11 of 18 (61%) mutated cases. Ectopic expression of the mutant in HEK293 cells showed increased SEAP levels compared to WT (534% vs. 100%) indicating STAT6 activation independent of cytokine stimulation. Introduction of the mutant into DEV cells showed IL4 independent hyperphosphorylation of JAK2, STAT5 and STAT6 proteins and upregulation of the B cell activation marker CD23 (963% vs. 100% in WT). Conclusions: IL4R is frequently mutated in PMBCL with a recurrent hotspot affecting the transmembrane domain of the protein. Functional analyses characterize these mutations as gain-of-function leading to constitutive activation of the JAK-STAT pathway and downstream target genes. These data suggest IL4R mutations as novel driver alterations in PMBCL and may provide a rational therapeutic target. Citation Format: Jay Gunawardana, Tessa Van Tol, Katina Mak, David Twa, Elizabeth Chavez, Bruce Woolcock, Robert Kridel, Anja Mottok, Shannon Healy, Adele Telenius, Merrill Boyle, Susana Ben-Neriah, Stacy Hung, Christoffer Hother, Randy Gascoyne, Christian Steidl. Recurrent IL4R mutations in primary mediastinal large B cell lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3941. doi:10.1158/1538-7445.AM2015-3941

Background Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype accounting for ∼40% of all non-Hodgkin lymphomas. Although R-CHOP has significantly improved outcome in DLBCL, 35% of patients still experience relapsed/refractory disease. Further investigation into the genomic architecture of DLBCL is needed to determine the biological correlates that underlie treatment failure. Immune cell signatures have been shown to impact survival in DLBCL (Lenz et al, NEJM 2008). Recurrent mutations and/or copy number alterations (CNAs) in genes that impact the tumor's immune-cell crosstalk have recently been described, but their clinical impact is unknown. Moreover, mutations and copy-number loss involving genes that foster immune escape has been implicated as a dominant oncogenic mechanism in DLBCL (Challa-Malladi et al, Can Cell 2011). We sought to determine the relationship between genetic alterations involving immune-cell crosstalk pathways and survival in a cohort of DLBCL patients treated with R-CHOP. Methods Following our initial analysis of 91 whole transcriptome sequencing (RNA-seq) including 40 with whole genome sequencing, we validated SNVs in the genes of interest by Sanger sequencing in the tumor and matched germline DNAs if available. RNA-seq-derived gene expression data were also used to determine the cell of origin (COO) distinction. Affymetrix SNP 6.0 microarrays were used to ascertain the CNAs in all 91 DLBCL samples. Clinical outcome correlations were analyzed by Cox regression and the log-rank test in the 82 patients treated with R-CHOP (median follow-up; 82 months). Results Using next-generation sequencing, we identified 97 SNVs in 16 genes (B2M, CD274, CD276, CD58, CD83, CIITA, NLRC4, NLRC5, RFXAP, RFXANK, TNFRSF14, TNFRSF25, TNFSF9, TAP1, TAP2 and TAPBP) that were selected based on an extensive literature review implicating these genes in immune-cell crosstalk pathways in DLBCL. Among them, eight genes were mutated in more than one case including B2M (7%), CD58 (7%), TNFRSF14 (7%), CIITA (4%), TNFSF9 (2%), NLRC5 (3%), CD83 (11%), and RFXAP (2%). These eight loci were further analyzed for CNAs, gene expression and outcome correlations. SNP 6.0 microarrays revealed either focal heterozygous or homozygous deletions in B2M (22%), CD58 (14%), TNFSF9 (10%), and heterozygous deletions in TNFRSF14 (12%), CIITA (3%), NLRC5 (7%), CD83 (2%), and RFXAP (7%). Amongst the 91 cases, 35 cases (38%) had no genetic aberration. 56 cases (62%) had at least one aberration, while 29 (32%) had multiple genetic aberrations indicating that genetic aberrations associated with immune-cell crosstalk are not mutually exclusive in DLBCL. Mutations and CNAs were not COO specific, with the exception of TNFRSF14, which was significantly restricted to the GCB-subtype (p=0.001). We evaluated outcome correlations for each genetic aberrations in the 82 cases with uniform therapy, including two recognized major histocompatibility class (MHC) groups; MHC class I (B2M and NLRC5) and MHC class II (CIITA, RFXAP and CD83). In univariate analysis, the presence of genetic aberrations in the MHCI group and NLRC5 individually were prognostic (5yr PFS, 59% vs 78%, p=0.025, 44% vs 77%, p=0.005, respectively), but not the MHCII group or other individual loci. In subgroup analyses according to COO, both MHCI and MHCII genetic aberrations were associated with inferior 5yr PFS in the ABC subtype (MHCI, 27% vs 79%, p Conclusions Recurrent genetic alterations involving genes related to immune-cell crosstalk in DLBCL are frequent (56%), co-occur and independently contribute to outcome in patients treated with R-CHOP. MHCI and MHCII perturbations are only prognostic in the ABC subtype. Disclosures: No relevant conflicts of interest to declare.

