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Background: In the 2017 version of the ELN recommendations (ELN2017), a comprehensive evaluation of karyotype and mutational status of specific genes (e.g. FLT3 and NPM1) allows to classify patients (pts) with Acute Myeloid Leukemia (AML) into 3 prognostically distinct risk groups (favorable, intermediate and adverse-risk). Before the publication of the ELN2017 guidelines, the Gruppo Italiano Malattie Ematologiche MAligne (GIMEMA) conducted a prospective trial (AML1310) in which prognostic classification relied on the risk assessment criteria (NCCN2009) at that time available. In this post-hoc analysis, we investigated the applicability of the ELN2017 risk stratification to the AML1310 study population. Methods: After induction and consolidation, pts in complete remission (CR) were to receive autologous stem cell transplant (AuSCT) if categorized as favorable-risk (FR) (CBF-AML, NPM1-mutated) or allogeneic stem cell transplant (ASCT) if adverse-risk (AR) (FLT3-ITD, complex karyotype). Intermediate-risk pts (IR) were to receive AuSCT or ASCT based on the post-consolidation levels of MRD as measured by flow-cytometry. Baseline genetic/cytogenetic, together with RUNX1/RUNX1T1, CBFb/MYH11, NPM1, FLT3 mutational status (including the FLT3 allelic ratio for those positive) were used to retrospectively classify pts according to the ELN2017. Results: All 500 pts, enrolled in the AML1310 trial, were included in the present analysis. Retrospective allocation was feasible in 445/500 (89%) cases and pts lacking crucial information for a proper ELN2017 assignment, defined a control group (ELN2017-NC). Median age was 49 (range 18-61). The re-assignmentaccording to the ELN2017, resulted in 186 pts (41.8%) belonging to the FR category (ELN2017-FR), 179 (40.2%) to the IR (ELN2017-IR) and 80 (18%) to the AR (ELN2017-AR) ones. Moreover, 55 (11%) pts were considered ELN2017-NC. Based on this process of re-assignment, 173 pts were reclassified according to ELN2017: 6 from NCCN FR (1 ELN2017-NC, 4 ELN2017-IR, 1 ELN2017-AR), 54 from NCCN IR (34 ELN2017-NC, 4 ELN2017-IR, 1 ELN2017-AR), and 113 from NCCN AR (20 ELN2017-NC, 38 ELN2017-FR, 55 ELN2017-AR) groups. After 1-2 cycles of induction, 361 (72%) pts obtained CR or CR incomplete (CRi): 163 (88.1%), 114 (65%), 45 (56.2%) and 39 (70%) in the ELN2017-FR, ELN2017-IR, ELN2017-AR and ELN2017-NC groups, respectively (p Summary/Conclusion: In this GIMEMA AML1310 post-hoc analysis, we confirmed that the ELN2017 classification is able to accurately define pts that can benefit from different post-remission strategies. Specifically, AuSCT granted longer survival in FR pts, while for IR pts AuSCT and ASCT performed equally when minimal residual disease was used as a driver for opting between one of the two. In conclusion, ELN classification is a reliable grouping system that, combined with MRD assessment, helps addressing pts to the most appropriate treatment. Such an hypothesis will be prospectively challenged in the next GIMEMA trial AML1819. Disclosures Luppi: Abbvie: Consultancy; Novartis: Consultancy, Speakers Bureau; Gilead Sci: Consultancy, Speakers Bureau; Sanofi: Consultancy; Daiichi-Sankyo: Consultancy; MSD: Consultancy. Voso:Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Venditti:Pfizer: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Speakers Bureau; Amgen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Jazz: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); AbbVie: Consultancy, Honoraria, Other: TRAVEL, ACCOMODATIONS, EXPENSES (paid by any for-profit health care company); Janssen: Consultancy, Honoraria, Other: TRAVEL, ACCOMODATIONS, EXPENSES (paid by any for-profit health care company); Novartis: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company).

The amplification of the 1q21 (amp1q21) region is one of the most acquired cytogenetic abnormalities (CA) in multiple myeloma (MM) associated with a worse patient outcome and disease progression. Moreover, different studies have demonstrated that the number of copies (CN) 1q21 (gain1q21: three copies or amp1q21: ≥ four copies) have a different impact in the response to anti-MM therapies. Particularly, it has been proposed that in MM patients, additional copies of 1q21 may be associated with the resistance to proteasome inhibitor (PI) treatment as bortezomib. A recent study showed that newly diagnosed MM (MMD) patients carrying amp1q21 but not gain1q21 receiving carfilzomib-based treatment have an early disease progression with shorter overall survival. Previous studies underlined that the amplification of 1q21 can lead to the overexpression and/or dysregulation of several candidate genes associated with cell proliferation, apoptosis, and drug resistance. Here we aim to identify 1q21 target genes possibly correlated to the response to PI therapy. We evaluated a total cohort of 29 primary plasma cells (PCs) purified from bone marrow (BM) blood aspirates from 11 smoldering MM (SMM) and 18 MMD. The median age of our cohort was 70 years (range: 38-86). Fluorescence in situ hybridization (FISH) analysis was performed to access the presence or absence of copy number alteration (CNA) in the 1q21 region in all patients. 14 out of 29 patients carried 1q21 CNA (5 with gain1q21 and 9 with amp1q21). A score reflecting the number of 1q21 copies was calculated based on the hybridization pattern. The transcriptional profiles of the 29 BM PCs samples were generated on GeneChip ClariomD Arrays (Affymetrix Inc., Santa Clara, CA, USA). The samr package was used in R for call genes as differentially expressed between 1q21 CN-altered and wild-type samples. The correlation between the 1q21 copy number score and the gene expression levels was performed. Moreover, we have evaluated by FISH the 1q21 CNA in a panel of human myeloma cell lines (HMCLs): OCY-MY5, JJN3, RPMI-8226, NCI-H929, and OPM2. JJN3 were transfected with a control vector and PSMB4 and PSMD4 short hairpin RNA (shRNA) lentivectors. The gene and protein expression levels of PSMB4 and PSMD4 in MM cell lines were analyzed by qRT-PCR and Western Blot, respectively. Cell viability and proliferation were assessed using MTT assay and flow cytometry. Our bioinformatic analyses highlighted the overexpression of different genes (IL6R, ILF2, BCL9, MCL1, CSk1B, ADAR1, ARNT, ANP32E) in the 1q21 CNA samples with respect to the controls, as already reported in the literature. Our analysis showed a significantly higher expression of two proteasome subunits (PSMB4 and PSMD4) in patients with 1q21 CNA when compared with patients without (PSMB4 p=0.0006; PSMD4 p= We have evaluated PSMB4 and PSMD4 mRNA and protein expression levels in a 1q21 wild-type cell line (OCY-MY5) and in a panel of MM cell lines carrying different degrees of 1q21 CN (in order: JJN3, U266, RPMI-8226, OPM2, and NCI-H929). The mRNA expression level of PSMB4 and PSMD4 was higher in cell lines carrying 1q21 amp, following a 1q21 copy number fashion. Similar results were obtained when protein levels of MM cell lines were analyzed by Western Blot. To further determine the potential role of both proteasome subunits in the pathogenesis of amp1q21, we generated a PSMB4-shRNA and PSMD4-shRNA knockdown stable MM cell lines. Functional studies showed that blockade of PSMB4 and PSMD4 decreased MM cell viability. In conclusion, our study identified proteasome subunits PSMB4 and PSMD4 to be significantly upregulated in MM patients carrying amp1q21, correlated with 1q21 copy number but not with disease stage. In addition, knockdown of both, PSMB4 and PSMD4 decreased MM cell proliferation. Therefore, targeting PSMB4 and PSMD4 could be a strategy to treat MM patients with ampq21 Disclosures Giuliani: Celgene: Membership on an entity's Board of Directors or advisory committees, Other: congress, Research Funding; Bristol Mayers Squibb: Other: congress; GSK: Other: clinical studies; Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Clinical studies, congress, Research Funding; Millenium Pharmaceutical: Other: clincial studies.

