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Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous cancer composed of at least 2 molecular subtypes that differ in gene expression and distribution of mutations. Recently, application of genome/exome sequencing and RNA-seq to DLBCL has revealed numerous genes that are recurrent targets of somatic point mutation in this disease. Here we provide a whole-genome-sequencing-based perspective of DLBCL mutational complexity by characterizing 40 de novo DLBCL cases and 13 DLBCL cell lines and combining these data with DNA copy number analysis and RNA-seq from an extended cohort of 96 cases. Our analysis identified widespread genomic rearrangements including evidence for chromothripsis as well as the presence of known and novel fusion transcripts. We uncovered new gene targets of recurrent somatic point mutations and genes that are targeted by focal somatic deletions in this disease. We highlight the recurrence of germinal center B-cell-restricted mutations affecting genes that encode the S1P receptor and 2 small GTPases (GNA13 and GNAI2) that together converge on regulation of B-cell homing. We further analyzed our data to approximate the relative temporal order in which some recurrent mutations were acquired and demonstrate that ongoing acquisition of mutations and intratumoral clonal heterogeneity are common features of DLBCL. This study further improves our understanding of the processes and pathways involved in lymphomagenesis, and some of the pathways mutated here may indicate new avenues for therapeutic intervention.

Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous cancer composed of at least 2 molecular subtypes that differ in gene expression and distribution of mutations. Recently, application of genome/exome sequencing and RNA-seq to DLBCL has revealed numerous genes that are recurrent targets of somatic point mutation in this disease. Here we provide a whole-genome-sequencing-based perspective of DLBCL mutational complexity by characterizing 40 de novo DLBCL cases and 13 DLBCL cell lines and combining these data with DNA copy number analysis and RNA-seq from an extended cohort of 96 cases. Our analysis identified widespread genomic rearrangements including evidence for chromothripsis as well as the presence of known and novel fusion transcripts. We uncovered new gene targets of recurrent somatic point mutations and genes that are targeted by focal somatic deletions in this disease. We highlight the recurrence of germinal center B-cell-restricted mutations affecting genes that encode the S1P receptor and 2 small GTPases (GNA13and GNAI2) that together converge on regulation of B-cell homing. We further analyzed our data to approximate the relative temporal order in which some recurrent mutations were acquired and demonstrate that ongoing acquisition of mutations and intratumoral clonal heterogeneity are common features of DLBCL. This study further improves our understanding of the processes and pathways involved in lymphomagenesis, and some of the pathways mutated here may indicate new avenues for therapeutic intervention.

Diffuse large B-cell lymphoma (DLBCL) accounts for 30% to 40% of newly diagnosed lymphomas and has an overall cure rate of approximately 60%. Previously, we observed FOXO1mutations in non-Hodgkin lymphoma patient samples. To explore the effects of FOXO1mutations, we assessed FOXO1status in 279 DLBCL patient samples and 22 DLBCL-derived cell lines. FOXO1mutations were found in 8.6% (24/279) of DLBCL cases: 92.3% (24/26) of mutations were in the first exon, 46.2% (12/26) were recurrent mutations affecting the N-terminal region, and another 38.5% (10/26) affected the Forkhead DNA binding domain. Recurrent mutations in the N-terminal region resulted in diminished T24 phosphorylation, loss of interaction with 14-3-3, and nuclear retention. FOXO1mutation was associated with decreased overall survival in patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (P= .037), independent of cell of origin (COO) and the revised International Prognostic Index (R-IPI). This association was particularly evident (P= .003) in patients in the low-risk R-IPI categories. The independent relationship of mutations in FOXO1to survival, transcending the prognostic influence of the R-IPI and COO, indicates that FOXO1mutation is a novel prognostic factor that plays an important role in DLBCL pathogenesis.

Diffuse large B-cell lymphoma (DLBCL) accounts for 30% to 40% of newly diagnosed lymphomas and has an overall cure rate of approximately 60%. Previously, we observed FOXO1 mutations in non-Hodgkin lymphoma patient samples. To explore the effects of FOXO1 mutations, we assessed FOXO1 status in 279 DLBCL patient samples and 22 DLBCL-derived cell lines. FOXO1 mutations were found in 8.6% (24/279) of DLBCL cases: 92.3% (24/26) of mutations were in the first exon, 46.2% (12/26) were recurrent mutations affecting the N-terminal region, and another 38.5% (10/26) affected the Forkhead DNA binding domain. Recurrent mutations in the N-terminal region resulted in diminished T24 phosphorylation, loss of interaction with 14-3-3, and nuclear retention. FOXO1 mutation was associated with decreased overall survival in patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (P = .037), independent of cell of origin (COO) and the revised International Prognostic Index (R-IPI). This association was particularly evident (P = .003) in patients in the low-risk R-IPI categories. The independent relationship of mutations in FOXO1 to survival, transcending the prognostic influence of the R-IPI and COO, indicates that FOXO1 mutation is a novel prognostic factor that plays an important role in DLBCL pathogenesis.