CH and PKR contributed equally to the work presented here and should therefore be regarded as equivalent PURPOSE. Ocular adnexal lymphoma (i.e., lymphoma with involvement of the orbit, eyelids, conjunctiva, lacrimal gland, and lacrimal sac), although rare, is common among malignant tumors involving the ocular adnexal region. The main subtypes are low-grade extranodal marginal zone lymphoma (EMZL) and aggressive diffuse large B-cell lymphoma (DLBCL). In rare cases, low-grade EMZL are reported to transform to DLBCL. It is unclear, however, which genetic events distinguish low-grade disease from aggressive, potentially fatal disease. METHODS. Using LNA-based arrays from Exiqon, we performed global microRNA (miRNA) expression profiling of 18 EMZLs and 25 DLBCLs involving ocular adnexal sites to investigate changes in the miRNA expression in low- versus high-grade disease. Findings were confirmed by real-time quantitative PCR (RTq-PCR). RESULTS. Our analysis revealed 43 miRNAs with altered expression profiles in DLBCL compared to EMZL. Seven of the miRNAs down-regulated in DLBCL relative to EMZL showed enrichment for a direct transcriptional repression by the oncoprotein MYC. We also report a possible loss-of-regulation of NFKB1 and its downstream miRNAs. In addition, our analysis identified a group of DLBCLs whose expression profiles resembled that of EMZL. Although transformation of EMZL to DLBCL in the ocular adnexal region is rare, we hypothesize that the intermediate group potentially may derive from transformation of EMZL that was not recognized by histology. CONCLUSIONS. We conclude that fundamental differences in miRNA expression exist between ocular adnexal EMZL and DLBCL, mainly due to differences in MYC and NF-+B regulatory pathways.

The purpose of this study was to describe the microRNA (miRNA) expression profiles of neutrophils and their precursors from the initiation of granulopoiesis in the bone marrow to extravasation and accumulation in skin windows. We analyzed three different cell populations from human bone marrow, polymorphonuclear neutrophil (PMNs) from peripheral blood, and extravasated PMNs from skin windows using the Affymetrix 2.0 platform. Our data reveal 135 miRNAs differentially regulated during bone marrow granulopoiesis. The majority is differentially regulated between the myeloblast/promyelocyte (MB/PM) and myelocyte/metamyelocyte (MC/MM) stages of development. These 135 miRNAs were divided into six clusters according to the pattern of their expression. Several miRNAs demonstrate a pronounced increase or reduction at the transition between MB/PM and MC/MM, which is associated with cell cycle arrest and the initiation of terminal differentiation. Seven miRNAs are differentially up-regulated between peripheral blood PMNs and extravasated PMNs and only one of these (miR-132) is also differentially regulated during granulopoiesis. The study indicates that several different miRNAs participate in the regulation of normal granulopoiesis and that miRNAs might also regulate activities of extravasated neutrophils. The data present the miRNA profiles during the development and activation of the neutrophil granulocyte in healthy humans and thus serves as a reference for further research of normal and malignant granulocytic development.

Abstract 1548 Introduction: The introduction of Rituximab as supplement to chemotherapy has significantly improved outcome in diffuse large B-cell lymphoma (DLBCL). Still, a fraction of patients are resistant or relapse shortly after treatment. Improved stratification of patients with DLBCL for standard immunochemotherapy or alternative treatment strategies is therefore urgently needed. Although DLBCL profiling based on mRNA expression may be helpful, this has not proven clinically efficient, and the prognostic value of immunohistochemical algorithms is controversial. In addition, novel therapeutic options are essential since the current alternative treatment modalities are often not curative. MicroRNAs (miRs) are particularly attractive molecules for clinical use since they are well conserved in formalin fixed paraffin embedded (FFPE) tissue, and novel data imply that they may be targeted directly in the patients. Materials and methods: RNA was extracted from diagnostic biopsies from DLBCL patients (n=97) treated uniformly with immunochemotherapy (R-CHOP n=80 or R-CHOEP n=17). GCB/non-GCB profiling was done by immunohistochemistry according to the Hans classification. MiR profiles were generated using Affymetrix microRNA version 1.0 arrays. Data analyses were performed using R/biocondutor and the webtool “SignS” that uses parallel computing for finding survival related genes and signatures from gene-expression datasets. Survival analysis was performed in R using the survival package. Univariate analysis was performed by comparing Kaplan-Meier survival estimates using Log-rank test. Cox proportional hazards regression model was used for multivariate analysis. Results and discussion: The median follow-up time for all patients was 3.4 years. The estimated 3-year over all survival probability was 82.8% (95% CI: 75.4%-90.9%). No difference in survivability was observed between the R-CHOP and the R-CHOEP treated cohort (P=0.145). High IPI (> 2) was significantly associated with inferior overall survival (OS, P = 0.038), but not progression free survival (PFS, P = 0.083). Univariate analysis showed that in this cohort the Hans classification was not prognostic (P=0.73; (52 GBC and 37 non-GCB subtypes; 8 NA)). Seven miRs were differentially regulated between GCB and non-GCB using a cutoff of P< 0.05. Five miRs were upregulated in non-GCB lymphomas: miR-625, miR-222, miR-221, miR-155 and miR-503, two were downregulated (miR-181a, miR-181b). For survival analysis, we initially applied a multivariate approach (Robust likelihood-based survival modeling, RBsurv), which identified a subset of miRs that significantly associates with poor survival. These include one upregulated miR, and four down regulated miRs. In order to obtain cross validated survival estimates, we applied three different algorithms; FCSM(SignS), TGD(SignS) and GLMboost(SignS) to the same sample set. These combined bioinformatic models identified a total of 17 deregulated miRs that significantly associate with survival. Among these, 9 are predicted by more that one algorithm, and interestingly, all 4 models identify a novel upregulated and potential oncogenic miR in patients treated by immunochemotherapy. When the cross-validated predictors were combined into a unified robust “miR-survival-predictor”, the performance is as good as, or even better, than the IPI. In addition, our model is a superior predictor of survival than the GCB/non-GCB classification according to Hans. Our data are currently being validated in a test set of 60 patients, and functional studies of the novel putative oncomiR are ongoing. Disclosures: No relevant conflicts of interest to declare.