INTRODUCTION Treatment of Diffuse Large B cell lymphoma (DLBCL) in the elderly population is challenging as many patients (pts) are not eligible to receive standard curative therapy, due to comorbid conditions and to a higher susceptibility to the side effects of standard anthracycline containing regimens. Among currently available active drugs, Lenalidomide has been used in the setting of relapsed/refractory DLBCL both as monotherapy and in combination with rituximab, showing a good activity and an acceptable safety profile. We started a prospective, multicenter, single arm, phase II trial to demonstrate activity and safety of a chemo-free combination of lenalidomide + rituximab in older (≥ 70 years) untreated pts with DLBCL who were prospectively defined as frail according to the simplified comprehensive geriatric assessment (sCGA) (Tucci A. et al., Leuk Lymphoma. 2015 Apr). PATIENTS AND METHODS Pts were eligible if they were previously untreated DLBCL patients, older than 69 years and defined as frail according to sCGA. The treatment consisted of a 28-day cycle (R2) combining oral Lenalidomide (20 mg on days 1 to 21) and i.v. Rituximab (375 mg/m2 on day 1); a maximum number of 6 cycles was planned; response assessment was performed after cycles 4 and 6. At the end of the 6 th cycle, patients with partial or complete response continued treatment with Lenalidomide 10mg/d on days 1 to 21 every 28 days, until cycle 12or unacceptable toxicity. Final response was evaluated within 28 days after the last study drug administration. Primary study endpoint was Overall Response Rate (ORR) after 6 R2 cycles, defined according to Lugano 2014 criteria; co-primary endpoint was the rate of extra-hematological toxicity with CTCAE grade >2 and of death for any cause during the treatment The study was planned according to a two stage Simon design. A total of 68 pts had to be enrolled to complete the study. With 34 responses the study would be able to demonstrate the initial hypothesis of an improvement of response rate from 45% (p0) to 65% (P1) RESULTS From August 2018 to June 2021, 68 newly diagnosed frail DLBCL were enrolled in 18 Italian centers. Median age was 83 years (range 70-91) and 73% had stage III/IV; 57% had High risk IPI (i.e. 3-5 risk factors). 64 pts were confirmed eligible and started R2 treatment. The planned 6 courses of R2 were completed in 36 pts (56%). The median number of R2 cycles was 6 (1-6). Treatment was discontinued in 28 pts due to lymphoma progression (11 cases), extra-hematological toxicity (7), hematological toxicity (1), lost after 1 cycle and investigator choice after 5 cycles in CR in 1 case each, death in 7 cases (1 each for infection, pancytopenia, cachexia, ischemia and bowel infarction, and from unknown causes in 2 cases). At the end of 6 th R2 cycle 26 patients achieved a response (ORR 41%), 13 CR and 13 PR. After a median follow-up of 17 months, the overall survival, and progression free survival at 12 months were 69% (95CI 56-79%) and 55% (95%CI 42-66%), respectively. The duration of remission at 12 months was 72%. Regarding safety 46 events were reported including 31 extra-hematological toxicities > CTCAE grade 2: 7 cardio-vascular (1 resulting in death), 4 nervous system disorders (1 resulting in death), 10 gastro-intestinal, 3 infections, 6 skin and subcutaneous tissue disorders, 5 respiratory events (all resolved). The rate of grade 3-4 adverse events was higher than the highest allowed limit of 28. CONCLUSIONS The Reri is the first study to evaluate activity and safety of a chemo-free therapy in patients with diffuse large B-Cell Lymphoma who are not eligible for conventional cytotoxic therapy. Even if this study was not able confirm the initial activity hypothesis, activity of the R2 combination was observed in a significant proportion of cases warranting further exploration of chemo-free approach of elderly frail patient with DLBCL. Disclosures Tucci: janssen: Membership on an entity's Board of Directors or advisory committees; Gentili: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Fabbri: Takeda: Honoraria; Servier/Pfizer: Honoraria; Takeda: Other: Travel, Accomodations, Expenses. Musuraca: janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Zilioli: Takeda: Other: travel expenses, accommodation; Gentili, Takeda, Gilead, Servier: Consultancy, Speakers Bureau; Roche, Italfarmaco: Consultancy, Honoraria; MSD, Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations. Luminari: Roche, Celgene, Teva Pharmaceuticals, Gilead Sciences, and Takeda Pharmaceuticals: Honoraria.

Glutamine (Gln) addiction has been recently described as a typical metabolic feature of MM by our group. In order to sustain high Gln demand, MM cells upregulate the expression of the Gln transporters ASCT2, SNAT1 and LAT1 and are characterized by fast Gln uptake. Currently, 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) is the standard tracer for the positron emission tomography/computed tomography (PET/CT) scan to detect both medullary and extramedullary disease in MM patients. However, FDG-PET has some limitations, since there is a portion of MM patients who are false-negative. For these reasons, the aim of our study is to characterize Gln metabolism in vivo in different MM mouse models and investigate the possible use of Gln as a PET tracer in MM. To this purpose, we have firstly synthesized enantiopure (2 S,4 R)-4-fluoroglutamine (4-FGln) and validated it in human MM cell lines (RPMI8226 and JJN3) comparing its uptake with that of 3H-labelled Gln. Both Gln and 4-FGln were actively accumulated by MM cells. Inhibition analysis revealed that ASCT2 was the major entry route of both compounds. We then carried out the radiosynthesis of [18F]4-FGln and tested its uptake for MM detection by PET in comparison with [18F]FDG. Firstly, we characterized [18F]4-FGln kinetics using immunodeficient NOD/SCID mice subcutaneously injected with JJN3. In this xenograft model, [18F]FDG- and [18F]4-FGln-PET were performed after plasmacytomas became palpable. In tumor, [18F]4-FGln uptake peaked at 25 min slowly declining thereafter. The Logan plot confirmed linearity starting at 15 min from injection, consistent with largely reversible tracer exchange. Thereafter, [18F]FDG and [18F]4-FGln distribution were assessed in both syngeneic (murine Vk12598 cells) and xenograft ( human JJN3 cells) MM models. C57BL/6 mice were injected intravenously with Vk12598 cells obtained from transgenic Vk*MYC mice. Mice were monitored weekly for M-spike and MM progression by retro-orbital bleeding. At weeks 3, 4 and 5, mice underwent PET with [18F]FDG and [18F]4-FGln on consecutive days. The expression of ASCT2, checked in the femur of Vk12598 MM bearing mice, increased along with MM progression. Uptake of both tracers was measurable in the spleen of MM mice by week 3, reached a peak at week 4 and declined thereafter. In JJN3 model, all the tumors were positive for [18F]FDG and displayed [18F]4-FGln uptake (18F]4-FGln: tumor to muscle ratios (T/M): 1.6 ± 0.1; [18F]FDG: T/M: 3.5 ± 1.1). Tumor volume (896.8 ±349.00 mm3), [18F]4-FGln uptake and [18F]FDG uptake (T/M: 2.3 ± 0.3 and 7.1 ± 2.6, respectively) increased after 1 week ([18F]4-FGln p = 0.0156; [18F]FDG p = 0.0167). Thereafter, the effect of bortezomib (BOR) was investigated to evaluate the potential use of [18F]4-FGln to monitor anti-MM treatment. JJN3-bearing mice were treated with BOR (1mg/kg) by intravenous injections twice weekly. Before and after treatment, animals performed the PET acquisitions with [18F]FDG and [18F]4-FGln on consecutive days and were sacrificed for post-mortem analysis on day 7. As expected, bortezomib treatment reduced tumor size compared with vehicles (172.0 ± 86.6 vs 687.9 ± 286.5 mm3; p = 0.0006). PET analysis performed after BOR treatment showed that BOR significantly reduced the uptake of both radiopharmaceuticals in comparison with vehicles (p< 0.05). BOR-treated mice were classified as responders and non responders according to the adapted RECIST score. Interestingly, responder mice showed a reduction of [18F]4-FGln T/M ratio and [18F]4-FGln-related tumor volume. On the contrary, all mice displayed increased [18F]FDG parameters independently from the response. Lastly, tracer overlap analysis showed that the area of relative exclusive uptake of [18F]4-FGln decreased in responders compared to non responders (9% versus 32%, respectively, p=0.04), while that of [18F]FDG increased in responders compared to non responders (82% versus 23%, respectively, p=0.04). Our data indicate that [18F](2 S,4 R)-4-FGln is a new PET tracer in pre-clinical MM models, providing the rationale to design studies in MM patients. Disclosures Giuliani: Celgene: Membership on an entity's Board of Directors or advisory committees, Other: congress, Research Funding; Millenium Pharmaceutical: Other: clincial studies; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Clinical studies, congress, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; GSK: Other: clinical studies; Bristol Mayers Squibb: Other: congress.