Pulmonary fibrosis is a progressive disease with unknown etiology that is characterized by extensive remodeling of the lung parenchyma, ultimately resulting in respiratory failure. Lymphatic vessels have been implicated with the development of pulmonary fibrosis, but the role of the lymphatic vasculature in the pathogenesis of pulmonary fibrosis remains enigmatic. Here we show in a murine model of pulmonary fibrosis that lymphatic vessels exhibit ectopic mural coverage and that this occurs early during the disease. The abnormal lymphatic vascular patterning in fibrotic lungs was driven by expression of platelet-derived growth factor B (PDGF-B) in lymphatic endothelial cells and signaling through platelet-derived growth factor receptor (PDGFR)–β in associated mural cells. Because of impaired lymphatic drainage, aberrant mural cell coverage fostered the accumulation of fibrogenic molecules and the attraction of fibroblasts to the perilymphatic space. Pharmacologic inhibition of the PDGF-B/PDGFR-β signaling axis disrupted the association of mural cells and lymphatic vessels, improved lymphatic drainage of the lung, and prevented the attraction of fibroblasts to the perilymphatic space. Our results implicate aberrant mural cell recruitment to lymphatic vessels in the pathogenesis of pulmonary fibrosis and that the drainage capacity of pulmonary lymphatics is a critical mediator of fibroproliferative changes.

Pulmonary fibrosis is a progressive disease with unknown etiology that is characterized by extensive remodeling of the lung parenchyma, ultimately resulting in respiratory failure. Lymphatic vessels have been implicated with the development of pulmonary fibrosis, but the role of the lymphatic vasculature in the pathogenesis of pulmonary fibrosis remains enigmatic. Here we show in a murine model of pulmonary fibrosis that lymphatic vessels exhibit ectopic mural coverage and that this occurs early during the disease. The abnormal lymphatic vascular patterning in fibrotic lungs was driven by expression of platelet-derived growth factor B (PDGF-B) in lymphatic endothelial cells and signaling through platelet-derived growth factor receptor (PDGFR)–β in associated mural cells. Because of impaired lymphatic drainage, aberrant mural cell coverage fostered the accumulation of fibrogenic molecules and the attraction of fibroblasts to the perilymphatic space. Pharmacologic inhibition of the PDGF-B/PDGFR-β signaling axis disrupted the association of mural cells and lymphatic vessels, improved lymphatic drainage of the lung, and prevented the attraction of fibroblasts to the perilymphatic space. Our results implicate aberrant mural cell recruitment to lymphatic vessels in the pathogenesis of pulmonary fibrosis and that the drainage capacity of pulmonary lymphatics is a critical mediator of fibroproliferative changes.

