ALBERT

Description: Background: Homopolymeric tracts, particularly poly dA.dT, are enriched within the intergenic sequences of eukaryotic genomes where they appear to act as intrinsic regulators of nucleosome positioning. A previous study of the incomplete genome of the human malarial parasite Plasmodium falciparum reports a higher than expected enrichment of poly dA.dT tracts, far above that anticipated even in this highly AT rich genome. Here we report an analysis of the relative frequency, length and spatial arrangement of homopolymer tracts for the complete P. falciparum genome, extending this analysis to twelve additional genomes of Apicomplexan parasites important to human and animal health. In addition, using nucleosome-positioning data available for P. falciparum, we explore the correlation of poly dA.dT tracts with nucleosome-positioning data over key expression landmarks within intergenic regions. Results: We describe three apparent lineage-specific patterns of homopolymeric tract organization within the intergenic regions of these Apicomplexan parasites. Moreover, a striking pattern of enrichment of overly long poly dA.dT tracts in the intergenic regions of Plasmodium spp. uniquely extends into protein coding sequences. There is a conserved spatial arrangement of poly dA.dT immediately flanking open reading frames and over predicted core promoter sites. These key landmarks are all relatively depleted in nucleosomes in P. falciparum, as would be expected for poly dA.dT acting as nucleosome exclusion sequences. Conclusions: Previous comparative studies of homopolymer tract organization emphasize evolutionary diversity; this is the first report of such an analysis within a single phylum. Our data provide insights into the evolution of homopolymeric tracts and the selective pressures at play in their maintenance and expansion.

Description: Background: In eukaryotic organisms, packaging of DNA into nucleosomes controls gene expression by regulating access of the promoter to transcription factors. The human malaria parasite Plasmodium falciparum encodes relatively few transcription factors, while extensive nucleosome remodeling occurs during its replicative cycle in red blood cells. These observations point towards an important role of the nucleosome landscape in regulating gene expression. However, the relation between nucleosome positioning and transcriptional activity has thus far not been explored in detail in the parasite. Results: Here, we analyzed nucleosome positioning in the asexual and sexual stages of the parasite's erythrocytic cycle using chromatin immunoprecipitation of MNase-digested chromatin, followed by next-generation sequencing. We observed a relatively open chromatin structure at the trophozoite and gametocyte stages, consistent with high levels of transcriptional activity in these stages. Nucleosome occupancy of genes and promoter regions were subsequently compared to steady-state mRNA expression levels. Transcript abundance showed a strong inverse correlation with nucleosome occupancy levels in promoter regions. In addition, AT-repeat sequences were strongly unfavorable for nucleosome binding in P. falciparum, and were overrepresented in promoters of highly expressed genes. Conclusions: The connection between chromatin structure and gene expression in P. falciparum shares similarities with other eukaryotes. However, the remarkable nucleosome dynamics during the erythrocytic stages and the absence of a large variety of transcription factors may indicate that nucleosome binding and remodeling are critical regulators of transcript levels. Moreover, the strong dependency between chromatin structure and DNA sequence suggests that the P. falciparum genome may have been shaped by nucleosome binding preferences. Nucleosome remodeling mechanisms in this deadly parasite could thus provide potent novel anti-malarial targets.

Description: A tight control of hematopoietic stem cell (HSC) quiescence, self-renewal and differentiation is crucial for lifelong blood production. The mechanisms behind this control are still poorly understood. Here we show that mitochondrial activity determines HSC fate decisions. A low mitochondrial membrane potential (Δψm) predicts long-term multi-lineage blood reconstitution capability, as we show for freshly isolated and in vitro-cultured HSCs. However, as in vivo both quiescent and cycling HSCs have comparable Δψm distributions, a low Δψm is not per se related to quiescence but is also found in dividing cells. Indeed, using divisional tracking, we demonstrate that daughter HSCs with a low Δψm maintain stemness, whereas daughter cells with high Δψm have undergone differentiation. Strikingly, lowering the Δψm by chemical uncoupling of the electron transport chain leads to HSC self-renewal under culture conditions that normally induce rapid differentiation. Taken together, these data show that mitochondrial activity and fate choice are causally related in HSCs, and provides a novel method for identifying HSC potential after in vitro culture. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 422 Background: Antibody-dependent cell-mediated cytotoxicity (ADCC), largely mediated by natural killer (NK) cells, is thought to play an important role in the efficacy of rituximab, an anti-CD20 monoclonal antibody (mAb) used to treat patients with B cell lymphomas. CD137 is a costimulatory molecule expressed on a variety of immune cells following activation, including NK cells. We hypothesized that the anti-lymphoma activity of rituximab could be enhanced by stimulation of NK cells with an anti-CD137 agonistic mAb. Methods: Rituximab induced upregulation of CD137 on NK cells was assessed using lymphoma cell lines and primary lymphoma patient samples. In-vitro NK cell degranulation and cytotoxicity were assessed by CD107a mobilization and chromium release. A murine lymphoma tumor model targeted by mouse anti-mouse CD20 mAb was used to assess in-vivo synergy of anti-CD20 and anti-CD137 mAbs. Mechanism of synergy was explored by T cell, NK cell, and macrophage depletion in the immune competent mouse model. A xenotransplant model in SCID mice with disseminated, luciferase-labeled lymphoma was used to demonstrate efficacy of anti-CD137 mAb and rituximab, and sufficiency of an innate immune response. Results: NK cells in human primary lymphoma samples do not express CD137 at baseline, however these cells highly upregulate CD137 when encountering rituximab-coated tumor B cells. Rituximab-induced NK cell degranulation and cytotoxicity as measured by CD107a mobilization (p=.006) and chromium release (p=.01) are enhanced by anti-CD137 agonistic mAb. In a murine lymphoma model, anti-CD137 mAb significantly enhances anti-tumor activity of anti-CD20 mAb leading to complete tumor resolution (p

