Abstract: Splenic marginal zone B-cell lymphoma (SMZL) is a heterogeneous clinico-biological entity. The clinical course is variable, multiple genes are mutated with no unifying mechanism, and essential regulatory pathways and surrounding microenvironments are diverse. We sought to clarify the heterogeneity of SMZL by resolving different subgroups and their underlying genomic abnormalities, pathway signatures, and microenvironment compositions to uncover biomarkers and therapeutic vulnerabilities. We studied 303 SMZL spleen samples collected through the IELSG46 multicenter international study (NCT02945319) by using a multiplatform approach. We carried out genetic and phenotypic analyses, defined self-organized signatures, validated the findings in independent primary tumor metadata and determined correlations with outcome data. We identified 2 prominent genetic clusters in SMZL, termed NNK (58% of cases, harboring NF-κB, NOTCH, and KLF2 modules) and DMT (32% of cases, with DNA-damage response, MAPK, and TLR modules). Genetic aberrations in multiple genes as well as cytogenetic and immunogenetic features distinguished NNK- from DMT-SMZLs. These genetic clusters not only have distinct underpinning biology, as judged by differences in gene-expression signatures, but also different outcomes, with inferior survival in NNK-SMZLs. Digital cytometry and in situ profiling segregated 2 basic types of SMZL immune microenvironments termed immune-suppressive SMZL (50% of cases, associated with inflammatory cells and immune checkpoint activation) and immune-silent SMZL (50% of cases, associated with an immune-excluded phenotype) with distinct mutational and clinical connotations. In summary, we propose a nosology of SMZL that can implement its classification and also aid in the development of rationally targeted treatments.

Abstract: Background and Aims. Prediction of treatment efficacy is an active and growing field of pharmacology. In the Fondazione Italiana Linfomi (FIL) MCL0208 phase III trial (NCT02354313), a 24 months lenalidomide maintenance (LM, 15 mg days 1-21 every 28 days) after high-dose immuno-chemotherapy followed by autologous transplantation (ASCT) in 300 frontline mantle cell lymphoma (MCL) patients showed substantial clinical activity in terms of Progression-Free Survival (PFS) vs observation (OBS). However, this benefit seemed not uniform across patient series. To deeper investigate the differential pattern of response to lenalidomide, a wide analysis of the host pharmacogenomics (PG) background was planned, in order to dissect whether specific germline polymorphisms of transmembrane transporters, metabolic enzymes or cell surface receptors (ABCB1, ABCG2, VEGFA, FCGR2A, NCF4, GSTP1, CRBN) might predict the drug efficacy. Actually, several single nucleotide polymorphisms (SNPs) of ABCB1 exert an effect on substrate affinity of lenalidomide for the transmembrane transporter. Moreover, VEGFA is involved in the anti-angiogenic activity of lenalidomide and might eventually upregulate ABCB1 expression, too. Patients and methods. Genotypes for SNPs were obtained through allele-specific (ASO) probes on germline DNA from peripheral blood. Minor allele frequencies (MAFs) were obtained and the Hardy-Weinberg equilibrium (HWE) was checked. Genotypes were used to infer individual haplotypes by Arlequin and Haploview softwares. Minimal residual disease (MRD) was assessed with ASO primers on either IGH or BCL-1/IGH rearrangements by RQ-PCR in bone marrow samples. TP53 disruption was identified by NGS targeting resequencing and copy number variation analysis. Clinical-biological correlations were screened by automated machine learning methods and validated by both Kaplan-Meier at univariate level and Cox models for multivariate analysis (MV). A logistic regression was implemented to investigate correlations between polymorphisms and MRD kinetics. Results. 278 out of 300 patients (93%) were fully genotyped. The MAF values of the SNPs were very similar to published data and the HWE was confirmed. Most notably, ABCB1 c.2677G & gt;T/A(W) and VEGFA c.2055A & gt;C were significantly associated to outcome and are thus described in this abstract. In the case of ABCB1, the three loci were in strong linkage disequilibrium (p & lt;0.001). 31% of patients were homozygous for ABCB1 wild type alleles (GG, "WT"), 53% heterozygous (GW, "HET") and 16% polymorphic on both chromosomes (WW, "POL"). 