Discovery of novel non cytotoxic drugs for cancer focuses on targets selectively expressed in malignant cells, only testing at the end if they are toxic to patients. We have developed a novel approach to discover these drugs starting at the end; we screen 2.000 approved drugs with proven safety, directly on freshly extracted (ex vivo) blood samples of patients with Chronic Lymphocytic Leukemia (CLL). These screens are enabled by a novel technology platform based on automated flow cytometry we call ExviTech for ex vivo technology.All screening studies were performed directly on either peripheral blood or bone marrow samples from 44 patients diagnosed with various subtypes of B-cell malignancies, after informed consent. Patient samples were diluted and plated with each of the 2.000 drugs individually, retaining the erythrocyte population and serum proteins to enable clinically relevant concentrations. The experimental assay was setup the same or a day after sample extraction. Each sample was diluted to achieve a leukemic cell concentration of approximately 3,000 cells/μl; then 45μl of the suspension is added to each well of 96-well plates that contain the pharmacological agents (final concentration of 30μM). The compound plates were then sequentially incubated for 24 hours at 37°C with 5% CO2 for screening (sterile conditions). After incubation, the erythrocytes were lysed and the leucocytes incubated with Annexin V-FITC, anti-CD45-APC and anti-CD19-PE added to each well. The plates were then transferred to an automated flow cytometry system where the contents of each well were aspirated and analyzed by a CyAn flow cytometer. Candidates from the primary screens were validated in additional samples with dose-responses, combinations with approved drugs, multiple incubation times, etc…Analyzing primary screens from 24 CLL patients, three related compounds (Vivia007, Vivia008 and Vivia009) were found to consistently induce apoptosis of nearly all leukemic B-cells from most of the patient samples diagnosed with B-cell chronic lymphocytic leukemia at levels equal to or greater than known CLL active cytotoxic agents. Notably, these candidates are equally effective against samples of p53 mutated patients. These 3 drugs are pharmacologically me-too drugs sharing the same target and mechanism of action, and are non cytotoxic drugs with a known and good safety profile, administered to millions of patients over many years. Validation experiments were done on 20 additional CLL patients and Vivia009 emerged as the most effective agent with an average EC50 of 18.2μM. The mechanism of action is different than the known mechanism of Vivia009 and its class members for their approved indications. Consistent with this observation, only 3 of 15 members of the same pharmacological drug class were efficacious against CLL malignant cells. All 3 Vivia′s candidates were equally efficacious against other B-Cell Malignancies such as B-ALL (pediatric and adult), and Multiple Myeloma. These drugs are not effective in their current oral formulation and require a novel intravenous formulation. Interestingly, kinetics of induction of apoptosis were faster for Vivia009 than for fludarabine, cyclophosphamide and mitoxantrone. Vivia009 requires only 1 hour of incubation with fresh cells to induce maximal apoptosis. This timeline is less than the 3 hours in which Vivia009 was found present at high concentrations in bone marrow of rats using a single intravenous bolus. Thus, Vivia009 seems to fulfill the pharmacokinetic criteria to eliminate all leukemic cells with a single intravenous bolus, which would be a major advantage over current treatments (5-days fludarabine or 3 days FCR). Animal models are ongoing to confirm the non cytotoxic nature of the candidates in the novel IV formulation and the fewer days needed to reach remission, both compared with fludarabine monotherapy.In summary, our results demonstrate the potential of the ExviTech technology platform as a successful model for the systematic search of new uses for already existing approved drugs directly on patient samples of hematological malignancies. A new drug candidate with excellent safety profile has been identified with similar efficacy ex vivo as the best approved cytotoxic drugs, which is a non-cytotoxic drug with fast kinetics that might enable significantly safer and shorter treatments.Bennett: Vivia Biotech: Employment. Sapia:Vivia Biotech SL: Employment. Primo:Vivia Biotech SL: Employment. Suarez:Vivia Biotech SL: Employment. Lago:Vivia Biotech SL: Employment. Matoses:Vivia Biotech: Employment. Espinosa:Vivia Biotech: Ana Espinosa, Employment. Tudela:Vivia Biotech SL: Employment. Arroyo:Vivia Biotech SL: Employment. Jackson:Vivia Biotech SL: Employment. Okun:Vivia Biotech SL: Research Funding. Lopez:Vivia Biotech SL: Employment. Gornemann:Vivia Biotech SL: Employment. Diez:Vivia Biotech SL: Employment. González:Vivia Biotech SL: Consultancy. Dominguez-Gil:Vivia Biotech SL: Consultancy. Troconiz:Vivia Biotech SL: Consultancy. Rodriguez de Fonseca:Vivia Biotech SL: Consultancy. Saunders:Vivia Biotech: Consultancy. Montejo:Vivia Biotech SL: Consultancy. Caveda:Vivia Biotech SL: Employment. Orfao:Vivia Biotech SL: Research Funding. Ballesteros:Vivia Biotech SL: Equity Ownership.

