ALBERT

Beschreibung: 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.

Beschreibung: 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

Beschreibung: 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.

Beschreibung: 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.

Beschreibung: 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

Beschreibung: 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.

Beschreibung: Abstract 2791 Introduction: Elderly mantle cell lymphoma (MCL) patients (pts) do not benefit from dose-intensive chemotherapy upfront.1 The GOELAMS group recently demonstrated that a regimen comprising Vincristine/Adriamycine/Dexamethasone plus Chlorambucil (VAD+C) was well tolerated, had a good efficacy/toxicity profile and induced similar PFS than R-CHOP (median PFS between 16 to 18 months2,3,4). Additionally, it has been shown that bortezomib (Velcade®), with or without Rituximab has efficacy in relapsed/refractory MCL patients5,6. These data prompted, our group to conducte a phase II prospective non randomized clinical trial evaluating the combination of Velcade plus Rituximab/Adriblastine/Dexamethasone/Chlorambucil (RiPAD+C) as a first line therapy for elderly MCL patients. Aims: To evaluate the overall response rate (ORR) and toxicity after 4 cycles of RiPAD+C regimen (main objective) and to evaluate prognostic factors for survival (secondary objective). Protocol: RiPAD+C : Rituximab 375 mg/m2 on d1 (and d8 for cycle 1); PS 341, Velcade® 1.3 mg/m2 on d1, 4, 8 and 11; Adriblastine 9mg/m2/d as a continuous infusion for 4 days; Dexamethasone 20 mgx2/d from d1 to d4; Chlorambucil 12 mg/d, d20 to d29. Repeat cycles every 35d. After 4 cycles, responding pts (Cheson 1999 criteria) received 2 additional cycles for a maximum of 6. Patients and methods: Inclusion criteria: All untreated elderly (65 to 80 years old) MCL patients (including blastoid forms) presenting with a stage II to IV disease with a good PS (ECOG

Beschreibung: Abstract 144 Introduction: Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin's lymphoma. Although newer treatment regimens including intensive chemo-immunotherapy followed by stem cell transplant have improved survival to a median approaching 6 years, MCL is still incurable in many cases. The use of flow cytometry to investigate stimulation induced signaling at the single cell level represents an opportunity to obtain each patient's signaling profile as well as to discover tumor cell heterogeneity and identify signaling events potentially responsible for poor outcomes. We recently used this method to characterize signaling in subpopulations of tumor samples from patients with follicular lymphoma (FL). In FL, we identified a lymphoma negative prognostic (LNP) cell subset with impaired B cell antigen receptor (BCR) signaling, the prevalence of which correlated with adverse clinical outcome (Irish et al., PNAS 2010). In the present study we used the same approach to identify signaling responses with large variation within MCL patient samples, and tested whether these signaling events might predict patients' clinical outcomes. By understanding the biology based on lymphoma signaling profile, we might identify prognostic significant factors such as the LNP subset observed in FL. Method: Single cell flow cytometry measurements of signaling were acquired for samples of MCL (n=25). Of these, 21 MCL specimens were obtained prior to any treatment. Median age was 61 years, and follow up time ranged from ½ until 15 years. Treatment regimens varied. Samples from diffuse large B cell lymphoma (DLBCL, n=12), FL (n=14), chronic lymphocytic leukemia (CLL, n=14) and tonsils from healthy donors (n=4) were also included for comparison. Phosphorylation of 14 signaling proteins was measured under 12 different stimulation conditions in every cell within the patient specimens, including lymphoma B cells and in tumor-infiltrating T cells. Stimulation conditions included BCR crosslinking, CD40 ligand, CpG oligodeoxynucleotides (CpG ODN) and several cytokines (IL-4, IL-10, IL-21, IFN-γ). Result: The magnitude of most cytokine induced signaling responses in lymphoma cells from MCL patients was higher than in lymphoma cells from FL. This included IL-10 induced phosphorylation of STAT3 (p