20% were VEGFA WT (AA), 47% HET (AC) and 33% POL (CC). PG did not impact on induction therapy and randomization rates of this trial, as superimposable polymorphism frequencies were described between the enrolled and randomized population. Conversely, both ABCB1 HET and POL and VEGFA HET/POL associated with higher MRD clearance rates vs WT after 6 months of LM (93% vs 71% and 91% vs 67%, respectively). Interestingly, the risk of MRD reappearance during LM was 86% lower for patients harboring either polymorphism vs WT (odds ratio 0.14, 95% CI 0.02-0.99; p & lt;0.05). Actually, ABCB1 HET/POL predicted for a more favorable PFS vs WT in LM (3yPFS 85% vs 69% p & lt;0.05, Fig.1A), as well as VEGFA HET/POL (3yPFS 85% vs 59% p & lt;0.01, Fig.1B). The two polymorphisms co-occurred in 57% of patients, being 12% ABCB1 HET/POL only, 23% VEGFA HET/POL and 8% ABCB1/VEGFA WT. Interestingly, patients with either polymorphism had superimposable outcome to patients in whom both co-occurred (Fig.1C). Finally, MV showed that either polymorphism was protective for PFS among randomized patients (HR=0.42; 95% CI 0.20-0.85; p & lt;0.05). According to this hypothesis, among the 17 ABCB1/VEGFA WT patients LM did not improved PFS vs OBS (Fig.1D), independently from TP53 disruption. Conclusions. The first PG data on LM after ASCT in MCL suggested that: 1) ABCB1 and VEGFA polymorphisms did not impact on the chemotherapeutic efficacy of FIL-MCL0208 trial; 2) both polymorphisms favored sustained MRD clearance during LM; 3) either polymorphism conferred a survival advantage during LM. Taken together, these observations hint that a variable excretion of lenalidomide through ABCB1 (heralded by SNPs), as well as an altered VEGFA pathway, could predict treatment efficacy. This observation might be very useful in the future to tailor lenalidomide therapy to MCL patients. Disclosures Ferrero: Servier: Speakers Bureau; Gilead: Research Funding, Speakers Bureau; EUSA Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Boccomini:SC Ematologia, ASOU Città della Salute e della Scienza di Torino, Turin, Italy: Current Employment. Maria:Roche: Consultancy, Other: travel, accomodations, expenses; Abbvie: Consultancy, Other: travel, accomodations, expenses; BMS: Consultancy; MSD: Consultancy; Janssen: Consultancy, Other: travel, accomodations, expenses; Gilead: Consultancy, Other: travel, accomodations, expenses, Research Funding. Ferreri:Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Morphosys: Research Funding; Hutchinson: Research Funding; BMS: Research Funding. Palumbo:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Galimberti:Novartis: Speakers Bureau; Incyte: Honoraria. OffLabel Disclosure: Lenalidomide maintenance in mantle cell lymphoma

Abstract: Background. The classic Ph-negative myeloproliferative neoplasms (MPN) are a group of clonal haematopoietic disorders, including polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF), whose shared and diverse phenotypic signatures are caused by a dysregulated JAK/STAT signal transduction because of acquired somatic mutations. It has been demonstrated that autoimmune diseases and MPN can be associated (Kristinsson et al., Haematologica. 2010 Jul;95(7):1216-20.), suggesting a common background of immune dysregulation (Barosi, Curr Hematol Malig Rep. 2014 Dec;9(4):331-9). SARS-CoV-2 infection displays extreme inter-individual clinical variability, ranging from silent infection to lethal disease. It has been described that at least 10% of patients with life-threatening coronavirus disease 2019 (COVID-19) pneumonia have neutralizing autoantibodies (AAbs) against type I IFNs, that precede SARS-CoV-2 infection (Bastard et al., Science. 2020 Oct 23;370(6515):eabd4585). In this study we searched for AAbs against type I IFNs in a cohort of MPN patients to evaluate the prevalence of these AAbs in the MPN population and to check for clinical correlations, including severity of COVID-19. Methods. Plasma samples from consecutively referred MPN patients were prospectively collected between November 2020 and June 2021 and frozen at -30°C immediately after collection. Levels of AAbs against type I IFN subtypes including IFNs alpha, beta and omega were measured using the enzyme-linked immunosorbent assay (ELISA) and a neutralization assay, as previously reported (Bastard et al., Science. 2020 Oct 23;370(6515):eabd4585; Moreews et al., Sci Immunol. 2021 May 25;6(59):eabh1516). Results. We included a total of 219 MPN patients (101 ET, 76 PV, 36 MF and 6 MPN unclassificable). Neutralizing AAbs to type I IFNs were detected in 29 patients (13.2%, 95%CI: 9.1% - 18.5%). Comparing patients with and without AAbs we observed a significant difference in terms of distribution of MPN diagnosis (P = 0.029) and driver mutations (P = 0.019), while we did not observe a difference in terms of age, sex, and treatment (Table 1). Overall, 29 patients (13%) got SARS-CoV-2 infection and 8 of them (28%) required hospitalization due to severe COVID-19. AAbs against type I IFNs were detected in 4 of the 29 SARS-CoV-2 infected patients. A higher rate of hospitalization for severe COVID-19 was observed in patients