Abstract 2136 Formation of polymorphonuclear neutrophils (PMN) is a tightly regulated process where the myeloid progenitor cells, myeloblasts (MBs), divide and mature in the bone marrow, along a well defined path. The cells pass through six well defined stages in differentiation ending up with the release of mature PMNs to peripheral blood (granulopoiesis). Expression of essential transcription factors such as RUNX1, C/EBP-a, and C/EBP-e during granulopoiesis has been shown to have great importance for correct neutrophil development. microRNAs (miRNAs) could be important players in the fine-tuning of transcription factor expression due to their ability to regulate protein synthesis. The function of neutrophils is to detect and destroy invading microorganisms. This involves activation of the PMNs in the blood stream causing a release of secretory vesicles and up-regulation of extracellular adhesion molecules followed by migration in the tissue towards the focus of inflammation. Expression of miRNAs might also be regulated during activation and diapedesis of the neutrophils in order to adapt the neutrophil to its new environment and function. A regulatory role for miRNAs has been demonstrated for several biological processes, such as proliferation, differentiation, inflammation and cancer, and dysregulation of miRNA expression has been shown to contribute to disease development. The purpose of this study was to determine the miRNA expression profiles during normal human granulopoiesis starting with the first identifiable granulocytic precursor cell (MB) and ending with activated neutrophils that have migrated into the tissue using an Affymetrix 2,0 miRNA microarray platform. We isolated four populations of cells: Myeloblasts (MB) and promyelocytes (PM), myelocytes (MC) and metamyelocytes (MM), and band cells (BC) and segmented cells (SC) from the bone marrow and PMNs from peripheral blood from three different donors. We found 135 differentially expressed miRNAs in granulopoiesis, which could be divided into six clusters according to their expression pattern. 87% of the 135 miRNAs were differentially regulated between the MB/PM (dividing cells) and the MC/MM stages (cessation of cell proliferation and initiation of terminal differentiation) and could imply a need for miRNA-mediated regulation of the many proteins involved in regulating this process. Interestingly, we also found two distinct clusters of miRNAs that were either up- or down-regulated only in the MC/MM population, indicating the importance of a specific temporary regulation of some proteins during neutrophil development. To determine miRNA expression profiles in activated granulocytes, we examined PMNs and activated neutrophils from skin window (i.e. PMNs migrated to a site of inflammation). We found seven differentially expressed miRNAs, - all of them up regulated in the activated neutrophils. Using microRNA target-prediction software, we found that miRNAs 155, 146a and 130a, all of which are strongly up-regulated in the MB/PM stage, have several targets in the IL1-receptor signalling cascade, indicating the importance of miRNA of dampening an innate immune response in immature neutrophil precursors. miR-146a, 155 and 130a also have predicted targets in either the TGF-βI or the TGF-βII receptor which inhibits proliferation when binding to TGF-β. This finding supports the proliferating profile for the MB/PM cells, and the shift towards cell cycle arrest when the cells differentiate to the next stage, where expression of these three miRNAs is low. miRNA-34c-3p is highly expressed only in the MC/MM stage and has verified targets in many different mRNAs involved in the regulation of cell cycle arrest. All the miRNAs that were up-regulated in the activated neutrophils have several predicted targets in the IL1R pathway, and some of them (miR-212, −132 and −297) have previously been shown to be important in regulating the inflammatory response. The study indicates that several different miRNAs have important roles in the regulation of normal granulopoiesis, and that miRNAs also might be part of a possible negative feed back loop in the inflammatory response in activated neutrophils. Grant acknowledgments: The Danish Cancer Society, Lundbeck foundation, Danish Medical Research Council, Brøchner Mortensen foundation Disclosures: No relevant conflicts of interest to declare.

