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Deregulated expression of lineage-affiliated transcription factors is a major mechanism of oncogenesis. However, how deregulation of non-lineage affiliated TF impacts chromatin to initiate oncogenic transcriptional programmes is not well known. To address this, we studied the chromatin effects imposed by oncogenic MAF as the cancer-initiating driver in the plasma cell cancer multiple myeloma. We found that the ectopically expressed MAF endows myeloma plasma cells with migratory and proliferative transcriptional potential. This potential is regulated by activation of enhancers and super-enhancers, previously inactive in normal B cells and plasma cells, and in co-operation of MAF with the plasma cell-defining TF IRF4. Forced ectopic MAF expression confirms the de novo ability of oncogenic MAF to convert transcriptionally inert chromatin to active chromatin with features of super-enhancers, leading to activation of the MAF-specific oncogenic transcriptome and acquisition of cancer-related cellular phenotypes such as CCR1-dependent cell migration. These findings establish oncogenic MAF as a pioneer transcription factor that can initiate as well as sustain oncogenic transcriptomes and cancer phenotypes. However, despite its pioneer function, myeloma cells remain MAF-dependent thus validating oncogenic MAF as a therapeutic target that would be able to circumvent the challenges of subsequent genetic diversification driving disease relapse and drug resistance.

Introduction: Pomalidomide (POM) plus low-dose dexamethasone (POM/LoDex) is a standard of care for patients with relapsed/refractory multiple myeloma (RRMM), who have received ≥2 prior therapies, including lenalidomide (LEN) and bortezomib (BORT). In view of the limited real-world evidence on POM/LoDex effectiveness, this study aimed to provide insight into progression-free survival (PFS), response to treatment and drug utilization patterns in the routine clinical practice in Greece. Methods: 'POWERFUL' (NCT03353545) was a non-interventional, multicenter, retrospective and prospective study of adult RRMM patients, initiated on POM/LoDex between 01-Jan-2016 and 28-Feb-2019 per the approved label. Patients who had received up to one POM/LoDex cycle were consecutively enrolled between 16-Nov-2017 and 21-Feb-2019 (prospective recruitment phase). One month prior to completion of this phase, aiming to facilitate recruitment of the target size, patients, whose treatment with POM/LoDex was ongoing but >1 cycles had been received or for whom treatment was discontinued, were consecutively enrolled in reverse chronological order, based on POM/LoDex start date (retrospective recruitment). Patients were observed until the earliest point of disease progression (PD), death, informed consent (IC) withdrawal, treatment discontinuation for reason other than PD or start of next antimyeloma therapy, physician's decision, or last patient enrolled plus up to 12 months of treatment. IC or consent waiver for deceased subjects was obtained. Safety data were collected prospectively. Results: Eligible patients (N=99; 75 with prospective and 24 with a purely retrospective follow-up) were recruited by 18 hematology departments. The median (IQR) observation period was 8.8 (4.2-15.4) months. Baseline patient characteristics are presented in Table 1. Fifty patients (50.5%) started POM/LoDex as third-line treatment. POM was initiated at 4 mg/day on days 1-21 every 28 days in 75.8% of the patients. A median of 8 (range: 1-38) cycles were received at a median POM dose of 4 mg/day (range: 1-4), over 8.3 (range: 0.3-47.6) months. The POM dose reduction and interruption rates were 28.3% and 59.6%, respectively. Of the patients, 37.4% were treated for ≥ one year. Treatment was discontinued in 81.8%, due to PD (56.8%), safety reasons (22.2%), death (6.2%), other reasons (9.9%), and due to loss of follow-up (4.9%). The investigator-assessed overall response rate (≥partial response [PR]) was 32.3%; best response rates were: stringent