The predictive power of measuring the effect of anticancer treatments on whole living tumor cells freshly removed from cancer patients, called Individualized Tumor Response Testing (ITRT), has been recently further validated in a clinical trial, the UK's LRF CLL4 trial (Bosanquet ASH 2007). It predicts resistance better than sensitivity. We present a novel approach to ITRT based on measuring drug induced apoptosis of tumor cells in whole blood ex vivo (in vitro using freshly extracted samples). It uses a novel automated flow cytometry platform (ExviTech) capable of evaluating hundreds of drugs and drug combinations used in current treatment protocols, and can address the significant scaling of potential future protocols induced by a number of new drug approvals in each indication.We evaluated 47 samples of peripheral blood or bone marrow from patients diagnosed with hematological malignancies: 20 chronic Lymphocytic Leukemia (CLL), 14 Acute Lymphoblastic Leukemia (ALL), 7 Multiple Myeloma (MM), and 6 Acute Myeloblastic Leukemia (AML). After informed consent, samples, collected into heparin, were processed the same or the next day. Whole blood was diluted and incubated with drugs for 24 and 48 hours. Whole blood was used to retain erythrocytes and serum proteins enabling more clinically relevant physiological conditions. Three types of drugs were tested: 1) Approved drugs for each indication, including all possible pair wise combinations, and combinations administered within current and experimental protocols as advised by the PETHEMA groups in Spain. 2) Concomitant medicines (Con-Meds), including alternative drugs within the same class of antacids, antiemetics, etc… to test whether they may also induce apoptosis 3) Drugs in clinical trials, preferentially Phase III drugs, alone and in combination with approved drugs, which may form the basis of future treatment protocols. Drugs were plated at a final concentration equivalent to their reported plasma Cmax concentration. Synergistic drug combinations were identified as one drug potentiating the effect of the other.The efficacy of each drug and combination tested was categorized as highly resistant, intermediate or highly sensitive. Highly resistant drug results were contraindicated. Among the highly sensitive treatments ex vivo, often those that effectively killed all malignant cells, we selected those whose drugs were significantly less toxic as treatment guidelines, highlighting those treatment protocols that act faster ex vivo (24 vs 48 hours) and/or show synergistic combinations. The final result was a set of multiple reasonable ex vivo options for hematologists. The efficacy of individual drugs varied notably from patient to patient, , as reported earlier by other methods. Drug-drug combinations show surprising results. Some combinations, effective at high doses, kill 80% of malignant cells when combined in low concentrations at which the individual drugs kill only 10%20% of these cells. On the contrary, many drug combinations were antagonistic, effectively turning them into cytoprotectors and the patient into potential resistance. Specific combinations that show consistent efficacy across samples are indicative of potential new protocols. Surprisingly, for a proportion of patients, some of the Con-Meds were highly efficient in killing malignant cells selectively. For example, in a particular CLL patient an antacid and an antiviral drug had similar efficacies as the best approved cytotoxic drugs. In other patients, drugs still in clinical trials showed high sensitivity and highly selective apoptosis – suggesting that those patients could be referred for inclusion into these trials, which could represent new alternatives especially for refractory patients with few therapeutic options available.We have developed a Personalized Medicine Multi-Drug ex vivo test, evaluating the efficacy of hundreds of drugs and drug combinations in whole blood. This scale could address the predictable expansion of multi-drug potential treatments as the existing extensive drug pipeline delivers new drug approvals, exploring hundreds of new protocols ex vivo. Promising results obtained ex vivo (in vitro using freshly extracted samples) need to be verified in clinical trials.Bennett: Vivia Biotech: Employment. Sapia:Vivia Biotech SL: Employment. Primo:Vivia Biotech SL: Employment. Suarez:Vivia Biotech SL: Employment. Lago:Vivia Biotech SL: Employment. Matoses:Vivia Biotech SL: Employment. Espinosa:Vivia Biotech SL: Employment. Tudela:Vivia Biotech SL: Employment. Arroyo:Vivia Biotech SL: Employment. Gorrochategui:Vivia Biotech SL: Employment. Jackson:Vivia Biotech SL: Employment. Okun:Vivia Biotech SL: Research Funding. Lopez:Vivia Biotech SL: Employment. Gornemann:Vivia Biotech SL: Employment. Diez:Vivia Biotech SL: Employment. Gonzáalez:Vivia Biotech SL: Consultancy. Bosanquet:Vivia Biotech SL: Consultancy. Orfao:Vivia Biotech SL: Research Funding. Ballesteros:Vivia Biotech SL: Equity Ownership.

Delforge: Amgen, Celgene, Janssen and Takeda: Consultancy; Celgene and Janssen: Research Funding.

INTRODUCTION:Major progresses have been achieved to identify older patients with malignant hemopathies who should be treated with standard doses of chemotherapy. However, a reliable frailty score remains urgently needed to better define the unsuspected vulnerable population that does not benefit from chemotherapy. In the literature, three clinical (functional decline, cognitive impairment (CI) and comorbidities) and two biological (low albumin level and high IL-6 level) factors are frequently associated with a poor overall survival (OS) and/or chemotherapy-related toxicities.

Díez Campelo: Novartis: Research Funding, Speakers Bureau; Janssen: Research Funding; Celgene: Research Funding, Speakers Bureau. Off Label Use: Use of erythropoietic stimulating agents for anemia in patients with myelodysplastic syndromes. Ramos:JANSSEN: Honoraria, Membership on an entity's Board of Directors or advisory committees; AMGEN: Consultancy, Honoraria; NOVARTIS: Consultancy, Honoraria; CELGENE: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Falantes:Celgene: Honoraria. Garcia:Celgene: Research Funding. Sanz:JANSSEN CILAG: Honoraria, Research Funding, Speakers Bureau.