Abstract 296 Background: In diffuse large B-cell lymphoma (DLBCL), a number of studies have shown that dual disruption of the p16/Rb and the ARF/p53 pathway, e.g. deletion of the INK4A/ARF locus, are negative prognostic factors for survival. Molecular studies have shown that the p53-miR34 axis may be another link between the ARF/p53 pathway and the p16/Rb pathway. In a complex circuit, p53 promotes transcription of MIR34A and MIR34B/C, and the miR34s' in turn act as mediators of p53 signaling. In addition, miR34s' have been shown to inhibit MYC and several proto-oncogens that counteract the p16/Rb tumor suppressor pathway. These observations place the miR34s' at the center of cell cycle and apoptosis regulation, and loss of miR34 expression has been associated with adverse response to therapy in several tumor types. In CLL it has been suggested that miR34A expression is a surrogate marker for TP53 disruption and the MDM2 SNP309 GG genotype, both associated with a poor prognosis. Design and methods: In CD19+ B-cells isolated from healthy donors (PBL-B) and reactive lymph nodes, the expression of the –5p and –3p species of miR34A, miR34B, and miR34C was analysed by qRT-PCR using LNA-based primers from Exiqon that discriminate the individual miR34 members. Histone H3K4 and H3K27 methylation status at the MIR34A and MIR34B/C promoters was analyzed by ChIP. In 120 newly diagnosed DLBCLs we studied the combined status of TP53 mutations, MIR34A and MIR34B/C promoter DNA methylation, and the MDM2SNP309 GG genotype using a panel of PCR based methods. Results: We initially determined the expression of the individual miR34 family members in PBL-B and in reactive lymph nodes. While miR34A-5p was expressed at low levels in normal PBL-B, no expression of miR34A-3p, miR34B-5p, miR34B-3p, miR34C-5p, and miR34C-3p was observed. In reactive lymph nodes the expression of miR34A-5p was induced in average 70 fold (P In 100 of the 120 (83%) DLBCLs one or more of the investigated genes carried (epi)-genomic alterations including TP53 mutations: 20(17%), MIR34A methylation: 34(28%), MIR34B/C methylation: 93(78%) and the MDM2 SNP309 GG-genotype 11(9%). All but one of the MIR34A methylated cases also had methylation of MIR34B/C. Seven cases carried combined TP53 mutation and methylation of both MIR34A and MIR34B/C. A borderline inferior overall survival for patients carrying TP53 mutations was found (p=0.058), while none of the other parameters influenced survival. However, the 7 cases with concomitant mutation of TP53 and methylation of both MIR34A and MIR34 B/C had a median survival of 14 months (p=0.02). In a DLBCL-cell line with concomitant TP53 mutation and methylation of both MIR34A and MIR34B/C, miR34s' were upregulated by 5-aza-2'deoxycytidine (5-aza-CdR). Conclusions: In normal B-cells miR34A seems to play a dominant role over miR34B/C, however in DLBCL the concomitant DNA methylation of MIR34B/C in the MIR34A methylated cases suggest they may compensate for each other. Our results suggest that in DLBCL the p53-miR34 axis is not linear, and that the TP53, MIR34A and MIR34B/C “triple-hit” DLBCLs may represent more aggressive lymphomas that may benefit from treatment with 5-aza-CdR. This is, to the best of our knowledge, the first study to address these issues in a large sample of diagnostic DLBCL cases. Disclosures: No relevant conflicts of interest to declare.

Abstract 1600 Introduction: Although rare, ocular adnexal lymphomas (i.e. lymphoma of the orbit, eyelids, conjunctiva, lacrimal gland and lacrimal sac), are among the most common malignant tumors involving the ocular adnexal regions. The main subtypes are low-grade mucosa associated lymphoid tissue (MALT) lymphoma and aggressive diffuse large B cell lymphoma (DLBCL). In rare cases low-grade MALT lymphoma are reported to transform to DLBCL. It is unclear, however, which genetic events distinguish low-grade disease from aggressive, potentially fatal, disease. Material and methods: A total of 18 MALT lymphomas and 25 DLBCLs involving ocular adnexal sites were included in the study. All sections were analyzed immunohistochemically by two independent pathologists (ER, SH) using the following panel of antibodies: bcl-2, bcl-6, CD3, CD5, CD10, CD20, CD23, CD79α, cyclin D1, MUM-1 and Ki-67. Confirmed cases of DLBCL were categorized as either germinal centre B-cell-like (GCB) or non-GCB types according to the algorithm by Hans et al. Using LNA-based arrays from Exiqon, we performed global miRNA expression profiling of RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. The most differentially expressed microRNAs were confirmed by RT-qPCR analyses. Microarray processing was performed using the R environment. Results: Of the18 MALT patients (pts.) 15 pts. (83%) presented with Stage I lymphoma and 3 pts. (17%) had Stage IV. The 5-year overall survival for the entire population was 77%. In the DLBCL group 13 pts. (52%) presented with Stage I lymphoma, 3 pts. (12%) Stage II lymphoma, 1 pt. (4%) Stage III and 8 pts. (32%) presented with Stage IV lymphoma. Nine of the DLBCLs were classified as GCB and 16 as non-GCB type. The 5-year overall survival for the entire group was only 13%. Our miR arrays and confirmatory qPCR analysis revealed 43 miRNAs with significantly altered expression profiles (41 down- and 2 upregulated) in DLBCL compared to MALT lymphoma. Seven of the miRNAs down-regulated in DLBCL relative to MALT lymphoma showed enrichment for a direct transcriptional repression by the oncoprotein MYC. Supervised hieracical clustering analysis identified tree clusters: Cluster 1: MALT (high expression), cluster 2: DLBCL (intermediate expression), cluster 3 DLBCL (low expression). Thus, apparently the DLBCLs in cluster 2 seem to resemble MALT more than DLBCLs in cluster 3. We also report loss of miRNAs involved in the regulation of NFKB1 and DNA methyltransferases in DLBCL vs MALT. Conclusion: We conclude that fundamental differences in miRNA expression exist between ocular adnexal MALT lymphoma and DLBCL. Among the possible consequences of altered miRNA expression are increased NF-kB signaling and DNA hypermethylation. However, in line with the recent observations in gastric MALT/DLBCL transformation1, we suggest the differences may at least in part be caused by MYC transcriptional regulation of miRNAs in aggressive cases. The fraction of DLBCL that is reported to arise from MALT is exceedingly low. However, in the current study we find a group of DLBCLs (cluster 2), whose level of MYC regulated miRNA expression is intermediate between MALT (cluster 1) and DLBCL (cluster 3). Thus it could be speculated whether cluster 2 DLBCLs have developed secondary to a preceding MALT lesion, which, however was not detected by histology. Disclosures: No relevant conflicts of interest to declare.