complete or complete response (7.1%), very good PR (8.1%), PR (17.2%), minimal response (11.1%), stable disease (21.2%), PD (12.1%), and non-evaluable response (23.2%). Median time to response and duration of response in patients achieving ≥PR was 3.2 [95% confidence interval (CI): 2.6-3.6] and 15.8 (95%CI: 11.3-not reached) months, respectively. Over a Kaplan-Meier estimated median follow-up of 13.8 months, the median PFS was 10.5 months (95%CI: 7.4-14.4) [13.0 vs. 8.8 months for patients treated in the third vs. the fourth- and beyond line (log-rank p=0.494), and 13.0 vs. 8.8 months for patients treated with POM/LoDex only vs. those co-administered other antimyeloma agents (log-rank p=0.411)]. The estimated 6-, 12-, 24- and 36-month PFS rates were 70.3%, 48.3%, 20.1% and 12.0%, respectively. Multivariate Cox regression analysis of the impact of baseline characteristics on PFS indicated male sex [hazard ratio (HR)=2.08; 95%CI: 1.12-3.89; p=0.021] and higher than normal serum lactate dehydrogenase levels (HR=2.84; 95%CI: 1.39-5.81; p=0.004) as negative predictors of PFS. Over a median safety data collection period of 7.6 months (range: 0.4-18.6), the POM-related adverse events (ADR) incidence rate in the safety-evaluable population (i.e., 75 patients with prospective follow-up) was 42.7% (73 events). Only neutropenia (13.3%) was reported at a frequency ≥10%. The serious ADR rate was 18.7%, whereas grade ≥ 3 hematological and non-hematological ADR rates were 8.0%, and 5.3%, respectively. Conclusion: In this Real-World dataset from Greek centers, POM/LoDex, administered in the third-line and beyond setting of RRMM, displayed a longer PFS than in controlled clinical trials, which was not impacted by the treatment line and concurrent receipt of other antimyeloma agents. About one-third of patients achieved at least PR with an about 16-month response duration. Disclosures Terpos: Amgen: Honoraria, Research Funding; Sanofi: Honoraria; Celgene: Honoraria; Takeda: Honoraria, Other: travel expenses , Research Funding; Janssen: Honoraria, Research Funding; Genesis pharma SA: Honoraria, Other: travel expenses , Research Funding; BMS: Honoraria. Repousis:Genesis pharma SA: Membership on an entity's Board of Directors or advisory committees, Research Funding. Hatjiharissi:Roche: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis pharma SA: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees. Assimakopoulou:Genesis pharma SA: Research Funding. Vassilopoulos:Abbvie: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Genesis pharma SA: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding. Pouli:Genesis pharma SA: Research Funding. Spanoudakis:Genesis pharma SA: Research Funding. Michali:Takeda: Research Funding; Genesis pharma SA: Research Funding. Pangalis:Genesis pharma SA: Research Funding. Poziopoulos:Genesis pharma SA: Research Funding. Kyrtsonis:Genesis pharma SA: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Pappa:Genesis pharma SA: Research Funding. Symeonidis:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GenesisPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck Sharp & Dohme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; WinMedica: Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Research Funding; Astellas: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Georgopoulos:Genesis pharma SA: Research Funding. Zikos:Genesis pharma SA: Membership on an entity's Board of Directors or advisory committees, Research Funding. Papadaki:Genesis pharma SA: Membership on an entity's Board of Directors or advisory committees, Research Funding. Dadakaridou:Genesis pharma SA: Research Funding. Karvounis-Marolachakis:Genesis pharma SA: Current Employment. Patos:Genesis pharma SA: Current Employment. Katodritou:Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Theagenion Cancer Hospital: Current Employment; Takeda: Honoraria, Other: Expenses, Research Funding; Genesis Pharma: Honoraria, Other: Expenses, Research Funding; Abbvie: Research Funding; Karyopharm: Research Funding.