INTRODUCTION

Older patients are increasingly prevalent in oncological practice. However, the evidence suggests that this group of patients is undertreated, mainly because of their advanced age, regardless of whether they are highly functional patients, do not present comorbidities, or could benefit from oncological therapies. The US National Comprehensive Cancer Network and the International Society of Geriatric Oncology have recommended that some form of geriatric assessment should be conducted to help Hematologists and Oncologists in order to identify current health problems and to guide interventions to reduce adverse outcomes and optimize the functional statusCurrently, the main tool for assessing older patients is a comprehensive geriatric assessment, although its complexity and duration may hinder its regular use in daily practice as a tool for clinical decision making. Several attempts have been made to assess comorbidities in the specific field of mielodysplasia, but mainly focused on organic damage rather than global assessment.We are in the process of developing and validating a comprehensive health status assessment scale (Geriatric Assessment in Hematology, GAH Scale) with eight dimensions in patients ≥ 65 years with: Myelodysplastic syndromes (MDS), acute myeloblastic leukemia (AML) and multiple myeloma (MM).After item-pool generation, stakeholder consultation and content validation, a brief scale of 8 dimensions with selected items has been created.Feasibility was confirmed in 83 patients. Afterwards, a multicenter, observational, prospective study has been carried out in 20 hospitals in Spain, enrolling 189 elderly naïve to treatment patients with newly diagnosed MDS, AML or MM.The scale validation process integrates the analysis of criterion and concept validity, internal consistency (Cronbach's alpha), test-retest reliability, as well as the evaluation of intraclass correlation coefficient (ICC) and factor analysis. After psychometric validation phase, further studies will be carried out in order to evaluate its clinical use for prognosis and clinical decision making.189 patients fulfilling inclusion criteria have been enrolled in the study, 54% women. Median age at diagnosis was 73.3 ± 6.64 years. According to diagnosis, 103 patients (54.5%) had MDS or AML and 86 (45.5%) had MM.Regarding feasibility, mean time for filling in the questionnaire was 12.1 ± 4.5 min. 83.6% of patients answered 100% of questions of the scale. Mean percentage of unanswered questions per patient was 1%.Test-retest was completed by 112 patients. GAH Scale showed satisfactory test-retest reliability. ICC was statistically significant for each dimension, being greater than 0.65 for 6 of the 8 dimensions (p<0.05), indicating that GAH Scale is independent of the observer and is stable in clinically stable patients along the time. Floor and ceiling effects were no detected.Internal consistency, content validity and factor analysis are being carried out and results will be presented in the forthcoming congress.This new GAH Scale is a valid, reliable and consistent tool, simple enough to assess health status in older patients with haematological malignancies. Further studies will have to stablish if it may be a tool to improve decision making in such patients.Bonanad: Celgene: Consultancy. Gonzalez:Celgene: Consultancy. Durán:Celgene: Employment. Marcos:Celgene: Employment. López:Celgene: Employment. Cruz-Jentoft:Celgene: Research Funding.

: Follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL) are the most common types of B-cell derived non-Hodgkin lymphomas (NHL). A significant proportion of patients with FL develop transformed disease reminiscent of DLBCL, which arises from the cells of FL. A common feature of FL and a subset of DLBCL is the presence of a balanced cytogenetic translocation t(14;18)(q32;q21). In addition to chromosome rearrangements, inappropriate somatic hypermutation of proto-oncogenes, including MYC, PIM1, ARHH and PAX5, have been proposed to contribute to DLBCL, but not to FL. Evidence for other mutations contributing to the development of DLBCL and FL is lacking. Our research group had previously detected a residue of the SET domain of the methyltransferase EZH2 (Y641) that is frequently mutated both in FL and the GCB subtype of DLBCL (Morin, R et al. 2010 Nature Genetics 42 (2):181-5). It has also been recently reported that specific gene expression signatures can reveal unique diagnostic and prognostic information about FL and DLBCL. The presence of mutations in genes that contribute to chromatin modification (from transcriptionally active to silent, and vice versa) can have an effect on the regulation of gene expression, playing an important role in cancer.We have deep-sequenced the whole transcriptome of DLBCL samples using Illumina second-generation sequencing to detect candidate mutations in different genes that may contribute to lymphoma development and progression. Based on our observations of these data, we subsequently PCR amplified and sequenced the entire MLL2 locus from genomic DNA isolated from FL and DLBCL tumor samples, and from matched germline DNA where available. Using an alternative approach, consisting of a targeted hybridization capture of MEF2B exons from genomic DNA, we re-sequenced the exons of MEF2B in an extended cohort of FL and DLBCL samples.Among many other mutated genes we have characterized the mutations found in two chromatin modifying genes, MLL2 and MEF2B. MLL2, a gene that encodes a histone methyltransferase, is somatically mutated in 89% of FL (n=35) and 32% of DLBCL (n=37) samples. The majority of these mutations were truncations and frame-shift insertions and deletions, likely inactivating MLL2. MEF2B, which encodes a MADS/MEF2 DNA binding protein involved in the regulation of gene expression, was found mutated in 12% of FL (n=274) and 9% of DLBCL (n=321) tumor samples. In contrast with the mutational pattern of MLL2, we did not detect non-sense mutations in MEF2B. All mutations in MEF2B affected a small number of residues. Several studies in MEF2 family members have shown the importance of some of these mutated residues for the correct function of the protein.After high-throughput sequencing of transcriptomes in a cohort of FL and DLBCL lymphoma samples, two targeted second generation re-sequencing approaches have enabled the screening of individual genes in a large cohort of samples. We chose two of the genes with strong evidence for recurrent somatic mutations and no previously known role in lymphoma, MLL2 and MEF2B, for detailed characterization. The high incidence of mutations in both of these genes suggests that these mutations might act as driver mutations of interest for further study.No relevant conflicts of interest to declare.