Hairy cell leukemia (HCL) is a rare lymphoproliferative disease with specific morphologic and molecular features and excellent prognosis. Although high rate of complete response (CR) has been reported after treatment with purine analogs, expecially cladribine (2CDA), relapse may occur during follow-up. The aim of the study is to review the efficacy, safety, long term remission rate and overall survival (OS) in those patients (pts) that received 2CDA as first line treatment. We retrospectively reviewed data of all HCL pts treated with 2CDA between March 1991 and May 2019 at 18 Italian Hematological centers. Among 553 pts reported, only 513 were evaluable because treated with 2CDA alone. Considering the clinical carachteristics, M/F ratio was 4.5 with a median age of 54 years (range 24-88) and ECOG 0 in 85% of cases. Splenomegaly and presence of circulating hairy cells recorded by morphology were reported in 241 (47%) and 138 (27%) pts, respectively. Thirty-seven (7%) pts presented with an infection. Other comorbidities were cardiovascular in 29 (6%) pts, a previous cancer or diabetes in 27 (5%) each, chronic hepatic disorders in 18 (3%), obstructive pulmonary disease in 16 (3%), chronic kidney disease in 3 (1%). Three hundred-thirty (64%) pts received 2CDA intravenously (253 as daily continuous infusion for 5-7 consecutive days and 77 as weekly infusion for 5-7 consecutive weeks) and 183 (36%) subcutaneously. Response criteria were defined as per recent consensus guidelines (Grever MR et al. Blood 2017). The overall response rate (ORR) was 83%: CR in 335 pts (65%) and partial response (PR) in 96 (19%); 40 (8%) pts obtained hematological improvement (HI) and in 42 (8%) no response was observed. Nine of 11 (82%) pts with HI and 18/25 (72%) non responders who received salvage therapy obtained a major response (fig. 1). A slightly higher hemoglobin value (12.4 vs 11.4 g/dl, p=0.044), a reduced frequency of circulating hairy cells (28.7% vs 31.8%, p=0.039), absence of palpable splenomegaly (p= 2CDA is greatly effective in treating HCL, with an ORR of 83%. Early and long term adverse events were rare and easily managed: although HCL-related mortality is still possible, OS at 15 years is higher than 80% Disclosures Motta: Roche: Honoraria; Janssen: Honoraria. Offidani:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Tedeschi:Abbvie: Honoraria, Speakers Bureau; Sunesis: Honoraria, Speakers Bureau; Acerta: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Beigene: Honoraria, Speakers Bureau. Trentin:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Octapharma: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Shire: Honoraria. Varettoni:Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Other: Travel/accommodations/expenses; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel/accommodations/expenses. Visentin:Janssen: Honoraria; Gilead: Honoraria; Abbvie: Honoraria. Falini:Roche: Research Funding. Pulsoni:Sandoz: Consultancy; Pfizer: Consultancy; Takeda: Consultancy; Gilead: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau; roche: Consultancy, Speakers Bureau; Merk: Consultancy. Tiacci:Roche: Research Funding; Abbvie: Other: Travel and meeting expenses. Zinzani:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Portola: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kirin Kyowa: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Verastem: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Immune Design: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Therapeutics, Inc.: Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sandoz: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Eusapharma: Consultancy, Speakers Bureau; Kyowa Kirin: Consultancy, Speakers Bureau; Immune Design: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; EUSA Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Despite the promising results of immune-checkpoint blockade in the treatment of many tumors, the use anti-PDL-1/PD-1 antibodies in multiple myeloma (MM) still remains debated and under observation for the high toxicity in combination with immunomodulatory drugs or for the lack of a clear evaluation of the PD-L1/PD-1 distribution in MM patients. Literature data on PD-L1/PD-1 expression by CD138+ and bone marrow (BM) cells in MM patients are discordant and none of them compared patients with active and smoldering myeloma (SMM). This suggests the need to better define PD-L1/PD-1 distribution in the BM immune-microenvironment in MM patients, to identify those that could benefit from PD-L1/PD-1 blockade. In this study, we isolated mononuclear cells from BM aspirates in a cohort of patients with monoclonal gammopathies (total number= 107) including 39 patients with SMM and 78 with active MM, including both newly diagnosed (MMD) and relapsed MM (MMR). We compared the expression profile of PD-L1/PD-1 axis on CD138+ cells, CD14+ monocytes and T cells (both CD4+ and CD8+), by flow-cytometry. Results were correlated with clinical parameters, as International Staging system (ISS), cytogenetic risk, bone disease. BM sera were also collected to measure the levels of different soluble factors known to regulate PD-L1 expression or to exert pro/anti-tumor activity in MM (IL-6, IL-10, IL-27, IFN-γ). Results from ELISA assay were examined in relation with flow-cytometry data. We found that neither PD-L1 expression on CD138+ cells nor PD-1 on CD4+/CD8+ cells significantly differ between SMM and MM patients; although, CD14+PD-L1+% increases with disease progression (SMM vs MMD vs MMR: median: 39.14 vs 59.49 vs 47.66, p=0.14) without reaching a statistical significance. Analysis on the total cohort revealed that PD-L1 is expressed at higher levels on CD14+CD16+ non-classical monocytes compared with classical monocytes (17.41 vs 23.09, p Focusing on patients with active MM, those with ISS=II and III showed increased PD-L1 expression on CD14+ cells (ISS II+III vs I, median MFI 20.35vs14.59, p=0.005) and higher CD8+PD-1+% (II+III vs I, 4.35vs2.58, p=0.022) compared with ISS=I patients. Analysis on PD-L1/PD-1 expression in relation with the cytogenetic features of our cohort of patients revealed that CD138+ cells from hyperdiploid patients express higher levels of PD-L1 compared with not-hyperdiploid ones (25.66 vs 13.96, p=0.001), suggesting that the expression of this immune checkpoint does not contribute as a high risk factor in MM disease. Finally, we investigated PD-L1/PD-1 distribution, in relation with the presence of bone lesions, and we found that not-osteolytic patients have higher CD8+PD-1+% compared with osteolytic ones (p=0.071). In conclusion, our data show a similar PD-L1/PD-1 expression pattern between SMM and MM patients, and higher PD-L1 intensity on the non-classical monocytes CD14+CD16+ compared with classical CD14+CD16- cells. On the other hand, an inverted CD4+/CD8+ ratio characterizes relapsed MM patients, together with high amount of MM pro-survival IL-6 and low anti- tumor IL-27 BM serum levels. Overall these data suggest that, despite the similarity in PD-L1/PD-1 expression, SMM and early MM patients rather than relapsed MM, could represent the potential subset which could better benefit of anti PD-L1/PD-1 therapy, in light of their less compromised immune-microenvironment. Disclosures Giuliani: Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Participation in congresses, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Other: Participation in congresses; GSK: Other: Clinical study sponsorship, Research Funding; Janssen Pharmaceutical: Membership on an entity's Board of Directors or advisory committees, Other: Clinical study sponsorship; participation in congresses, Research Funding; Millennium Pharmaceutical: Other: Clinical study sponsorship, Research Funding.