Abstract 3383 MicroRNAs (miRNA) are noncoding RNA molecules that regulate the synthesis of proteins and, if dysregulated, can result in development of various form of cancers. We have found that miR-130a is highly expressed in immature proliferating granulocytic precursors. In more mature granulocytes the miR-130a expression is significant lower. In acute myeloid leukemia the granulocyte precursors have lost the ability to undergo terminal maturation, leading to accumulation of non-functional, immature granulocytes (myeloblasts). We hypothesize that a sustained high expression of miR-130a during granulopoiesis may sustain continuous cell proliferation. TGF-β is a strong inhibitor of cell proliferation and lack of TGF-β expression is associated with various form of cancer. Smad4 is an essential compound in the TGF-β signaling pathway. Using microRNA target-prediction software, we identified Smad4 as a putative target for miR-130a. This was confirmed experimentally by demonstrating that transient overexpression of miR-130a results in reduction in the amount of Smad4 protein. Luciferase reporter constructs with the 3`-UTR of Smad4 also respond to miR-130a – an effect that is abolished by point mutations in the miRNA–binding site. In agreement, we observed that stable overexpression of miR-130a in a granulocytic cell line reduces the level of Smad4 protein, and render the cells less sensitive to TGF-β-induced growth inhibition. This was also confirmed with cell cycles analysis. Furthermore, the effect was diminished when transfecting the same clones with SMAD4 lacking the 3-‘UTR. In line with our hypothesis, the most immature granulocyte precursors demonstrate the highest expression of miR-130a is highest, and the lowest expression of Smad4 protein. As the granulocyte precursors mature, the expression of miR-130a decreases dramatically whereas the level of Smad4 protein expression increases demonstrating inverse correlation between miR-130a and Smad4 protein. The level of Smad4 mRNA is comparable at all stages of granulopoiesis. High miR-130a levels and low or no expression of Smad4 was found in primary cells from patients with acute myeloid leukemia and in a cell line (Kasumi-1) with the t(8;21)(q22;q22) chromosomal translocation. The level of Smad4 increased in Kasumi-1 cells when the endogenous level of miR-130a was inhibited by anti-miR-130a LNA. Our data indicate that miR-130a is involved in cell cycle regulation of normal and malignant granulocytic cells through engagement of Smad4 in the TGF-β-pathway. Grant acknowledgment: Lundbeck foundation, Carlsberg foundation, Swedish Research Council. Disclosures: No relevant conflicts of interest to declare.

Abstract 3450 Introduction: Deletions of chromosome 5q are associated with poor outcomes in acute myeloid leukemia (AML), suggesting the presence of tumor suppressor(s) at the locus. Two different critical deleted regions (CDRs) have been identified on 5q. One region is linked to de novo AML and high-risk MDS (CDR1 at 5q31), and a second region to the low-risk MDS 5q- syndrome (CDR2 at 5q32–33). However, definitive identification of putative tumor suppressor genes remains controversial. Since it has recently been shown that several types of hematological malignancies have globally altered expression of non-coding RNAs (ncRNAs), we therefore searched for candidate ncRNA genes in the CDR1 region. Methods: Cell lines were treated with either 5-azacytidine or 5-aza-2`-deoxycytidine. Upregulated microRNAs (miRs) were identified by microarray analysis and confirmed by RT-qPCR analysis. The transcription start site was determined by 5`RACE and promoter methylation status by methylation specific melting curves, pyrosequencing and bisulfite sequencing. ncRNA expression and processing were analysed by RT-qPCR, Northern blotting, and siRNA mediated knock down of Drosha. Results: We identified the putative miR886, which became induced by azanucleoside treatment in an AML cell line, suggesting that it was regulated by promoter methylation. We found that the processing of miR886 was independent of Drosha, and northern blotting showed that this was a different type of longer ncRNA, annotated vtRNA2-1. These observations were supported by the results from three others groups (Nandy, J Mol Biol 2009; Stadler, Mol Biol Evol 2009; Lee, RNA 2011). The gene that encodes VTRNA2-1 is embedded in a CpG island. The CpG island is fully methylated in 4 different myeloid cell lines (HL60, NB4, U937 and F36P) and these cell lines have no expression of vtRNA2-1. Treatment with 5-azacytidine and 5-aza-2`-deoxycytidine derepressed expression of vtRNA2-1 in the cell lines. vtRNA2-1 is expressed in hematopoietic tissue (including CD34+ cells) from healthy individuals. Surprisingly, 75% of these carry a monoallelicly methylated VTRNA2-1 promoter while 25% carry an unmethylated VTRNA2-1 promoter. The methylation status of VTRNA2-1 was examined in bone marrow mononuclear cells from 101 AML patients taken at the time of diagnosis. 38 (38%) of these patients carried a hypomethylated promoter, 53 (52%) an intermediate methylated promoter and 10 (10%) a hypermethylated promoter. AML patients with hypomethylation of VTRNA2-1 have a significant better prognosis than patients with intermediate- or hypermethylation of the promotor (p=0.001). VTRNA2-1 methylation was independently associated with a poor survival in Cox proportional-hazards analysis (p=0.043), when testing against age, cytogenetic risk classification and leukocyte count at diagnosis. Discussion: It has previously been shown that vtRNA2-1 may be involved in the regulation of the double stranded RNA dependent kinase, PKR. Down regulation of vtRNA2-1 leads to activation of PKR and its downstream targets including NFkB (Lee, RNA 2011). Furthermore, PKR has previously been shown to alter response to chemotherapeutic agents by promoting cell survival (Pataer, Cancer Biol Ther 2009). Since constitutive PKR and NFkB activity are well documented features in AML, we speculate whether this may at least in part be mediated via loss of vtRNA2-1 expression. Here, we show that VTRNA2-1 may be directly implicated in AML, that expression of vtRNA2-1 is regulated by promoter methylation. Interestingly, we found that the majority of the healthy Danish population (∼75%) carry a monoallelically silenced VTRNA2-1 in normal hematopoietic cells. Our data suggest that the gene dosage of this particular type of ncRNA may play an important role in tumor progression or response to therapy since patients with hypomethylation of both alleles of the VTRNA2-1 promoter have a significantly better prognosis, while those that gain hypermethylation of the second VTRNA2-1 copy have a poorer outcome. Our data, combined with the previous findings, suggest that VTRNA2-1 is a novel tumor suppressor, located on chromosome 5q31.1, which probably acts through PKR. Disclosures: Jones: Eli Lilly: Consultancy.