Abstract 632

Abstract 473

Follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL) are the two most common types of non Hodgkin lymphoma (NHL). It is widely accepted that DLBCL can be divided into two major subtypes using gene expression profiling: germinal center B-cell (GCB) and activated B-cell (ABC). Both FL and the GCB subtype of DLBCL derive from germinal center B cells and have been found to share some common mutational events such as translocations leading to the deregulation of the BCL2 oncogene and mutations affecting a single tyrosine (Y641) in the histone methyltransferase EZH2. In contrast, ABC DLBCL tumors are characterized by mutations leading to the constitutive activity of NFkB. The clear differences in treatment response between subtypes allow this distinction to be used as a prognostic indicator and may ultimately lead to therapies that target individual features of each subtype. However, besides the gene expression and mutational signatures that differentiate the DLBCL subtypes, there is a paucity of molecular prognostic markers in these NHLs. Further, there is limited knowledge about the genetic events that drive the GCB subtype of DLBCL, which, if better understood, may enable the design of targeted therapeutics.To identify mutations driving lymphomagenesis and in particular, aggressive cases of NHL, we applied Illumina second-generation sequencing technology to the analysis of tumor genomes and constitutional DNAs from a FL and a DLBCL tumor and the exomes from two additional DLBCLs. In these “omes”, we identified somatic protein-altering point mutations in more than 250 genes including genes known to be involved in cancer, for example TP53, FAS and TNFAIP3 (A20). Many of these mutations may represent passenger rather than driver mutations, the latter of which are involved in disease progression. To identify the likely driver mutations, we sought to identify the genes that are recurrent targets of somatic mutation in these cancers. To this end, we further analyzed the transcriptome sequences we generated using RNA-seq from 95 primary DLBCLs,13 FL cases and 10 DLBCL-derived cell lines.105 of the genes found mutated in the FL and DLBCL genomes were observed to be recurrent targets of somatic mutation in these diseases. Some of these were known targets of aberrant somatic hypermutation (SHM) including BCL2, PIM1, and IRF4 and others have been previously identified as targets of recurrent mutation in lymphoma, such as EZH2, CD79B and CARD11. One of the most frequently mutated genes was MLL2, a histone methyltransferase never before implicated in lymphomagenesis. MLL2 showed a pattern of mutation characteristic of a dosage-sensitive tumor suppressor gene. Another frequently mutated gene was MEF2B, a calcium-regulated transcriptional co-activator/repressor that cooperates with histone modifying enzymes to epigenetically regulate the expression of genes. We found that mutations affecting MEF2B occur in 11.7% of FL and 9% of DLBCL, with the majority (73%) of these mutations affecting three amino acids (K4, Y69, and D83). Analysis of these 105 recurrently mutated genes for prognostic signatures is ongoing.High-throughput sequencing platforms have enabled the identification of recurrent targets of somatic mutations never suspected to be involved in lymphoma. Some of these mutated genes may have prognostic value while others may represent targets for the rational design of novel therapeutics.No relevant conflicts of interest to declare.