Metabolic alterations of cancer cells, aimed at sustaining their growth, may also influence the behavior of the tumor microenvironment. Our group has recently demonstrated that multiple myeloma (MM) is a highly glutamine(Gln)-addicted tumor that utilize huge amounts of Gln to fuel its metabolism through the enzyme glutaminase (GLS). For this reason, MM cells exhibits increased Gln uptake, mainly through the ASCT2 transporter. Interestingly, lower bone marrow (BM) plasma Gln concentration (down to a median value of 0.4 mM vs. a median value of 0.6 mM) was found in MM patients as compared with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS). The main feature of MM BM microenvironment is the suppression of osteoblastic (OB) differentiation leading to the development of osteolytic bone lesions, the hallmark of MM. Most recently, it has been demonstrated that Gln metabolism is needed to sustain bone mass formation in murine models and that GLS inhibition decreases OB differentiation of human mesenchymal stromal cells (hMSCs). However, no information is yet available on the role of Gln depletion imposed by MM cell metabolism on OB differentiation into the BM. This topic has been investigated in the present study. Firstly, human MM cells were co-cultured with BM hMSCs, and Gln medium concentration was evaluated with mass spectrometry (MS), demonstrating a MM-induced depletion of the amino acid. Upon Gln depletion, MSC exhibited a sustained induction of Glutamine Synthetase (GS). On the contrary, when differentiated in osteogenic medium (D-MEM + 5% Fetal Bovine Serum, supplemented with 2 mM Gln, ascorbic acid and dexamethasone), GS was suppressed. Conversely, GLS (both KGA and GAC isoforms) and SLC38A2, the gene for the concentrative Gln transporter SNAT2, were induced. These data suggest that hMSCs differentiation in OBs is associated with an increased dependence upon extracellular Gln. Consistent with this conclusion, the activity of SNAT2 was absent in undifferentiated hMSCs but well detectable after 14 days of OB differentiation, when total Gln uptake was also increased. Under the same conditions, OB differentiation markers (RUNX2, COL1A1, ALPL expression and ALPL activity or staining) were significantly induced but their expression was blunted by incubation in low-Gln (0.4 mM) medium or in the presence of the SNAT2 inhibitor MeAIB. The incubation in Gln-free D-MEM suppressed the induction of GLS and SLC38A2 along with OB differentiation, which was restored by the supplementation of Non-Essential Amino Acids (NEAA). Among NEAA, only asparagine (Asn) was able to rescue OB differentiation in the absence of Gln. The determination of intracellular amino acids with MS indicated that OB differentiation was associated with the increase of cell Asn, without significant changes of Gln, glutamate (Glu) or aspartate (Asp). Asparagine Synthetase (ASNS), the Gln-dependent enzyme that accounts for Asn synthesis, was also found induced during OB differentiation of hMSCs. Gene Expression Profiles of primary BM hMSCs and OBs from bone biopsies of both healthy donors (n=7) and MM patients (n=16) indicated that GLS, ASNS, and SLC38A2 are more expressed in OBs, while the expression of GLUL, the gene for GS, is higher in undifferentiated hMSCs from healthy donors. Overall, these results indicate that (1) OB differentiation of hMSCs is Gln-dependent; (2) the partial Gln depletion, imposed by Gln-addicted MM cells in the BM microenvironment, contributes to the impairment of osteoblastic differentiation of hMSCs; (3) hindrance of differentiation may depend on the limited availability of intracellular Asn derived from Gln-dependent ASNS. These results support the evidence that Gln addiction of MM cells affects bone microenvironment leading to the inhibition of OB differentiation and, consequently, to the development of MM bone disease. Disclosures Giuliani: Janssen: Research Funding.

The identification of risk factors for progression is critical in the clinical management and appropriate follow up of patients with pre-malignant Asymptomatic Monoclonal Gammopathies (AMG) including Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM). The development of prognostic score and consequently the early identification of patients with possible short-term progression to Multiple Myeloma (MM) could lead to anticipate the treatment. In this study, we retrospectively evaluated possible risk factors of short-term progression to active MM in a large cohort of MGUS and SMM patients admitted to a single haematological center (Hematology and BMT Unit, University Hospital of Parma) between 2010 and 2018. We analysed a total cohort of 235 patients diagnosed with AMG (81 MGUS and 154 SMM) according to the IMWG recently updated diagnostic criteria. All patients analysed underwent to Bone Marrow (BM) examination; moreover, imaging evaluation was performed in 22 MGUS and 123 SMM patients, in order to exclude the presence of bone disease. In a subgroup of AMG patients (n=50), bone mineral density (BMD) evaluation by Dual-energy X-ray Absorptiometry (DXA) was also available. Median age of the AMG patients analysed was 68 years (range 35-93 years). Median percentage of BM plasma cells (BMPCs) was 12% (range 2-55%) in the entire population, 7% (range 2-9) in MGUS and 15% (range 10-55) in SMM patients. Median serum M-protein was 1.7 g/dL (range: 0.17-4.5), 1.5 g/dL (range 0.17-4.5) in MGUS and 1.8 g/dL (range 0.4-2.7) in SMM patients. An abnormal free light chain (FLC) ratio was found in 70% of AMG patients, among the ones that performed the analysis; regarding SMM patients, FLC ratio value was available in 97 patients: in 72 (76%) the ratio was unbalanced, 37 (39%) had a FLC ratio ≤ 0.125 or ≥ 8 and in 14 (15%) it was > 20; among MGUS patients, value was collected in 41 patients and in 21 (51%) it was 1.65. The presence of immunoparesis in one or two uninvolved immunoglobulins occurred in 59% of the entire population. The median follow up time was 18 months (range 0 - 111 months) for whole population. Overall 44 patients of the entire cohort progressed to MM (41 SMM and 3 MGUS) with a median TTP of 14.5 months. By univariate analysis we found that percentage of BMPCs, entity of M-protein and presence of immunoparesis were significantly correlated with progression to active MM (p 8 (as used in Mayo scoring system for SMM) showed a relationship at the limit of statistical significance in this subgroup of patients (p=0.052). Any significant correlation was not observed with age, sex, Ig isotype, light chain's type and the BMD values (p=NS). Afterwards, we applied Kaplan Meier method on risk factors resulted significant in univariate analysis demonstrating that they also significantly influenced the time to progression to MM. Finally, through a binomial logistic regression, we developed a new prognostic score for whole population. By combining the values of M-protein (< 2, score=0 or ≥ 2 g/dL, score=1) and the percentage of BMPC (20%, score=2), we obtained six groups at different probability of progression to active MM (Table 1). Given that result, we stratified patients in 3 groups: low-risk (score=0), intermediate-risk (score=1) and high-risk (score≥2); log-rank test confirmed that high-risk patients had a significantly shorter time to progression to symptomatic MM as compared to intermediate and low-risk patients (p In conclusion, our results show that in patients with AMG the clinical factors, which mostly impact on the short-term risk of progression to active MM, are the entity of the PCs infiltrate and the MC related to the tumoral mass. The development of a clinical score based on BMPCs and M-protein will permit to overcome the traditional distinction between MGUS and SMM in the evaluation of the progression of AMG patients to active MM. Disclosures Giuliani: Janssen: Research Funding.