Abstract 1364 Introduction: Cytosine methylation (mC) is a major DNA modification in higher eukaryotic genomes, which is involved in transcriptional silencing. A large amount of data has shown that patterns of DNA methylation are perturbed in hematological cancers including diffuse large B-cell lymphoma (DLBCL). The discovery that the TET hydroxylases convert mC to hydroxymethylcytosine (hmC) is a major break through for our understanding of how DNA methylation is deregulated. Multiple reports describe TET2 (Ten-Eleven Translocation 2) loss-of-function mutations in myeloid malignancies, and a recent study shows that TET2 inactivation perturbs both myeloid and lymphoid development in the mouse, and identifies TET2 mutations in ∼2% of human B-cell lymphoma (Quivoron et al, Cancer Cell 20, 1–14, 2011). Aims: In the present study our aims are to determine the frequency and clinical impact of TET2 mutations in DLBCL, to identify TET2 target genes in CD34+ cells, normal- and malignant B-cells, and evaluate the role of TET2 mutations on the methylation pattern at TET2 targets genes in normal and malignant hematopoiesis. Methods: DNA was isolated from fresh frozen DLBCL (n=110), normal CD34+ cells and B-cells, and a TET2 mutant DLBCL-cell line. Mutation scanning was performed by denaturing gradient gel electrophoresis (DGGE) and automated sequencing. Global methylation profiling was done by Illumina Infinium microarrays, methylation at individual genes by methylation specific melting curve analysis and pyrosequencing. Global mC and hmC patterns were determined by DNA immunoprecipitation and promoter array analysis in cell lines, B-cells and CD34+ cells. TET2 target genes were identified by ChIP followed by deep sequencing. Gene expression by Nimblegen custom made arrays and RT-qPCR. Results: We identified TET2 mutations in 15% of primary diffuse DLBCL, including missense mutation in the catalytic domain (n=8, 2 of which showed allelic loss), loss-of-function mutations (n=7, one of which showed allelic loss), and missense mutation outside the catalytic domain (n=1 with allelic loss). Somatic origin of these mutations was verified in 11 of the 16 cases where matched normal tissue was available. No difference in overall survival was observed between TET2mut and TET2wt cases (P=0.17). To a large extent, the TET2 targets genes identified by ChIP seq analysis were overlapping in CD34+ cells, normal- and malignant B-cells. Gene ontology analysis showed that TET2 target genes are mainly involved in DNA metabolism and repair, metabolic processes and cell cycle homeostasis. Global methylation in TET2mut and TET2wt cases and gene expression data are being analyzed in DLBCL samples. In addition, the distribution patterns of hmC and mC at TET2 target genes and the relation to gene expression is being analyzed in a TET2 mutant DLBCL cell line, normal B-cells and CD34+ cells. Conclusion and further analyses: Here, we show that TET2 mutations are frequent in DLBCL, and identify the TET2 target genes in CD34+ cells, and in normal and malignant B-cells. The role of TET2 mutations for global methylation and for the methylation patterns at TET2 target genes will be presented at the meeting. By investigating the clinical implications of TET2 mutations we aim to identify DLBCL subsets that may benefit from hypomethylating therapy. Furthermore, the identification of hypermethylated TET2 target genes will hopefully contribute to molecular understanding of how TET2 mutations induces malignant transformation. Disclosures: Christensen: EpiTherapeutics: cofounder of EpiTherapeutics and have shares and warrants in the company. Helin:EpiTherapeutics: cofounder of EpiTherapeutics and have shares and warrants in the company.