Non-Hodgkin lymphomas (NHL) are the most common type of lymphoma and can be broadly classified as indolent (slow-growing) diseases, progressing over many years; and aggressive (fast-growing) diseases, which progress rapidly. The latter class includes diffuse large B-cell lymphoma (DLBCL), which accounts for approximately 30% of all NHL diagnoses. Three DLBCL subtypes have been identified based on gene expression profiling, namely: germinal center B-cell (GCB), activated B-cell (ABC) and primary mediastinal B-cell lymphoma (PMBCL). These subtypes show substantial differences in response to treatment and ultimate disease outcome, suggesting that molecular subtyping is an important prognostic indicator and that each subtype may benefit from a distinct treatment regimen. Despite recent advances in cancer genomics revealing molecular and mutational differences between these subtypes, further studies focused on the common NHL subtypes are required to identify critical players in the pathogenesis of DLBCL that may be targeted by pharmacological intervention to improve patient outcome.Using ultra-high throughput whole genome shotgun sequencing (WGSS) and whole transcriptome shotgun sequencing (WTSS/RNA-seq) we have discovered protein-coding mutations in NHL genomes. With a focus on recurrent and likely gain-of-function mutations we have established procedures to model the three-dimensional structures of mutant proteins and using a computational “molecular docking” pipeline have identified candidate molecules with specificity for the mutant protein. These small molecule compounds are acquired and tested in cell proliferation assays against a suite of DLBCL cell lines characterized for target mutations.Mutations affecting a single key tyrosine in the catalytic site of enhancer of zeste, homolog 2 (EZH2), a member of the Polycomb-group family involved in transcriptional repression were identified (Morin, R. et al. 2010 Nature Genetics 42(2):181-5). This mutation, in a gene previously unknown to be mutated in cancer, is restricted to the GCB subtype of lymphomas and is highly prevalent in patient samples and DLBCL cell lines. Mutations have also been observed in other proteins involved in epigenetic regulation and thus afford potentially novel therapeutic targets. In proof-of-principle experiments small molecule inhibitors were identified using molecular docking approaches to target the effect of EZH2 mutations in both mutant and wild-type DLBCL cell lines. We identified and imported 96 compounds from the Developmental Therapeutic Program NCI/NIH repository. These compounds were tested in alamarBlue cell proliferation assays revealing three with activity at 10uM concentration in EZH2 mutant but not wild-type cells. Computational optimization of these compounds is underway to identify related compounds with improved activities at reduced concentrations.High-throughput sequencing platforms have enabled the identification of recurrent, non-synonymous protein mutations in tumor genomes and transcriptomes. Such a catalogue of mutations provides new avenues of exploration for targeted therapy including small molecule inhibitors. Despite intensive efforts launched in recent years to determine the crystal structure for every human protein, many (including EZH2) do not currently have three dimensional structures. This poses a challenge to novel drug discovery but can be overcome using homology modeling and/or targeting other members of a pathway. Our observations also demonstrate the importance of epigenetic regulation in NHL tumorigenesis and thus provide potential new therapeutic targets.No relevant conflicts of interest to declare.

Abstract 804

Abstract 4414

Abstract 2655

Anemia is the main symptom in patients (pts) with myelodysplastic syndromes (MDS) which usually makes subjects with this pathology dependent on transfusions, resulting in a very limited quality of life. Darbepoetin alfa (DA) is a highly glycosylated erythropoiesis-stimulating agent (ESA) with a pharmacokinetic profile that includes a longer serum half-life and allows it to be administered less frequently than rHuEPO, thereby increasing patient convenience. This single-arm, open-label, multi-center, phase 2 study evaluated the efficacy and safety of DA in pts with low risk MDS. Eligible pts had low or intermediate-1 risk MDS [International Prognostic Scoring System (IPSS) criteria], anemia [Hemoglobin (Hb) 10 g/dL], FAB (French–American–British) classification of refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS), or refractory anemia with excess blasts (RAEB) (with marrow blasts ≤ 10%), serum erythropoietin levels < 500 IU/L and transfusion requirements < 2 packed red blood cells cells/month over the preceding 2 months. Pts were evaluated for erythroid response after 8 weeks (wks) and 16 wks of treatment. Major erythroid response (MaR) was defined as an increase in Hb values ≥2 g/dl on baseline values and/or suppression of transfusion requirements. A minor erythroid response (MiR) was defined as a > 1 g/dL increase in Hb values but < 2 g/dl on baseline values or a reduction in transfusion requirements of at least 50%. DA treatment was initiated at a fixed dose of 300 mcg weekly (QW). For pts achieving a MaR at wk 8, DA could be administered at the same dose every two wks (Q2W). For pts who did not achieve a MaR by 8 weeks, filgrastim 300 mcg QW was added for an additional 8 wks. If at any time during the treatment period Hb values of > 12g/dL were obtained, DA was discontinued until Hb falls to values of < 11 g/dL. At this time, DA treatment was reintroduced with half of the previous dose (150 mcg). A number of 44 evaluable pts were included in the study. Median age 75, 8 years (range 41, 3–92, 4), 53, 5% male and 90,7% with ECOG Performance Status (0–1). FAB diagnoses were 30,2% RA, 62,8% RARS and 7% RAEB; divided into IPSS low (76,7%) and intermediate-1 (23,3%). Baseline mean (SD) Hb was 9,2 g/dL (0.8) and serum erythropoietin 122,1 IU/L (124,7).72,7% of the pts were transfusion independent. An erythroid response was achieved by 31 of 44 pts (70,5%) at wk 8 (43,2% MaR, 27,3% MiR, 29,5% no response) and by 30 of 43 pts (69,8%) at week 16 (51,2% MaR, 18,6% MiR, 30,2% no response). A total of 19 pts received filgrastim between wk 8 and 16. Erythroid response at wks 8 and 16 depending on the transfusion dependence were 68,8% (59,1% MaR and 40,9% MiR) and 71.0% (68,2% MaR and 31,8% MiR) in the transfusion independent group and 75.0% (66,7% MaR and 33,7% MiR) and 66,70% (87,5% MaR and 12,5% MiR) in the transfusion dependent group. Baseline serum erythropoietin levels were the only predictive factor of response found in the study population. DA was generally well tolerated. Three mild adverse events were considered related to DA (iron deficiency, flu syndrome and headache), one severe (iron deficiency) and one fatal (thromboembolic event). Results from this study indicate that a fixed dose of 300 mcg DA QW (+/− filgrastim) is an effective and well-tolerated treatment of anemic patients with low- and intermediate-1 risk MDS.