INTRODUCTION Treatment of Diffuse Large B cell lymphoma (DLBCL) in the elderly population is challenging as many patients (pts) are not eligible to receive standard curative therapy, due to comorbid conditions and to a higher susceptibility to the side effects of standard anthracycline containing regimens. Among currently available active drugs, Lenalidomide has been used in the setting of relapsed/refractory DLBCL both as monotherapy and in combination with rituximab, showing a good activity and an acceptable safety profile. We started a prospective, multicenter, single arm, phase II trial to demonstrate activity and safety of a chemofree combination of lenalidomide + rituximab in elderly (≥ 70 years) untreated pts with DLBCL who were prospectively defined as frail according to a simplified comprehensive geriatric assessment (sCGA) (Tucci A. et al., Leuk Lymphoma. 2015 Apr). PATIENTS AND METHODS Pts were eligible if they were previously untreated DLBCL patients, older than 69 years and defined as frail according to sCGA. The treatment consisted of a 28-day cycle (R2) combining oral Lenalidomide (20 mg on days 1 to 21) and i.v. Rituximab (375 mg/m2 on day 1); a maximum number of 6 cycles was planned; response assessment was performed after cycles 4 and 6. At the end of the 6thcycle, patients with partial or complete response continued treatment with Lenalidomide 10mg/d on days 1 to 21 every 28 days, until cycle 12or unacceptable toxicity. Final response was evaluated within 28 days after the last study drug administration. Primary study endpoint was Overall Response Rate (ORR) after 6 R2 cycles, defined according to Lugano 2014 criteria; co-primary endpoint was the rate of extra-hematological toxicity with CTCAE grade >2 and of death for any cause during the treatment The study was planned according to a two stage Simon design. A total of 68 pts had to be enrolled to complete the study: 23 pts were required in the first stage. Second stage could be activated withat least 12 patients showing a Partial or Complete Response (PR/CR) in stage I. According to the Ray and Rai method less than 15/23 adverse events were also required for the safety coprimary endpoint. RESULTS From January 2017 to December 2017, 24 newly diagnosed frail DLBCL were enrolled in 8 Italian centers. Median age was 83 years (range 76-89) and 79% had stage III/IV; 42% of pts were male, and 44%, had elevated LDH, 45% had High risk IPI (i.e. 3-5 risk factors). All pts were confirmed eligible and started R2 treatment. The planned 6 courses of R2 were completed in 13 pts (54%). The median number of R2 cycles was 6 (1-6). Treatment was discontinued in 11 pts for the following reasons: lymphoma progression in 4 cases, second malignancy in 2, extra-hematological toxicity in 3 cases, consent withdrawal and investigator choice after 4 cycles in CR in 1 case each. Response assessment after 6 R2 cycles showed12 responding pts (ORR 50%), 4 CR and 8 PR, that was higher than by the inferior limit required by the Simon optimal design. Regarding safety coprimary endpoint 13 events were reported including 9 extra-hematological toxicities > grade 2 CTCAE (3 cardio-vascular and 6 respiratory events, all resolved) and 4 deaths (2 patients due visceral arterial ischemia and 2 due infectious disease). The rate of adverse events was lower than the superior limit of 15 allowed the first stage of the study.Since August 2018 the enrollment in the stage II of the trial has been resumed and it is currently ongoing with 45 patients enrolled. CONCLUSIONS The ReRi is the first study to evaluate activity and safety of a chemo-free therapy in patients with diffuse large B-Cell Lymphoma who are not eligible for conventional cytotoxic therapy. The results of the planned interim analysis of our study confirmed the initial efficacy and safety hypotheses of R2 combination in untreated elderly pts with DLBCL. Treatment of elderly frail DLBCL pts with R2 holds promise in terms of both ORR and safety. Disclosures Bassan: Amgen Inc.: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; Shire: Honoraria. Merli:Sandoz: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses; Janssen: Honoraria; Takeda: Honoraria, Other: Travel Expenses; Gilead: Honoraria; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Research Funding; Mundipharma: Honoraria. Liberati:AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Clinical trial support; Roche: Other: Clinical trial support; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Clinical trial support; Celgene: Honoraria, Other: Clinical trial support; Bristol-Myers Squibb: Honoraria; Takeda: Membership on an entity's Board of Directors or advisory committees; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Janssen: Honoraria; Novartis: Other: Clinical trial support. Luminari:ROCHE: Other: Role as Advisor ; CELGENE: Other: Role as Advisor & Travel Grant; TAKEDA: Other: Travel Grant; GILEAD: Other: Lecturer .

Alterations of the bone marrow (BM) immune-microenvironment characterize the progression of monoclonal gammopathies and the development of osteolytic bone disease in multiple myeloma (MM). MM patients exhibit immune dysfunctions as impaired dendritic, NK and T cells, whereas the onset of MM osteolytic lesions is associated to an increased prevalence of Th17 cells. Recently, the pathophysiological role of the programmed cell death protein 1 (PD-1)/PD-1 ligand (PD-L1) pathway together with an increase of myeloid derived suppressor cells (MDSCs) in the induction of tumor tolerance and immune evasion has been underlined with a therapeutic relevance. However, unclear data on the expression profile of PD-1/PD-L1 axis in MM patients have been reported and it is not known if this axis could be related with the presence of osteolytic bone disease. In order to elucidate these aspects, we analyzed a total cohort of 80 consecutives patients with monoclonal gammopathies, including 15 monoclonal gammopathy of undetermined significance (MGUS), 23 smoldering MM (SMM), 21 newly diagnosed MM (MMD) and 21 relapsed/refractory MM (MMR) patients. The presence of bone disease was checked by low-dose computerized tomography (CT) with or without positron emission tomography (PET) scan and by X-rays skeletal survey in 11 MM patients. 87% of MM patients displayed osteolytic lesions. High bone disease (HBD) was defined by the presence of 3 or more osteolytic lesions (62% of our cohort). Patients without bone lesions or with minus of 3 lesions were defined as low bone disease (LBD). BM mononuclear cells were analyzed by flow cytometry, evaluating plasma cells (PCs) (CD138+), monocytes (CD14+) and T cells (total CD3+, CD3+CD4+ and CD3+CD8+). PD-L1 (CD274) expression was evaluated on CD138+ and CD14+ cells, and PD-1 (CD279) on CD3+, CD4+ and CD8+ cells. Lastly, in a subgroup of patients we analysed MDSC populations, including both granulocytic (gMDSCs) (CD11b+HLA-DR-CD14-CD15+) and monocytic MDSCs (mMDSCs), (CD11b+HLA-DR-/lowCD14+CD15-). The percentage of BM CD3+PD-1+ cells increased from MGUS to MMR patients with a significant trend (p=0.004). Indeed, patients with active MM (MMD and MMR) showed both higher % of CD3+PD-1+ cells (median value: 48.5% vs 40.6%, p=0.001) and PD-1 median fluorescence intensity (MFI) on CD3+ (median value: 18.9 vs 16.7 MFI, p=0.024) as compared to patients with SMM and MGUS. CD4+PD-1+, but not CD8+PD-1+ cells are increased in active MM compared to SMM and MGUS patients (p=0.023). On the other hand, any significant difference was not observed in the PD-L1 expression on both BM CD138+ and CD14+ cells across patient groups. The percentage of BM MDSC populations did not significantly change across the different monoclonal gammopathies (p=0.14); moreover, comparing MM with SMM and MGUS patients, the % of gMDSCs was significantly reduced (median %: 12.5% vs 17%, p=0.04) and the % of mMDSCs was increased (median %: 0.36% vs. 0.24%) without reaching a statistical significance. Focusing on MM bone disease, we found that osteolytic MM patients displayed higher CD4+/CD8+ ratio compared to non-osteolytic ones (p=0.043). Regarding the PD-1 expression, the % of CD3+PD-1+ cells did not change (p=0.192), whereas the % of CD8+PD-1+ cells was significantly reduced in osteolytic patients compared to non-osteolytic ones (p=0.016). Consistently, among the BM CD8+ cells, a significant decrease of %PD-1+ cells was found in HBD vs LBD MM patients (median value: 46.9% vs 57.4%, p=0.045). Interestingly, when compared to LBD MM patients, HBD MM patients displayed a significant reduction of PD-L1 expression on both BM CD138+ PCs (median MFI value: 13.3 vs 21.6 MFI, p=0.007) and CD14+ cells (median MFI value: 15.4 vs 23.8 MFI, p=0.007). In a multivariate analysis, PD-1+ expression on CD8+ cells and PD-L1 MFI on CD138+ were significant independent factors related to the presence of HBD. In conclusion, our study indicates that the frequency of PD-1+ T cells increases across the progression of the monoclonal gammopathies. On the other hand, for the first time, we show in MM patients a significant relationship between the presence of extensive osteolytic bone disease and a reduced expression profile of BM PD-1/PD-L1 axis on CD8+ and CD138+ cells. We hypothesize that a less immune-suppressive profile could be related to the development of osteolysis consistent with the negative cross talk existing between PD-1/PD-L1 axis and Th17 cells. Disclosures Aversa: Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Basilea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Giuliani:Takeda Pharmaceutical Co: Research Funding; Janssen Pharmaceutica: Other: Avisory Board, Research Funding; Celgene Italy: Other: Avisory Board, Research Funding.