Description: Several gene-expression signatures predict survival in diffuse large B-cell lymphoma (DLBCL), but the lack of practical methods for genome-scale analysis has limited translation to clinical practice. We built and validated a simple model using one gene expressed by tumor cells and another expressed by host immune cells, assessing added prognostic value to the clinical International Prognostic Index (IPI). LIM domain only 2 (LMO2) was validated as an independent predictor of survival and the “germinal center B cell–like” subtype. Expression of tumor necrosis factor receptor superfamily member 9 (TNFRSF9) from the DLBCL microenvironment was the best gene in bivariate combination with LMO2. Study of TNFRSF9 tissue expression in 95 patients with DLBCL showed expression limited to infiltrating T cells. A model integrating these 2 genes was independent of “cell-of-origin” classification, “stromal signatures,” IPI, and added to the predictive power of the IPI. A composite score integrating these genes with IPI performed well in 3 independent cohorts of 545 DLBCL patients, as well as in a simple assay of routine formalin-fixed specimens from a new validation cohort of 147 patients with DLBCL. We conclude that the measurement of a single gene expressed by tumor cells (LMO2) and a single gene expressed by the immune microenvironment (TNFRSF9) powerfully predicts overall survival in patients with DLBCL.

Description: Abstract 2093 Background: Adoptive immunotherapy is a promising novel approach to the treatment of cancer. However, clinical translation of adoptively transferred CD4 T cells is limited by cotransfer of an inhibitory population of regulatory CD4 T cells (Tregs). We identified a method of isolating viable antitumor CD4 T cells while excluding Tregs based on two surface markers—CD44 and CD137. Methods: We have developed a model for adoptive cell therapy of lymphoma whereby anti-tumor T cells are generated in vivo through vaccination with a CpG-loaded whole cell vaccine (CpG/H11). These vaccine-induced cells can protect from lethal tumor challenge when isolated and transferred into lethally irradiated, syngeneic recipient mice. We investigated the subsets of T cells involved in the anti-tumor response through a combination of in vitro and in vivo assays. Results: Adoptive transfer of CD137negCD44hi CD4 T cells, but not other sub-populations, provided protection from B cell lymphoma. We demonstrate that the population of CD137posCD44hi CD4 T cells consists primarily of activated Tregs. In vitro, these CD137pos cells suppressed the proliferation of effector cells in a contact-dependent manner. We observed that this CD137pos Treg population persisted following adoptive transfer and maintained expression of FoxP3 as well as CD137. Moreover, the addition of CD137posCD44hi CD4 cells to CD137negCD44hi CD4 cells suppressed the antitumor immune response. In the presence of CD137posCD44hi CD4 T cells, homing of other T cell populations to tumor sites was disrupted. These results suggest that CD137 expression on CD4 T cells defines a population of activated Tregs that prevent antitumor immune responses. Conclusions: Our findings identify two surface markers that can be used to facilitate the enrichment of anti-tumor CD4 T cells while depleting an inhibitory Treg population. This approach has direct applicability to clinical trials for patients with lymphoma. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1582 Background Lymphoma occurring in very elderly patients (i.e. 〉90 years of age) is not uncommon and its incidence is expected to increase due to prolongation of life expectancy. Its management raises therapeutic as well as ethical considerations. However, no reports are currently available in this population of patients rendering their management particularly challenging. The purpose of our study is to describe the clinical presentation, management and outcome of patients diagnosed with lymphoma at the age of 90 or over. Methods In a retrospective, multicenter study, we analyzed 234 patients with Non-Hodgkin (NHL, n=227) and Hodgkin (HL, n=7) lymphoma diagnosed at the age of 90 or over, between 1990 and 2012. Results Histologic incidence of lymphoma subtypes was evaluated using 3 epidemiologic cancer registries, accounting for 128 patients. In this cohort, 55% of the patients presented with aggressive NHL, 42% with indolent NHL and 3% with HL. The most frequent histologies were diffuse large B cell lymphoma (46%), follicular lymphoma (9.5%), lymphoplasmacytic lymphoma (8%) and marginal zone lymphoma (5.5%). Clinical characteristics, management and outcome were collected for 169 patients among several institutions. At diagnosis, median age was 92 [range 90–100]. Most patients were female (66%) and still living at home (73%). Charlson index was low (i.e. ≤2) in 68% of the patients. Forty-five percent of the patients had a low (i.e. ≤1) performance status (PS). The majority of the patients (63.5%) received a treatment (Fig. 1). Median overall survival (OS) for the entire cohort was 6.9 months. OS was significantly shorter in patients with aggressive lymphoma (OS = 5.2 vs 19.2 months, p