Anemia is the main symptom in patients (pts) with myelodysplastic syndromes (MDS) which usually makes subjects with this pathology dependent on transfusions, resulting in a very limited quality of life. Darbepoetin alfa (DA) is a highly glycosylated erythropoiesis-stimulating agent (ESA) with a pharmacokinetic profile that includes a longer serum half-life and allows it to be administered less frequently than rHuEPO, thereby increasing patient convenience. This single-arm, open-label, multi-center, phase 2 study evaluated the efficacy and safety of DA in pts with low risk MDS. Eligible pts had low or intermediate-1 risk MDS [International Prognostic Scoring System (IPSS) criteria], anemia [Hemoglobin (Hb) 10 g/dL], FAB (French–American–British) classification of refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS), or refractory anemia with excess blasts (RAEB) (with marrow blasts ≤ 10%), serum erythropoietin levels < 500 IU/L and transfusion requirements < 2 packed red blood cells cells/month over the preceding 2 months. Pts were evaluated for erythroid response after 8 weeks (wks) and 16 wks of treatment. Major erythroid response (MaR) was defined as an increase in Hb values ≥2 g/dl on baseline values and/or suppression of transfusion requirements. A minor erythroid response (MiR) was defined as a > 1 g/dL increase in Hb values but < 2 g/dl on baseline values or a reduction in transfusion requirements of at least 50%. DA treatment was initiated at a fixed dose of 300 mcg weekly (QW). For pts achieving a MaR at wk 8, DA could be administered at the same dose every two wks (Q2W). For pts who did not achieve a MaR by 8 weeks, filgrastim 300 mcg QW was added for an additional 8 wks. If at any time during the treatment period Hb values of > 12g/dL were obtained, DA was discontinued until Hb falls to values of < 11 g/dL. At this time, DA treatment was reintroduced with half of the previous dose (150 mcg). A number of 44 evaluable pts were included in the study. Median age 75, 8 years (range 41, 3–92, 4), 53, 5% male and 90,7% with ECOG Performance Status (0–1). FAB diagnoses were 30,2% RA, 62,8% RARS and 7% RAEB; divided into IPSS low (76,7%) and intermediate-1 (23,3%). Baseline mean (SD) Hb was 9,2 g/dL (0.8) and serum erythropoietin 122,1 IU/L (124,7).72,7% of the pts were transfusion independent. An erythroid response was achieved by 31 of 44 pts (70,5%) at wk 8 (43,2% MaR, 27,3% MiR, 29,5% no response) and by 30 of 43 pts (69,8%) at week 16 (51,2% MaR, 18,6% MiR, 30,2% no response). A total of 19 pts received filgrastim between wk 8 and 16. Erythroid response at wks 8 and 16 depending on the transfusion dependence were 68,8% (59,1% MaR and 40,9% MiR) and 71.0% (68,2% MaR and 31,8% MiR) in the transfusion independent group and 75.0% (66,7% MaR and 33,7% MiR) and 66,70% (87,5% MaR and 12,5% MiR) in the transfusion dependent group. Baseline serum erythropoietin levels were the only predictive factor of response found in the study population. DA was generally well tolerated. Three mild adverse events were considered related to DA (iron deficiency, flu syndrome and headache), one severe (iron deficiency) and one fatal (thromboembolic event). Results from this study indicate that a fixed dose of 300 mcg DA QW (+/− filgrastim) is an effective and well-tolerated treatment of anemic patients with low- and intermediate-1 risk MDS.