A staging system was developed a decade ago for patients with Waldenstrom’s macroglobulinemia (WM), however, since then WM treatments have changed. A revised staging system could better capture prognosis of WM patients in the chemoimmunotherapy era. We developed a revised system based on data from 492 symptomatic patients with at least 3 years and a median of 7 years of follow up while an independent validation cohort included 229 symptomatic patients. We identified age (≤65 vs 66–75 vs ≥76 years), b2-microglobulin ≥ 4 mg/L, serum albumin 65 years and

Introduction The use of lenalidomide in frontline therapy for patients (pts) with newly diagnosed multiple myeloma (NDMM) has increased the number of pts who become refractory to lenalidomide at second line. Thus, there is an ongoing need for new treatment options for lenalidomide refractory patients at first relapse. Daratumumab, an anti-CD38 monoclonal antibody, has shown significant activity and acceptable safety in pts with relapsed/refractory multiple myeloma (RRMM) as monotherapy or in combination with other agents. Ixazomib, the first oral proteasome inhibitor, in combination with lenalidomide and dexamethasone also improves key survival endpoints in RRMM. This study assessed the efficacy of daratumumab in combination with ixazomib and dexamethasone (Dara-Ixa-dex) as second-line therapy in pts with RRMM who have been previously treated with a lenalidomide-based regimen. Methods DARIA is an ongoing, prospective, open-label, multicenter, phase 2 study. Eligible adult pts with RRMM had measurable disease after one prior line with a lenalidomide-based regimen and a Karnofsky Performance Status (KPS) score of ≥70. Exclusion criteria included previous anti-CD38 or ixazomib treatment, treatment with CYP3A inducers or use of St. John's wort within 14 days before C1D1, anti-myeloma treatment within 2 weeks or 5 pharmacokinetic half-lives of the treatment prior to C1D1 and allogenic or autologous stem cell transplantation (ASCT) within 12 weeks before C1D1. Treatment with Dara-Ixa-dex comprises an induction phase of nine 28-days cycles and a maintenance phase. In induction, pts received DARA 16mg/kg (weekly for cycles 1-2, bi-weekly for cycles 3-6, and every 4 weeks thereafter) administered intravenously until November 2020 and subcutaneously at a fixed dose of 1800 mg thereafter; 4mg oral ixazomib (days 1, 8, and 15 of each cycle); and 40mg oral dexamethasone (weekly, each cycle). In maintenance, Dara-Ixa were administered every 4 weeks until disease progression or unacceptable toxicity, with dexamethasone being discontinued. The primary endpoint was overall response rate (ORR). Secondary endpoints included progression-free survival (PFS), the toxicity profile of Dara-Ixa-dex, and the effects of the combination on serum bone metabolism markers (C-terminal telopeptide of type 1 collagen [CTX], tartrate-resistant acid phosphatase isoform 5b [TRACP-5b], bone-specific alkaline phosphatase [bALP], and osteocalcin [OC]) from baseline until disease progression. Results The study has completed accrual and 50 pts have been enrolled (mean [range] age: 69.0 (50.0-89.0) years; female: 28 [56.0%]). At screening, 24 (48.0%) pts had a KPS score ≥90, and most pts were at stage ≤II by the International Staging System (ISS; 42, 84.0%) or by the revised ISS (47, 94.0%). Thirty-two (64.0%) pts were refractory to lenalidomide, and 15 (37.5%) had prior ASCT. The median (range) number of study treatment cycles was 9 (1-26). ORR was 50.0% (25 pts); one (2.0%) pt had a complete response, 13 (26.0%) a very good partial response (VGPR), and 11 (22.0%) pts had a PR. The median (range) time from first Dara-Ixa-dex dose until first response (≥PR) is 1.0 (0.9-3.7) month. The median PFS was 8.1 months (95% CI: 5.8-15.6) (Figure). After a median (range) follow-up of 12.4 (0.9-28.4) months, 14 (28.0%) pts are still on treatment; reasons for treatment discontinuation were progressive disease (28 pts, 77.8%), physician's decision and fatal serious adverse event [SAE] (3 pts, 8.3% each), and adverse event [AE] (2pts, 5.6%). Following 15 months of treatment, the median change from baseline for CTX, TRACP-5b, bALP and OC were significant (p Overall, 20 (40.0%) pts have ≥1 grade 3/4 AE, the most common being thrombocytopenia (10 pts, 20.0%), and 14 (28.0%) pts have ≥1 SAE, the most common being acute kidney injury and pneumonia (2 pts [4.0%] each condition). Four fatal SAEs were reported (pneumonia, infection, urinary tract infection, and lower respiratory tract infection). Conclusions Second-line treatment with Dara-Ixa-dex in pts with RRMM who were pre-treated with a lenalidomide-based regimen resulted in rapid ( Figure 1 Figure 1. Disclosures Terpos: Genesis: Consultancy, Honoraria, Research Funding; BMS: Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; GSK: Honoraria, Research Funding; Janssen-Cilag: Consultancy, Honoraria, Research Funding; Novartis: Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding. Gavriatopoulou: Sanofi: Honoraria; Genesis: Honoraria; GSK: Honoraria; Karyopharm: Honoraria; Amgen: Honoraria; Janssen: Honoraria; Takeda: Honoraria. Katodritou: GSK, Amgen, Karyopharm, Abbvie, Janssen-Cilag, Genesis Pharma, Sanofi: Honoraria, Research Funding. Hatjiharissi: Gilead: Honoraria; Genesis: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Verrou: Karyopharm: Research Funding; Takeda: Honoraria; Genesis: Honoraria; Abbvie: Honoraria, Research Funding; Janssen Cilag: Honoraria, Research Funding; Roche: Honoraria; Amgen: Honoraria. Leonidakis: Health Data Specialists: Current Employment. Delimpasi: Amgen: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Takeda: Honoraria, Speakers Bureau. Symeonidis: MSD: Consultancy, Research Funding; GSK: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Consultancy, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GenesisPharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi/Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Demo: Research Funding; WinMedica: Research Funding. Dimopoulos: Amgen: Honoraria; Janssen: Honoraria; BMS: Honoraria; Beigene: Honoraria; Takeda: Honoraria.

Introduction Despite the availability of novel agents in treating multiple myeloma (MM), renal impairment (RI) remains a poor prognostic factor, and the median survival of patients (pts) with MM and RI is approximately half of that for MM pts with normal renal function. RI can affect up to 50% of pts with MM at presentation, highlighting the need for effective treatment options for this patient population. Daratumumab, an IgG1 κ human monoclonal antibody that targets CD38, has shown efficacy and a favorable safety profile in pts with relapsed or refractory MM (RRMM). The DARE study assessed the safety and efficacy of daratumumab with dexamethasone in pts with RRMM and severe RI or requiring hemodialysis. Methods DARE is a prospective, open-label, phase 2 study, conducted in eight sites in Greece and Italy. Eligible pts were adults with documented RRMM and severe RI (estimated glomerular filtration rate [eGFR] Results The study has completed accrual and 38 pts were enrolled. The pts' median (range) age was 72 (40-89) years, and most pts were male (29, 76.3%). At baseline, 37 (97.4%) pts had ECOG PS ≤1 and 34 (89.5%) were at International Staging System (ISS) stage III. By revised ISS, 20 (52.6%) and 16 (42.1%) pts were at stages II and III, respectively. Pts had a median (range) of 3 (2-6) prior systemic therapies; thirteen (34.2%) pts had undergone prior autologous stem cell transplantation. The median eGFR at baseline was 13.0 mL/min/1.73m 2 and seventeen (44.7%) pts were on dialysis at the time of enrollment. The median (range) number of cycles given was 8.0 (1.0-38.0), and the median (range) follow-up was 11.3 ( Overall, 19 (50.0%) pts had ≥1 grade 3/4 adverse event (AE), and 10 (26.3%) had ≥1 serious AE (SAE). The most common grade 3/4 AEs were anemia (6 pts, 15.8%), hyperglycemia (5 pts, 13.2%), and hypercalcemia (3 pts, 7.9%). The most common SAE was septic shock (3 pts, 7.9%). Conclusions The administration of daratumumab with dexamethasone in pts with RRMM and severe RI