with AAbs to type I IFNs (2 of 4 patients, 50%) compared to those without these AAbs (6 of 25 patients, 24%), although the difference did not reach a statistical significance (P = 0.300). Conclusions. In this study, we detected a prevalence of AAbs against type I IFNs which is much higher in our MPN cohort (13%) than in the general population (2-3%). We also found a correlation between the presence of AAbs to type I IFNs and both the hematological diagnosis and the driver mutation. Despite a comparable prevalence of SARS-CoV-2 infection between MPN patients with or without AAbs to type I IFNs, we observed a different rate of hospitalization due to severe COVID-19 which is almost twice in those with AAbs against type I IFNs compared to those without these AAbs. However, this difference did not reach a statistical significance, probably because of the low number of SARS-CoV-2 infection in the subgroup of patients with AAbs against type I IFNs. Thus, further studies to analyse the prevalence of AAbs against type I IFNs in patients with MPN, their association with other forms of auto-immunity and severe COVID-19 are warranted. Figure 1 Figure 1. Disclosures Arcaini: Gilead Sciences: Research Funding; Bayer, Celgene, Gilead Sciences, Roche, Sandoz, Janssen-Cilag, VERASTEM: Consultancy; Celgene, Roche, Janssen-Cilag, Gilead: Other: Travel expenses; Celgene: Speakers Bureau. Rumi: Novartis, Abbvie: Consultancy.

Abstract: According to evidence- and consensus-based practice guidelines (Haematologica2002;87:1286–306), red cell transfusion is the therapy of choice for the majority of patients with myelodysplastic syndrome (MDS) and symptomatic anemia. Previous studies have shown that widespread organ dysfunction can result from transfusion iron overload developing in non-thalassemic adults (Schafer et al, N Engl J Med1981;304:319–24; Cazzola et al, Blood1988;71:305–12). In this study, we evaluated the effect of transfusion dependency and secondary iron overload on survival of MDS patients classified according to WHO criteria. Four hundred and sixty-seven consecutive patients with a diagnosis of de novo MDS made at the IRCCS Policlinico San Matteo, University of Pavia Medical School, Pavia, Italy, between 1992 and 2002 were retrospectively evaluated and reclassified according to the WHO criteria. The effects that developing transfusion dependency or secondary iron overload had on survival were evaluated by applying Cox proportional hazards regression with time-dependent covariates. Transfusion-dependent patients had a significantly shorter overall survival (OS) and leukemia-free survival (LFS) than did patients who did not become transfusion-dependent (HR=2.16, P & lt;.001 and HR=2.02, P & lt;.001, respectively). Transfusion burden, calculated as the number of transfusions per month, was found to have a significant effect on both OS (HR=1.35, P & lt;.001) and LFS (HR=1.75, P & lt;.001). These effects were maintained after accounting for cytogenetics. We then assessed the survival of transfusion-dependent and non-transfused patients considering non-leukemic death as an end-point. In the first 50 months of follow-up transfusion-dependent patients had a significantly worse survival than those who did not require transfusions (HR=1.98, P=.01). Cardiac failure was significantly more frequent in transfusion-dependent patients (P=.01). Focusing the analysis on WHO subgroups, transfusion requirement affected both the OS and LFS of patients with RA, RARS or MDS with del(5q) (HR=1.54, P & lt;.001 and HR=1.44, P=.05, respectively), and of those with RCMD or RCMD-RS (HR=1.87, P=.04 and HR=1.54, P=.02, respectively), while it showed no effect on the survival of RAEB patients. Finally, we evaluated the prognostic value of patients with MDS developing iron overload during their follow-up. The development of secondary iron overload significantly affected survival (P & lt;.001) with a HR of 1.36 for every 500 ng/mL increase in serum ferritin. The effect of iron overload was maintained after adjusting for transfusion burden (HR=1.30, P=.003). With respect to WHO subgroups, the effect of secondary iron overload was still present in patients with RA/RARS (HR=1.51, P & lt;.001), while it was not significant in those with RCMD/RCMD-RS (HR=1.34, P=.20). In conclusion, these findings show that the development of transfusion dependency significantly worsens the survival of MDS patients. Although this poor prognosis partly reflects the severity of bone marrow failure, our observations also suggest that development of secondary iron overload per se can worsen survival of transfusion-dependent patients. These individuals may therefore considerably benefit from therapeutic approaches aimed at reducing transfusion needs and/or at preventing secondary iron overload.