Background Gastric cancer is the fourth most common cancer in the world and the second most prevalent cause of cancer related death. The development of gastric cancer is mainly associated with H. Pylori infection leading to a focus in pathology studies on bacterial and environmental factors, and to a lesser extent on the mechanistic development of the tumour. MicroRNAs are small non-coding RNA molecules involved in post-transcriptional gene regulation. They are found to regulate genes involved in diverse biological functions and alterations in microRNA expression have been linked to the pathogenesis of many malignancies. The current study is focused on identifying microRNAs involved in gastric carcinogenesis and to explore their mechanistic relevance by characterizing their targets. Results Invitrogen NCode miRNA microarrays identified miR-449 to be decreased in 1-year-old Gastrin KO mice and in H. Pylori infected gastric tissues compared to tissues from wild type animals. Growth rate of gastric cell lines over-expressing miR-449 was inhibited by 60% compared to controls. FACS cell cycle analysis of miR-449 over-expressing cells showed a significant increase in the sub-G1 fraction indicative of apoptosis. ß-Gal assays indicated a senescent phenotype of gastric cell lines over-expressing miR-449. Affymetrix 133v2 arrays identified GMNN, MET, CCNE2, SIRT1 and CDK6 as miR-449 targets. Luciferase assays were used to confirm GMNN, MET, CCNE2 and SIRT1 as direct targets. We also show that miR-449 over-expression activated p53 and its downstream target p21 as well as the apoptosis markers cleaved CASP3 and PARP. Importantly, qPCR analyses showed a loss of miR-449 expression in human clinical gastric tumours compared to normal tissues. Conclusions In this study, we document a diminished expression of miR-449 in Gastrin KO mice and further confirmed its loss in human gastric tumours. We investigated the function of miR-449 by identifying its direct targets. Furthermore we show that miR-449 induces senescence and apoptosis by activating the p53 pathway.

Abstract 3629 Introduction: A tumor suppressor has long been sought for in the commonly deleted region on chromosome 5q in myelodysplastic syndrome and acute myeloid leukaemia (MDS/AML). MicroRNAs (miRs) may have tumor suppressor function since they may repress the expression of proto-oncogenes through post-transcriptional regulation by binding to the 3′UTR of mRNA. It has recently been shown that several types of haematological malignancies have globally altered expression of miRs. DNA methylation cause targeted down regulation of individual tumor suppressor genes that may be directly involved in the pathogenesis of MDS/AML and we speculated whether epigenetic downregulation of miRs also plays a role in MDS/AML pathogenesis. Methods: Cell lines were treated with the hypomethylating agent 5-azacytidine. Upregulated miRs were identified by microarray (Exiqon) analysis and confirmed by RT qPCR analysis. Transcription start site was determined by 5′RACE and promoter methylation by methylation specific melting curve analysis. MiR expression and processing were analysed by qPCR, Northern blotting, and si-RNA mediated knock down of Drosha. Clinical data were collected from patient files. Results: We focused on one upregulated miR that locates to the commonly deleted region of AML/MDS. We found that the processing of this “miR” was independent of Drosha, and northern blotting showed that this was in fact a different type of non-coding RNA (ncRNA). The gene that encodes this ncRNA has a CpG island at the transcription start site. This CpG island is unmethylated in normal CD34+ cells, normal peripheral blood lymphocytes and methylated in three different myeloid cells lines (HL60, NB4, F-36P). Normal CD34+ cells and normal peripheral blood lymphocytes expressed the ncRNA, while no expression was seen in the cell lines. However, treatment by 5-azacytidine derepressed expression of this ncRNA. Bone marrow mononuclear cells from 101 high-risk MDS and AML patients, isolated at the time of diagnosis, were examined for methylation of the ncRNA promoter. Methylation was detected in 48% of the patients. Patients with no methylation have a significant better survival as compared to patients with methylation of the promotor. Discussion: This is the first example of targeted methylation of this type of RNA that has not previously been implicated in cancer. The preliminary results indicate that methylation of this ncRNA may be used as a prognostic marker for survival in high-risk MDS- and AML patients. We are currently investigating the function of this ncRNA by performing stable transfections into MDS/AML cell lines. Disclosures: Jones: Lilly: Consultancy; Millipore: Consultancy.