Multiple myeloma (MM) cells are characterized by tight dependence on the bone marrow (BM) microenvironment that exerts a permissive role on cell growth and survival. In turn, MM cells markedly modify their microenvironment leading, in particular, to the development of osteolytic bone lesions. Recently, we demonstrated that metabolic alterations is a major feature of MM cells showing that BM plasma of MM patients is characterized by lower levels of Glutamine (Gln) and higher levels of Glutamate (Glu) and ammonium when compared with patients with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS). In the majority of MM patients MM cells are Gln-addicted since they strictly depend on extracellular Gln, do not express Glutamine Synthetase (GS), the enzyme that synthetizes Gln from Glu and ammonium, and are endowed with high levels of the Gln transporter ASCT2. Based on this evidence, we have hypothesized that the peculiar Gln metabolism of MM cells may have a significant impact on the relationship with the bone microenvironment and contribute to the development of osteolytic lesions. We firstly characterized a panel of human MM cell lines (HMCLs) for their GS expression and response to decreasing levels of Gln. The majority of HMCLs, which did not express GS, consumed large amounts of extracellular Gln but secreted nearly half of the amino acid as Glu. Two HMCLs, MM1.S and U266, with a sizable GS expression, were less sensitive to Gln deprivation and secreted less Glu in the extracellular space compared with GS-negative HMCLs. Consistently, the activity of the Glu exchanger x-CT (the product of SLC7A11 gene) was lower in GS-positive than in GS-negative cells. The response to Gln starvation was then studied in mesenchymal stromal cell line (MSC), as well as in osteoblastic (HOBIT) and pre-osteocytic cells (HOB-01). HOBIT and HOB-01 were more sensitive to Gln depletion than MSC. Indeed, while MSC showed a low EC50 for Gln (0.064mM), which is 10-times lower than the physiological blood Gln concentration (around 0.6 mM), the EC50 values of HOBIT and HOB-01 cells were 0.250 mM and 0.297mM, respectively. Furthermore, L-methionine sulfoximine (MSO), an irreversible inhibitor of GS, emphasized the effects of Gln deprivation on all the cell lines tested. Indeed, Gln deprivation enhanced the expression of GS, suggesting that both stromal and osteoblastic cells exploit the enzyme to counteract Gln deprivation. On the basis of these data, we assessed the effects of Gln and Glu on osteogenic differentiation by incubating MSC, either immortalized or primary, with an osteogenic medium containing different concentrations of Gln and Glu. After 2 weeks, compared with cells differentiated in high Gln/high Glu conditions, MSC incubated in the presence of decreased Gln and increased Glu showed lower osteogenic ability, as assessed by real time PCR and ALP staining. Lastly, MSC co-cultured for 72 hours with GS-negative, but not with GS-positive HMCLs, showed reduced viability and increased GS expression. Lastly, to put in a translational perspective these in vitro observations, we analyzed the BM plasma levels of Gln and Glu in a cohort of 41 patients with newly diagnosed MM, including 9 smoldering MM (SMM) and 32 active MM patients (20 of them with osteolytic bone disease, 12 of them without bone disease). All 20 osteolytic MM patients had more than three osteolytic lesions. We found that MM patients had lower Gln levels and higher Glu levels than SMM patients. Moreover, when compared with MM patients without bone disease, MM patients with bone disease showed lower levels of Gln and higher levels of Glu. The results of these analyses are being continuously updated increasing the number of samples tested. Overall, these results indicate that MM cells are able to create a low-Gln/high-Glu bone marrow microenvironment that sustains GS expression in bone cells and impairs their differentiation and viability. Thus, the peculiar metabolic milieu in the MM bone microenvironment affects the relationship between neoplastic and bone cells and may contribute to the development of osteolytic bone disease in MM patients. Disclosures Aversa: Astellas: Honoraria; Merck: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Basilea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Giuliani:Takeda Pharmaceutical Co: Research Funding; Celgene Italy: Other: Avisory Board, Research Funding; Janssen Pharmaceutica: Other: Avisory Board, Research Funding.

Recently, the introduction of anti-CD38 monoclonal antibody, Daratumumab (DARA), in multiple myeloma (MM) therapy has improved the response rate of relapsed MM patients. However only a fraction of the DARA-treated patients respond, thus further studies on DARA mechanisms of action are needed. Because the antibody dependent cellular phagocytosis (ADCP) mediated by monocyte, is one of the mechanisms through DARA exerts its anti-MM activity, an ex-vivo approach was established in order to investigate which mechanisms or patient's immunological characteristics could influence DARA-mediated killing of MM cells. Bone marrow mononuclear cells (BM-MNCs) obtained from 25 MM patients (12 newly diagnosed and 13 relapsed MM) were analyzed at time 0 (T0) by flow cytometry. We checked the % of plasma cells (PCs) (CD138+ cells), % of total monocytes (CD14+ cells) and their two subsets (CD14+CD16- or CD14+CD16+ cells), the ratio between % of CD14+ and % of CD138+ cells (CD14+:CD138+ ratio) and the median fluorescence intensity (MFI) of CD38 and of the immuno-check points CD47 on PCs and CD172a (SIRPα) on monocytes. Subsequently, BM-MNCs were treated with control IgG (10µg/ml), DARA (10µg/ml) and the F(Ab)2 portion of DARA (DARA F(Ab)2) (10µg/ml) for 48 hours and then the % variation of surviving 7AAD- CD138+ cell, the modification of the % of monocyte or subsets analyzed and the % of PCs that are attached to monocyte (identified as CD138+ cells also positive to CD14) were evaluated by flow cytometry. Firstly, we found that DARA significantly exploited its anti-MM activity (median % variation of surviving CD138+ cells: 69.05%, p=0.0007) compared to IgG control while DARA F(Ab)2 did not have any killing effect (median % variation of surviving CD138+ cells: 97.33%) compared to the control. Secondly, we detected that the % of total of monocytes and their subsets (CD14+CD16+ or CD14+CD16-) composition were not modulated by DARA or DARA F(Ab)2 treatment compared to IgG. Indeed, only in presence of DARA, we observed that a double positive population of CD138+CD14+ cell significantly increased compared to IgG control (mean %: 15.76 vs 3.14, p=69.05% of surviving PCs). Interestingly, in the HR group compared to LR group were significantly increased the % of double positive CD138+CD14+ (median % HR:21.59 vs LR: 7.41, p=0.035), the % of CD138+ cells bonded to CD14+CD16+ (median % HR:13.28 vs LR: 3.93, p=0.048) and the CD14+:CD138+ ratio measured at T0 (median HR:0.63 vs LR: 0.32, p=0.0158). Moreover, in 5 relapsed MM patient, we have correlated the ex-vivo response to DARA with the type of in vivo response after DARA single-agent treatment. In addition in our cohort of patients, we observed that the % of surviving CD138+ cells after DARA treatment negatively correlate with the CD14+:CD138+ ratio at T0 (r:-0.628, p=0.0023), with the % of CD138+CD14+ population (r:-0.602, p=0.0039) and with the % of CD138+CD14+CD16+ population (r:- 0.657, p=0.0238) but did not correlate with the MFI of CD38 expression on PCs and the % of CD14+CD16+ at T0. Finally, to go further inside the mechanism involved in DARA response, we have explored the role of the inhibitory axis CD47-CD172a in the ADCP DARA-induced in 5 ex-vivo samples from our cohort of MM patients. We found that the MFI of CD47 strongly positively correlated with the % of surviving CD138+ cells (r: 0.9897; p=0.010) after DARA treatment; on the other hand, we have not reported any correlation with the MFI of CD172a on monocytes. The results of these analyses are continuously updating increasing the number of samples tested. In conclusion, these data highlight that monocyte binding to PCs, in particular those CD14+CD16+, plays a central role in DARA killing effect independently of % subset composition in the pre-treatment samples, suggesting that there are mechanisms that regulate the effectiveness of the monocyte-based killing effect on malignant PCs, as the immune-suppressive axis CD47-CD172a, giving the rationale design to identify new strategies to increase the efficiency of DARA-based therapeutic regimen. Disclosures Malavasi: Takeda Pharmaceutical Co: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceutica: Membership on an entity's Board of Directors or advisory committees, Research Funding. Aversa:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Basilea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Giuliani:Takeda Pharmaceutical Co: Research Funding; Celgene Italy: Other: Avisory Board, Research Funding; Janssen Pharmaceutica: Other: Avisory Board, Research Funding.