Abstract: Indolent non follicular B-Cell Lymphomas (INFL) are a heterogeneous group of lymphomas and include small lymphocytic lymphoma (SLL), lymphoplasmacytic lymphomas (LPL), and marginal zone lymphomas (MZL). Combination chemo-immunotherapy regimens are used in the majority of patients (pts) with advanced stage INFL who require treatment. Bendamustine in combination with anti-CD20 monoclonal antibody therapy has been shown to be highly active and well tolerated. The purpose of this phase 2, multicenter study was to determine the activity and safety of bendamustine in combination with rituximab in first-line treatment of patients with INFL. Methods Untreated patients with SLL, nodal MZL and LPL who met predefined need-for-treatment criteria, had adequate hematologic function and stage II-IV disease, were eligible for study entry. Patients were scheduled to receive 6 cycles BR: bendamustine (90 mg/m2 on days 1 and 2 every 28 days) and 6 rituximab doses (375 mg/m2 on day 8 of cycle 1, and on day 1 of subsequent cycles) + 2 rituximab doses every 2 weeks. The primary endpoint of the study was the complete response rate (CRR) based on 2007 International Working Group criteria (Cheson 2007). The secondary endpoints were the rate of adverse events, the overall response rate (ORR), the progression free survival (PFS) and the overall survival (OS). Sample size was defined at 67 patients evaluable for response assuming a CR rate of 60%, a power of 80% and p value of 5%. Results The study was conducted from February 2011 to march 2012. Seventy-two patients were enrolled, and 3 patients were excluded from the study due to violation of inclusion criteria (1 patient) and due to treatment interruption before completion of cycle one (2 patients). Based on local pathology report 17 patients had SLL, 20 had nodal MZL, and 32 had LPL. Median age was 65 years (45-75) and 65% were male. LDH value was increased in 17% of pts and β2-microglobulin in 67%. B-symptoms were present in 16% of pts, 91% percent had bone marrow involvement and 99% had stage III/IV disease. Fifty-nine out of the 69 patients received at least 6 cycles of bendamustine/rituximab (BR, 85,5%). Based on the intention to treat analysis and on local assessment of response, ORR was 84% including 40 patients in CR in (58%), and 18 in PR (26%); 1 patients had stable disease and 5 patients had lymphoma progression; in 5 patients response was not assessed due to adverse event or early withdrawal. Safety analysis was available for all 69 patients and for 472 cycles. Overall, the combination of BR was well-tolerated. The most common grade 3/4 adverse events were neutropenia (43% of pts), febrile neutropenia (3%), thrombocytopenia (9%), gastrointestinal disorders (3%), and skin reaction (3%). One patient died during treatment due to a severe infection after cycle 2. At time of current analysis after a median follow-up of 15.6 months (range 1 to 25 months), 2 disease progressions and 2 deaths (due to lymphoma) were observed. As a result 1 year PFS was 90% (CI95% 80-95%) and 1 year OS was 95% (CI95% 87-98%). Conclusions Based on this preliminary analysis of the INFL09 study, BR combination is active and well tolerated in patients with previously untreated INFL. The ORR of 85% and CRR of 58% compare favorably with previously reported response rates observed in patients with indolent lymphoma. Disclosures: Federico: MedImmune: Research Funding.