Beschreibung: Background: Mantle cell lymphoma (MCL) is an aggressive lymphoma with variable 18Fluorine-Deoxyglucose (FDG) avidity. FDG - Positron Emission Tomography/Computed Tomography (FDG-PET/CT) is not yet recommended in MCL but many retrospective studies have underlined its prognostic impact either before any treatment or at interim or final time points of R-chemotherapy. The French LYSA/GOELAMS group published the results of a multicenter prospective phase II clinical trial evaluating the RiPAD + C front-line combination including one proteasome inhibitor (PS341-Velcade®) for elderly MCL patients(Houot, Le Gouill et al. 2012). The aim of the present study was to determine the prognostic value of FDG-PET/CT in this prospective cohort with a long follow up. Method: Patients between 65 and 80 years old were enrolled. They received 4 cycles of RiPAD+C regimen (every 35 days: Rituximab 375 mg/sqm D1, Velcade® 1.3 mg/sqm D1,4,8 and 11, Doxorubicin 9 mg/sqm D1 to D4, Dexamethasone 40 mg D1 to 4 and Chlorambucil 12 md D20 to D29) and 2 additional cyclesif they responded (IWR criteria). Three FDG-PET scans were performed: an initial pre-treatment, after 4 cycles (interim) and after 6 cycles (post-treatment). All available FDG-PET/CT were centrally reviewed by two experts, using visual international response assessment criteria proposed by IHP in Lymphoma and the Deauville five-point scale. The maximal standardized uptake value (SUVmax) and maximal standardized uptake value reduction (ΔSUVmax) of the most intense pathological area were measured. Results: From June 2007 to December 2008, 39 patients from 21 French centers were recruited. After 64 months follow-up for the 22 surviving patients, median overall survival (OS) has not been reached (the 3 year OS was 63,5%). Median progression free survival (PFS) is 22 months. Seventeen patients died either from lymphoma (n=13) or due to toxicity (n=4). Seventy-eight FDG-PET/CT were performed (31 initial FDG-PET/CT; 28 interim, 19 post-treatment), in 39 patients. We reviewed 24 initial FDG-PET/CT, 27 interim, and 16 post-treatment. By univariate analysis: neither initial, interim nor post-treatment FDG-PET/CT were predictive of OS or PFS. The ΔSUVmax (〉 65% vs ≤ 65%, or 〉 50% vs ≤ 50%) was also not predictive for OS or PFS (p= 0.48 to 0.92). However high SUVmax (〉10 vs ≤10) and clinical prognostic scores (MIPI or the Goelams index) correlated with OS (p=0.09, p= 0.054 and p=0.16, respectively). In a multivariate analysis patients with a high prognostic score at diagnosis combined with a positive post-treatment FDG-PET/CT had very poor OS compared to other profiles (high index with a negative post-treatment FDG-PET/CT or low-intermediate index with a negative or positive FDG-PET/CT). Conclusion: This is the first prospective study evaluating the prognostic impact of FDG-PET/CT in a cohort of homogeneously treated MCL patients with a long time of follow up. Neither initial, interim or post-treatment FDG-PET/CT were predictive of PFS or OS. However we confirm, as previously described in a retrospective analysis (Bodet-Milin, Touzeau et al. 2010), that both high SUVmax at initial FDG-PET/CT and the MIPI score were prognostic for OS. Interestingly, a negative post-treatment FDG-PET/CT seemed to erase the adverse prognostic significance of a high MIPI score before treatment. These observations warrant further confirmation in large prospective clinical trials. Disclosures Le Gouill: Roche: Consultancy; Janssen: Consultancy. Dartigeas:Roche: Consultancy. Tournilhac:mundipharma: Honoraria, Other, Research Funding; GSK: Honoraria, Other, Research Funding; Roche: Honoraria, Other, Research Funding. Gressin:MundiPharma: Other.

Beschreibung: This prospective phase II trial (Nov 2011- Dec 2012), supported by the LYSA group, aimed to evaluate the impact on PFS of the RiBVD regimen in newly diagnosed, previously untreated, elderly MCL patients (〉65 years or not eligible for ASCT) (NCT01457144). Inclusion criteria were: WHO 2008 MCL not previously treated, CD20 positive, ECOG 0-2, AA stage II-IV, no CNS involvement or active HBV/HCV/HIV infection. Patients were scheduled to receive a total of 6 cycles of the RiBVD regimen, if they responded (IWG criteria) after 4 cycles. The regimen was administered every 4 weeks with Rituximab 375 mg/m² IV on day(D)1, Bendamustine 90 mg/m² IV on D1 and 2, Dexamethasone 40 mg/m² IV on D2 and bortezomib (Velcade®) 1.3 mg/m² subcutaneously on D1, 4, 8 and 11. Primary prophylaxis with acyclovir was mandatory for Herpes virus reactivation, but there was no recommendation for bacterial prevention. Herein we present preliminary analysis of the trial after 4 RiBVD cycles. Results: A total of 76 patients were included, one was excluded because of HBV active disease and 5 had insufficient data reported in the database. To date we analyzed 70 patients. Patients characteristics: sex ratio M/F 49/21, median age 72 years (y) [64-83] (2 patients were 64 y old), AAstage II/III-IV 5/65, ECOG 0-1/2 59/11, MIPI score low/intermediate/high 3/19/48. Response: 61 responded (ORR=87%), with 19 in PR (26%) and 42 in CR/CRu (60%). Four patients died from pneumonia (n=1), cardiac arrest (n=2) and one following Progressive Multifocal Leukoencephalopathy. Three patients have progressed after 3 cycles. Sixty one patients were analysed by PETscan after 4 cycles, 39 (64%) reached a CR (30 were in CR/CRu and 9 in PR) and 22 remained PET positive (11 patients were in CR/CRu, 10 in PR and 1 stable). RiBVD cycles: 271 cycles were administered out of 280 planned (97%). Twenty four (9%) were delayed, 10 for toxicity. All but one planned Bendamustine doses (n=542) were administered with dosing modified 17 times (3%), mostly for hematological toxicity (n=14). Regarding Bortezomib, 79% (1028/1084) of planned doses were administered, it was prematurely stopped (56, 4%) for neurotoxicity (10 instances) or hematological side effects (46). Rituximab was not administered in 4 instances. Hematologic toxicities: Over the 271 cycles administered, neutropenia was reported in 104 cycles [56 grade 3/4 (g3/4) (21%)], 2.5% with fever; thrombopenia in 181 cycles [41 g3/4 (15%)]; anemia in 210 cycles, [6 g3/4 (2%)]. Non-hematologic toxicities:Reported in 〉10% of the cycles were : allergic reactions (10.3%, g3/4