The 1q21 amplification, which occurs in approximately 40% of de novo and 70% of relapsed MM, is among the most frequent chromosomal aberrations in multiple myeloma (MM) patients and is considered a very high-risk genetic feature that is especially correlated with disease progression and drug resistance. To uncover novel 1q21 MM-critical genes, we first identified a list of 78 potential 1q21 drivers, which were located in the minimal common region of amplification of 254 MM samples and showed copy number-driven expression. These 78 candidates were then subjected to an shRNA screen to identify those genes involved in selective death and/or growth inhibition of MM cells carrying the 1q21 amplification. Using this approach, we identified and functionally validated the Interleukin-2 enhancer binding factor 2 (ILF2) as one of key 1q21 amplification-specific genes. ILF2 downregulation in 1q21-amplified MM cells resulted in multinucleated phenotypes and abnormal nuclear morphologies, findings that are consistent with the DNA damage-induced genomic instability that is associated with DNA repair defects that occur during cellular replication. Correspondingly, ILF2 downregulation was associated with a significant increase in the activation of the ATM (but not ATR or DNA-PK) pathway and accumulation of gH2AX foci, which are indicative of double-strand DNA breaks, and resulted in caspase 3-mediated apoptosis. Therefore, we sought to determine whether ILF2 is involved in the genome damage repair that occurs during cellular replication. To this end, we evaluated whether ILF2 depletion could affect the efficiency of non-homologous end joining (NHEJ) or homologous recombination (HR), the two major repair pathways in mammalian cells. We observed a profound impairment of HR in ILF2-depleted cells (p=0.038), whereas NHEJ was unaltered after ILF2 downregulation. Conversely, enforced ILF2 expression significantly enhanced HR efficiency in MM cells (p=0.008). To further support the role of ILF2 in the regulation of the DNA repair pathway in MM cells, we evaluated whether ILF2 downregulation increased MM sensitivity to DNA-damaging agents routinely used in the treatment of MM. Employing the interstrand crosslinker melphalan as an instigator of double-strand DNA breaks, we found that ILF2-depleted MM cells subjected to continuous melphalan treatment showed increased accumulation of γH2AX and apoptosis. Consistent with these findings, elevated ILF2 expression significantly correlated with poor survival in MM patients treated with high-dose melphalan followed by tandem autologous transplantation (n=256, p=0.01). Mechanistically, mass spectrometry analysis showed that ILF2 interacted with numerous RNA binding proteins directly involved in the regulation of DNA damage response by modulating alternative splicing of specific pre-mRNAs. RNA-sequencing experiments confirmed that ILF2 depletion resulted in aberrant splicing of genes involved in the DNA repair pathway, including ERCC1, FANCD2, and EXO1. RNA immunoprecipitation sequencing experiments showed that ILF2 directly bound to transcripts involved in the regulation of the HR pathway, including components of BRCA1 protein complex. Furthermore, in an attempt to dissect the ILF2 protein interacting network involved in the DNA repair regulation in response to DNA damage activation, we found that ILF2 mediated drug resistance in a dose-dependent manner by modulating YB-1 nuclear localization and interaction with the splicing factor U2AF65 to promote mRNA processing and stabilization of DNA repair genes, including FANCD2 and EXO1, in response to DNA damage. In conclusion, our study reveals an intimate relationship among 1q21 amplification, mRNA splicing, and DNA repair in the control of DNA damage response in MM. Given that 1q21 amplification is one of the most frequent copy number alterations in cancer, synthetic lethality approaches based on targeting gain-of-functions associated with ILF2 may have a broad spectrum of applications to potentiate the sensitivity of cancer cells to chemotherapeutic agents. Disclosures Giuliani: Janssen: Research Funding; Celgene: Research Funding.

Multiple Myeloma (MM) is a plasma cell malignancy characterized by the tight dependence to the bone marrow (BM) microenvironment that supports MM cells survival. Despite significant therapeutic progress in the recent years with the introduction of several new drugs, MM remains an incurable disease. Oncolytic virotherapy is an alternative therapeutic technology in the cancer treatment exploiting naturally or genetically engineered viruses able to infect, transduce and consequently kill cancer cells directly or indirectly through the delivery of the microenvironment cells. Several oncolytic viruses have shown promising pre-clinical results for the treatment of MM, in particular Measles virus and Reovirus. However, the use of human viruses such as Measles could be limited by the antiviral immune response of the patients due to vaccination or natural infection. In order to avoid these potential limits of the human viruses, the aim of this study was to investigate the development of bovine viruses as an alternative oncolytic strategy in MM by checking these viruses that showed an anti-tumoral activity in different solid tumors. Thus, we investigated the potential lytic effect on human MM cells of the Bovine Viral Diarrhea Virus (BVDV), known to bind CD46 as reported for human measles virus, and the Oncolytic Bovine Herpesvirus type 4 (BoHV-4). Firstly, we treated several human myeloma cell lines (HMCLs) with BVDV or the heat-inactivated virus for 24, 48 and 72 hours. The infection efficiency was verified by nested multiplex PCR. We showed a significant increase of cell mortality, checked by trypan blue count and flow cytometry analysis, already after 48 hours of infection in JJN3 (mean±SD % of dead cells: BVDV 45±11 % vs inactive virus 16±2.5 %, p=0.013), and in OPM2 (BVDV 43±1.4 % vs inactive virus 28±2.1 %, p= 0.015) but not in U266 (BVDV 25±23 % vs inactive virus 18±12 %, p=NS. However, BVDV pre-treatment for 12 hours and followed by 48 hours bortezomib (bor) treatment (concentration ranging: 5-10nM) significantly restored bor sensitivity in U266 resistant cells (mean±SD % of dead cells: BVDV plus bor 10 nM 69±8 % vs inactive virus + bor 10 nM 36±1 %, p= 0.031). Interestingly, the cytotoxic effect of BVDV treatment in HMCLs was associated by a significantly increase of apoptotic markers evaluated by flow cytometry. Subsequently, we infected BM primary purified CD138+, showing a significant increase of the mortality rate after treatment with BVDV as compared to the inactivated virus. On the contrary, BVDV was not able to infect human BM mesenchymal stromal cells (MSCs) not showing any lytic effect. Thereafter the capacity to induce MM cell lysis by a recombinant BoHV-4 virus, delivering a Red Fluorescent Protein (RFP) expression cassette as reporter gene, was also evaluated. As observed by the percentage of RFP-positive cells, BoHV-4 was unable to infect and consequently to kill several HMCLs tested. Then we used BM MSCs as in vitro model for oncolytic virus delivery in co-culture systems with MM cells. BoHV-4 infected hTERT-MSCs, expressing RFP at 24, 48 and 72 hours. Consistently, hTERT-MSCs viability was progressively reduced at 24 and 48 hours after infection, as compared to controls, (mean±SD % reduction of cell viability: -22±8 %, p=0.0254 and -49±2 %, p=0.0001, respectively), reaching the highest effect at 72 hours (-70±1.5 %, p=0.0003). Thus we evaluated the effect of BoHV-4 in a co-culture system between human MSCs and two stroma-dependent HMCLs as INA-6 and saMMi. In both cases the percentage of dead HMCLs increased in co-culture with BoHV-4 infected hTERT-MSCs, as compared to hTERT-MSCs untreated controls (INA-6: BoHV-4 61±2.1 % vs control 12±2.1 %, p= 0.0018; saMMi: BoHV-4 48±1.9 % vs control 14±1.4 %, p= 0.0027). Overall our data indicate both direct and MSC-mediated oncolytic effects of bovine viruses on MM cell, suggesting their possible use as novel alternative anti-MM virotherapy strategy. Disclosures Giuliani: Celgene: Research Funding; Janssen: Research Funding.

The malignant plasma cells (PC) dependence on the bone marrow (BM) microenvironment is a main feature of multiple myeloma (MM), mainly due to high expression of cell adhesion molecules including CD44, CD54 (ICAM1), CD56, CD49d and chemokines receptors such as CXCR4. At the end stage of the disease, a rare MM complication, that may occurs, is a secondary extramedullary involvement without a leukemic phase and, among the extramedullary localizations, the skin is one of the possible sites, due to the physiological homing of PCs. However, the mechanisms behind the extramedullary spread are not completely understood. In a patient with refractory resistant MM, who has developed a cutaneous localization after 16 months from the diagnosis under Bortezomib (BOR) treatment, we analyzed the gene expression profiles (GEPs) by GeneChip U133 Plus 2.0 (Affymetrix), the immunophenotypic and immunohistochemistry (IHC) profiles of MM cells, across the course of the disease. To confirm the results, IHC profiles on selected molecules were then analyzed in other six MM patients with secondary skin involvement without PC leukemia. On BM CD138+ PCs at diagnosis and BM relapse, by GEP analysis, 742 genes resulted differentially expressed. Moreover, a more stringent analysis identified 19 down-regulated and 42 up-regulated genes; data were confirmed by Real Time PCR on selected genes mainly involved in PC homing. At the BM relapse, we found that MM cells expressed CXCR4 and were negative for CD56; moreover, a significant down-regulation of ICAM1 (CD54) and ALCAM was observed together with the up-regulation of MAGE family genes, DKK1 and SEMA3A in the BM relapse sample compared to diagnosis one. At the cutaneous involvement development, 4 months after BM relapse, by IHC analysis, the MM cells localized in the skin showed the expression of both CD56 and CD54. On the other hand, the BM MM cells immunophenotype confirmed the presence of CD56 but showed the lack of CXCR4 expression. IHC for CD54, CD56 and CXCR4 expression was then performed on both skin and bone biopsies of the other patients analyzed. To go further inside on the role of CXCR4 we treated with BOR (5-10nM) for 24-48h human myeloma cell lines (HMCLs) RPMI-8226 and U266, known to be resistant to BOR, evaluating the CXCR4 expression at both mRNA and protein level, by real-time PCR and by cytofluorimetry, respectively. CXCR4 resulted downregulated after BOR treatment in BOR resistant HMCLs. Consistently, the loss of CXCR4 expression was recently associated to BOR resistance and extramedullary disease in a mouse model of MM suggesting that CXCR4 loss can be correlated with BOR resistance and PCs mobilization from BM, leading to an extramedullary disease. In conclusion, our data indicate that the loss of CXCR4 expression by MM cells across the disease progression, together with CD54 and CD56 down-regulation and their re-acquisition at the extramedullary site, are involved driving the escape of PCs from BM to the extramedullary localization in the skin in the context of Bortezomib resistance. Disclosures No relevant conflicts of interest to declare.