Abstract: Abstract 2720 Introduction: Bendamustine is a promising agent that has been showed high efficacy in combination with Rituximab in indolent lymphomas; however experiences on its use in combination with other cytotoxic agents are scant, particularly in FL. A previous FIL trial showed that a brief R-FND induction chemoimmunotherapy with only 4 courses of R-chemotherapy followed by 4 rituximab doses as consolidation can achieve high CR and PFS rates in FL patients, supporting the feasibility of this regimen in the elderly (Vitolo et al, ASH 2011). These promising results prompted FIL to investigate safety and efficacy of a similar combined brief regimen substituting Bendamustine for Fludarabine aimed at reducing toxicity and maintaining efficacy. Patients and methods: From September 2009 to November 2011, 76 elderly patients (age 65–80) with advanced stage FL at diagnosis were enrolled. Inclusion criteria were: untreated grade I, II and IIIa FL; advanced disease requiring treatment (stage III/IV) or stage II including at least one of the following conditions: bulky disease ( 〉 7 cm), active disease with rapid lymph node progression, lactate dehydrogenase or β2 microglobulin above normal, systemic symptoms and extranodal involvement. A comprehensive geriatric assessment (ADL, I-ADL, CIRS) was also performed at diagnosis and only “FIT” patients were enrolled into the study. Treatment plan was: 4 monthly courses of R-BM (375 mg/m2 Rituximab day 1, 90 mg/m2 Bendamustine days 1–2, 8 mg/m2 Mitoxantrone day 1), followed by consolidation with 4 weekly Rituximab infusions. Polymerase chain reaction (PCR) for BCL2/IgH rearrangement was performed on bone marrow samples at diagnosis, after R-BM and after consolidation. Results: At the time of analysis, 69/76 patients are evaluable for clinical characteristics, response to treatment and toxicity. Median age was 71 years (range 65–80); 26 males, 43 females; WHO grading was as follow: I 16%, II 55% and IIIa 29%; 19% had advanced stage II disease, 27% stage III and 54% stage IV; 49% had BM involvement, 19% B symptoms and 7% leukemic dissemination; 58% patients had no comorbidity, 20% one and 22% two or more comorbidities. According to FLIPI patients were: 12% at low, 30% at intermediate and 58% at high risk. PCR analysis was carried out in 64 patients at diagnosis: 58% were Bcl-2 positive. Sixty three patients completed the planned treatment and the whole program was completed according to the planned time in 60/63 patients. Six patients did not complete the treatment: 1 for progressive disease, 4 for adverse events (2 haematological toxicity with prolonged neutropenia; 1 CMV colitis and 1 for infection and concomitant worsening of pre-existing oral pemfigo) and 1 patient for worsening of performance status. Overall response after R-BM + Rituximab was observed in 64 (96%) patients with 52 (75%) patients in complete remission and 12 (18%) in partial remission (PR). Response to 4 cycles of R-BM was as follow: 27 (39%) in CR, 37 (54%) in PR and 5 (7%) patients in stable (SD) or progressive disease. Of the 40 patients in PR or SD after 4 R-BM, 26 (65%) converted to CR following further Rituximab consolidation. At the time of this analysis, of the 37 patients Bcl-2 rearranged at diagnosis, 19 (51%) were evaluable for molecular response during and after treatment. PCR negativity was achieved in 16/19 (84%) patients after R-BM and in 18/19 (95%) patients at the end of treatment. A total of 521 courses of R-BM and Rituximab were given. The most frequent severe toxicity (CTC grade 3–4) was neutropenia reported in 18% of the courses; nevertheless only four serious infections were recorded (all due to bacterial agents). Pulmonary and cardiac toxicities were 〈 1%. Two deaths were recorded: one due to lymphoma after second line treatment and one due to hepatic carcinoma occurred 3 months after completion of therapy. Conclusions: A brief course of chemo-immunotherapy R-BM followed by Rituximab consolidation is safe and effective with a high CR rate in elderly patients with untreated advanced stage FL. Planned future analysis of the entire study will give further information on late toxicity and outcome. Disclosures: Off Label Use: In Europe use of Bendamustine in first-line treatment of follicular lymphoma is off-label. Drug was provided free by Mundipharma. Vitolo:Roche: Membership on an entity's Board of Directors or advisory committees.