Diffuse large B-cell lymphoma (DLBCL) accounts for approximately 40% of non-Hodgkin lymphomas, with an overall 3-year relapse-free survival of 53%. DLBCL consists of a germinal center B-cell (GCB-like) subgroup and an activated B-cell (ABC-like) subgroup exist, associated with good and poor prognosis, respectively. In addition to differential expression of coding genes, ABC-type lymphoma cells express high levels of the non-coding microRNA, miR-155, an onco-miR whose ectopic expression in the mouse has been shown to give rise to B-cell malignancies. DLBCL feature a dysregulated PI3 kinase pathway, which can promote proliferation and survival. PI3K is regulated by phosphatases PTEN (phosphatase and tensin homolog deleted on chromosome ten) and SHIP (SH2 domain-containing inositol 5-phosphatase). PTEN is a known tumor suppressor and is mutated in human cancer with a frequency approaching that of p53. cDNA array analyses of DLBCL patient specimens indicate that low mRNA expression level of SHIP can be used as a negative prognostic marker for survival. We have now identified SHIP as the first bona-fide target of miR-155. RT-PCR analysis of micro-dissected lymphoma samples confirmed high levels of miR-155 and low levels of SHIP in poor-prognosis ABC-cells as compared to GCB-cells. We further demonstrate that elevated levels of miR-155, and consequent abrogation of SHIP expression, is not due to point mutations or promoter hypermethylation of SHIP in primary DLBCL cells, but rather the result of a TNFa-mediated autocrine loop, a pro-inflammatory cytokine whose serum levels are elevated in DLBCL patients. Consequently, anti-TNFa regimens (Etenercept (Enbrel®), Infliximab (Remicade®)) are sufficient to reduce miR-155 levels, restore expression of SHIP, decrease p-Akt levels, and slow tumor cell growth of ABC-sub-type DLBCL cell lines. Furthermore, we observe a highly significant decrease in tumor growth when DLBCL-xenograft mice are treated with Etenercept, suggesting a novel approach for restoring tumor suppressor activity and inducing growth inhibition of these aggressive tumors. Our findings offer the first direct evidence that specific miRs can function as a the link between inflammation and cancer. Finally, our hypothesis, that TNFa promotes survival of DLBCL via miR-155, SHIP and pAkt, opens up a novel and immediately accessible (co)treatment for poor prognosis DLBCL.

The gene encoding the lysine-specific histone methyltransferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma and diffuse large B cell lymphoma; however, the biological consequences of KMT2D mutations on lymphoma development are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and that its genetic ablation in B cells promotes lymphoma development in mice. KMT2D deficiency also delays germinal center involution and impedes B cell differentiation and class switch recombination. Integrative genomic analyses indicate that KMT2D affects methylation of lysine 4 on histone H3 (H3K4) and expression of a set of genes, including those in the CD40, JAK-STAT, Toll-like receptor and B cell receptor signaling pathways. Notably, other KMT2D target genes include frequently mutated tumor suppressor genes such as TNFAIP3, SOCS3 and TNFRSF14. Therefore, KMT2D mutations may promote malignant outgrowth by perturbing the expression of tumor suppressor genes that control B cell-activating pathways.

Introduction Disruption of miR34a, -b, and -c has been implicated in lymphomagenesis, and in CLL it has been suggested that miR34a expression is a surrogate marker for TP53 disruption associated with a poor prognosis. P53 is a transcription factor for the miR34s, and overexpression of miR34s in p53 deficient cells can reinstate p53 functions (Chang et al, Mol Cell 26, 2007, He et al, Nature 447, 2007). However, in cellular senescence miR34a may be activated independently of p53 (Christoffersen et al, Cell Death Differ17, 2010). A recent multicenter study shows that in diffuse large B-cell lymphoma (DLBCL), TP53 disruption is still a negative prognostic factor for survival after the implementation of Rituximab (Xu-Monette et al, Blood 19, 2012). Information on the role of the miR34s in normal CD19+ B-cells (PBL-B), activated B-cells, and de novo diffuse large B-cell lymphoma (DLBCL) is limited. Aims Given that MIR34A and MIR34B/C locate to regions of allelic loss in DLBCL (1p36.23 and 11q23.1, respectively), and the importance of the miR34 targets in DLBCL pathogenesis, we investigated a large panel of newly diagnosed cases of DLBCLs for MIR34A and MIR34B/C promoter methylation, TP53 mutational status, clinical presentation patterns, and outcome. Methods MIR34A/B/C promotor methylation was performed by MsMCA and bisulfite sequencing. Histone modifcations at the MIR34A/B/C promotor was examined by ChIP RT-qPCR. Expression of miR34s was performed using miRCURY LNA™ Universal RT-qPCR. The coding sequences and splice sites of exons 5-9 of the TP53gene were scanned for mutations by PCR and denaturing gradient gel electrophoresis (DGGE). Differences in clinical characteristics using the one-way Anova, the Pearson chi-square, or Fisher’s exact tests. Overall survival was estimated using the Kaplan-Meier method and log-rank test. For assessment of independent predictors of survival a multivariate Cox regression hazard model with backward stepwise (likelihood ratio) entry was applied. Effects not meeting a p-value < 0.05 were removed from the model. Results We show that only miR34a-5p is expressed in PBL-B, and significantly induced in activated B-cells (P=0.017) and reactive lymph nodes (P3 years, while all but one of those Rituximab treated patients with concommitant disruption of TP53 and MIR34A died within the first 13 months from diagnosis. In 2 DLBCL-cell lines with concomitant TP53 mutation and methylation of MIR34A,miR34a-5p was upregulated by 5-aza-2’deoxycytidine. Conclusion Studies of many different cancer cell types including the present study suggest that miR34s for the larger part are downregulated by promoter hypermethylation, and we show that miR34a-5p can be upregulated by a hypomethylating agent in DLBCL cells with a methylated MIR34A promoter in cells with and without TP53 mutations. Thus we believe, we have identified a novel rare, aggressive, but treatable “double-hit” DLBCL. Disclosures: No relevant conflicts of interest to declare.