The relationship between multiple myeloma (MM) cells and osteoblast (OB)s and osteoclast (OCL)s into the bone marrow (BM) niche has a critical role in the pathophysiology of MM and in the development of bone disease in MM patients. Recently, Daratumumab (DARA), a human anti-CD38 monoclonal antibody has been developed with broad-spectrum killing activity including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and, at least in part, modulation of CD38 enzymatic activity. In pre-clinical studies, DARA has been shown to effectively kill MM cells and clinical trials are ongoing. However, the effects of DARA in the context of the MM bone niche and on MM-induced bone remodeling alterations are unknown. In this study, we have firstly evaluated the expression profile of CD38 and its related ectoenzymes by bone niche cells and, thereafter, we investigated the effect of DARA on OCL and OB formation and bone remodeling. The study design included immunohistochemistry for CD38, CD73, CD39, CD203a (PC-1), CD157 and CD31 on bone biopsies in a cohort of 38 patients with newly diagnosed MM and 14 patients with monoclonal gammopathy of uncertain significance (MGUS). Moreover, the expression profile of these antigens was performed by flow cytometry on primary BM CD138+ cells (sample number=16), mesenchymal stromal cells, OBs, monocytes and OCLs and on the related human cell lines. As expected, we found that CD38 was highly expressed by MM cells that were also positive for CD203a and for CD39 and CD31 at variable level but not for CD157 and CD73. CD38 was also expressed by BM monocytes but not by OBs, OCLs and BM stromal cells. Interestingly, we found that OBs expressed CD73 and CD203a. Any significant difference was not observed in the expression of CD38 and related ectoenzymes between MM and MGUS patients. In line with CD38 expression profile by MM cells and the niche, we further demonstrated that DARA binds both MM cells and monocytes, but not OBs and OCLs. Consistently, we lack to find any significant effect of DARA on OB formation from BM stromal cells or OBs proliferation and survival. Thus, we investigated the effect of DARA (1-25 ug/ml) as compared to human IgG isotype control on OCL formation and activity in the presence of RANKL and M-CSF, using either CD138- cell fraction or purified CD14+ cells from MM BM samples. OCLs were evaluated by both TRAP staining and a fluorimetric osteolysis assay. We found that DARA, between 10 and 25 ug/ml, with a dose dependent effect, significantly inhibited OCL formation and activity from BM mononuclear cells and from the CD138- cell fraction, but not from purified CD14+ cells. The inhibitory effect on OCL formation by DARA was observed when the antibody was present for all the culture period (21-28 days). On the other hand DARA did not show any effect on late OCL progenitors and mature OCLs. Accordingly CD38 expression by monocytes cultured in a pro-osteoclastogenic medium disappeared after 7 days. In conclusion, our data indicate that DARA inhibit osteoclastogenesis, likely mediated by ADCC, targeting monocytes and early OCL progenitors. This evidence supports the use of an anti-CD38 based approach as a treatment for MM bone disease. Disclosures Malavasi: Janssen: Honoraria, Research Funding. Giuliani:Celgene Italy: Other: Research Grant; Janssen Pharamceutical: Other: Research Grant.

The immunomodulatory drugs (IMiDs®) exert an anti-myeloma effect by cereblon-dependent destruction of IKZF proteinseither through a direct action on multiple myeloma (MM) cells or through indirect immunomodulatory and anti-angiogenic effects.Previous data indicated that MM cells overexpress hypoxia inducible factor (HIF)-1α and that HIF-1α suppression significantly blocks MM-induced angiogenesis and reduces in vivo tumoral burden in MM mouse models. Interestingly, it has been recently reported that HIF-1α knock-down in MM cells potentiates the in vitro effect of Lenalidomide (LEN) on cell proliferation without changing cell viability and that LEN is not able to suppress HIF-1α expression in MM cells. These evidences give the rationale design to investigate the in vivo effect of HIF-1α stable suppression in MM cells on LEN sensitivity. Thus, in this study, we assessed the effect of LEN in vivo in combination with HIF-1α inhibition in a non-obese diabetic/severe combined immunodeficiency (NOD/SCID) subcutaneous mouse model using JJN3, a cell line known to be resistant to the cytotoxic effect of LEN. Different groups of animals were injected with JJN3-pLKO.1 (empty vector) or JJN3-anti-HIF1α, obtained by anti-HIF1α lentiviral shRNA pool. When tumors became palpable, mice were treated with LEN (5mg/kg), using the intraperitoneal route. After three weeks, we evaluated tumor volume and weight. Moreover, by immunohistochemistry on ex vivo plasmacytomas, we evaluated the expression of p27 and the microvascular density (MVD), checked by CD34 immunostaining. In addition, the expression of a p27 inhibitor, the S-phase kinase-associated protein 2 (SKP2), the expression of the HIF-1α target key mediator of glycolysis and tumoral growth, Hexokinase II (HK2), and the levels of pERK 1/2, and total Caspase-3 (Casp-3) were evaluated in the ex vivo plasmacytomas lysates by western blot. We found that LEN treatment induced a significant weight and volume reduction of the tumor burden in mice injected with JJN3 anti-HIF1α as compared to JJN3-pLKO.1. The p27 nuclear expression was significantly increased by LEN treatment in JJN3-anti-HIF1α as compared to JJN3 pLKO.1 mice and compared to JJN3-anti-HIF1α mice treated with vehicle. In addition, we demonstrated that LEN in combination with HIF-1α inhibition significantly reduced in vivo the expression of p-ERK1/2, total Casp-3, HK2 and the p27 inhibitor, SKP2. Because it is known that LEN exerts its anti-MM effect targeting the IKZF proteins, we further checked in vitro whether the effect of HIF-1α suppression and LEN treatment combination could be mediated by IKZF proteins modulation. Interestingly, after LEN (2-10µM) treatment we found that both IKZF1 and IKZF3 were not differentially expressed, whereas IRF4 was down regulated, in JJN3-anti-HIF1α as compared to JJN3 pLKO.1. Finally, regarding a possible combinatory effect on the in vivo angiogenesis, we found that both the number of CD34 positive vessels and the MVD were reduced in mice colonized by JJN3-anti-HIF1α as compared to JJN3-pLKO.1. On the other hand, LEN treatment did not further significantly reduce the number of CD34 positive vessels and the MVD. Accordingly, we did not find any significant inhibitory effect by LEN treatment on the main pro-angiogenic molecules in JJN3 anti-HIF1α as compared to JJN3 pLKO.1 even after 72 hours. Overall, our data indicate that HIF-1α suppression in MM cells significantly increases the anti-MM effect of LEN in vivo, mainly through the inhibition of proliferation signaling including the modulation of p27 pathway and the IKZF target protein IRF4, rather than to an anti-angiogenic effect. These data suggest that the combination of LEN and HIF-1α inhibition has a therapeutic rationale in MM. Disclosures No relevant conflicts of interest to declare.