Description: BACKGROUND: Lenalidomide exerts anti-proliferative activity on lymphoma cells, enhances T- and NK-cell function, and improves ADCC. The combination of lenalidomide and rituximab (R2) has been shown to be synergistic in pre-clinical models and in patients with relapsed and first-line follicular lymphoma (FL). Obinutuzumab, a unique type II glycoengineered monoclonal anti-CD20 antibody with increased ADCC and increased direct cell death induction compared to rituximab, has shown promising efficacy in NHL. Combining obinutuzumab with lenalidomide might be even more efficient than R2 combination. In 2012, we started a phase Ib/II study to assess the safety and efficacy of this combination (ClinicalTrials.gov: NCT01582776, GALEN) for relapsed/refractory lymphoma patients. Here, we report the phase Ib part whose primary objective was to determine the maximum tolerated dose (MTD) of lenalidomide in combination with a fixed dose of obinutuzumab. METHODS: To identify the MTD of lenalidomide, four patient cohorts received escalating doses (10, 15, 20, and 25 mg) of daily oral lenalidomide on days 1-21 of a 28-day cycle for the first cycle and on days 2-22 of a 28-day cycle from cycles 2 to 6. Intravenous infusions of obinutuzumab at a flat dose of 1000mg were given on Days 8, 15, and 22 of the first cycle and at D1 of cycles 2 to 6 (total of 8 infusions). Steroid premedication was mandatory before first obinutuzumab infusion. All subjects were required to take daily aspirin (100 mg) for deep vein thrombosis (DVT) prophylaxis during study period. Subjects who were unable to tolerate aspirin and subjects with prior history of DVT or at high risk received low molecular weight heparin therapy or warfarin (coumadin) treatment. Dose was escalated in a 3+3 design based on dose limiting toxicity (DLT) assessment during cycle 1 RESULTS: 20 patients with FL ([10 mg: 7; 15 mg: 3; 20 mg: 6, 25 mg: 4] QD) were enrolled between Oct 2012 and Feb 2014 at 7 centers, 10 men/10 women, median age 64 (range, 39–80) years, 15 with Ann Arbor stage IV, median number of prior systemic therapies 2 (range, 1–5) and 8 were rituximab refractory. One patient was withdrawn before receiving any treatment due to neutropenia occurring at baseline screening. Median number of cycles was 6 (range, 1–6). We observed 163 AEs (87 grade 1; 53 grade 2; 21 grade 3; one grade 4 and one grade 5). The most common AEs (All grades, ≥20% patients) were neutropenia (10/19; 53%), constipation (10/19; 53%) asthenia (7/19; 37%), upper respiratory tract infection (7/19; 37%), rash/cutaneous eruption (5/19; 26%), cough (5/19; 26%), diarrhea (4/19 ; 26%) and fever (4/19, 21%). Grade (G) 3/4 AEs occurring in ≥2 patients only consisted in neutropenia (8/19; 42%). Infusion related reactions occurred in 3 patients and did not exceed grade 2. Four DLTs have occurred in 2 patients: one unexplained death (G5) at 10 mg in the setting of G3 worsening pleural effusion; another patient treated at 20 mg had G3 pulmonary infection with G3 hypokalemia, both deemed unrelated to study treatment. The MTD was not reached. Among 19 evaluable patients, 13 (68%) had an overall response according to Cheson 1999 criteria: 7 achieved CR, 3 CRu and 3 PR. CONCLUSION: Oral lenalidomide plus obinutuzumab is well tolerated and effective in patients with relapsed or refractory FL. Recommended dose of lenalidomide was established at 20mg based on the increased incidence of grade 3/4 neutropenia between cycle 2 and 6 at 25mg. The Phase II partis currently assessing theefficacy of lenalidomide (at 20 mg) in combination with obinutuzumab in 2 separate populations of patients: relapsed/refractory follicular lymphoma in one cohort and relapsed/refractory aggressive lymphoma (diffuse large B-cell lymphoma and mantle cell lymphoma) in another cohort. Disclosures Morschhauser: Celgene: advisory boards, advisory boards Other, Honoraria; Genentech/roche: Honoraria, travel grants Other. Off Label Use: obinutuzumab and lenlidomide in relapsed follicular lymphoma. Salles:celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; roche: advisory boards, advisory boards Other, Honoraria, Research Funding. Le Gouill:Roche: Consultancy. Tilly:Roche/Genetech: advisory boards, advisory boards Other, Honoraria; Celgene: advisory boards Other, Honoraria. Cartron:roche: Consultancy, Honoraria; celgene: Honoraria.