or requiring hemodialysis is safe and effective therapy associated with a median PFS of approximately 12 months; hematologic responses were rapid and observed within one month from treatment initiation and approximately one-fifth of pts achieved a major renal response. Almost half of the pts were on dialysis and the combination was active and safe also for those on dialysis. No new safety signals were observed with daratumumab in pts with RRMM and severe RI or in need of dialysis. Figure 1 Figure 1. Disclosures Kastritis: Janssen: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Genesis Pharma: Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Terpos: Sanofi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Novartis: Honoraria; Janssen-Cilag: Consultancy, Honoraria, Research Funding; GSK: Honoraria, Research Funding; Genesis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Symeonidis: Demo: Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; WinMedica: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GenesisPharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Consultancy, Research Funding; Sanofi/Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Consultancy, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Delimpasi: Janssen: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Takeda: Honoraria, Speakers Bureau. Cavo: Bristol-Myers Squib: Consultancy, 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, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Speakers Bureau; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive Biotechnologies: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Accommodations, Speakers Bureau; Novartis: Honoraria. Zamagni: Janssen: Honoraria; Bristol-Myers-Squibb: Honoraria; Takeda: Honoraria; Amgen: Honoraria. Katodritou: GSK, Amgen, Karyopharm, Abbvie, Janssen-Cilag, Genesis Pharma, Sanofi: Honoraria, Research Funding. Kyrtsonis: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/Genesis Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria; Sanofi: Membership on an entity's Board of Directors or advisory committees. Hatjiharissi: Gilead: Honoraria; Genesis: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Leonidakis: Health Data Specialists: Current Employment. Manousou: Health Data Specialists: Current Employment. Gavriatopoulou: Sanofi: Honoraria; Genesis: Honoraria; Amgen: Honoraria; Janssen: Honoraria; GSK: Honoraria; Karyopharm: Honoraria; Takeda: Honoraria. Dimopoulos: Amgen: Honoraria; Beigene: Honoraria; Janssen: Honoraria; BMS: Honoraria; Takeda: Honoraria.

Introduction: Osteolytic bone disease is the most common clinical feature of multiple myeloma (MM), affecting approximately 80% of patients (pts) at diagnosis. Non-invasive biomarkers of bone resorption and formation are indicative of bone dynamics and have been used to assess bone metabolism during anti-myeloma treatment. Daratumumab (dara), an IgG1 κ human monoclonal antibody targeting CD38, is approved as treatment of patients with relapsed and/or refractory MM (RRMM). Moreover, dara inhibits in vitro osteoclastogenesis and bone resorption. The present study investigated the impact of dara monotherapy on bone remodeling in pts with RRMM who have had ≥2 prior lines of therapy. Methods: REBUILD was a prospective, non-comparative, open-label, phase 2 study, conducted in six centers in Greece. Eligible pts were adults with documented RRMM, who have had ≥2 prior lines of therapy (including lenalidomide and a PI), evidence of progressive disease by International Myeloma Working Group criteria, a Karnofsky Performance Status score ≥70, and creatinine clearance ≥30 mL/min. Exclusion criteria included previous treatment with an anti-CD38 antibody, such as dara. Pts received intravenous dara at a 16 mg/kg dose weekly for 8 weeks, then every 2 weeks for an additional 16 weeks, followed by 4 weeks interval thereafter. The primary endpoint was the change from baseline in the bone resorption markers C-terminal cross-linking telopeptide of type 1 collagen (CTX) and tartrate-resistant acid phosphatase isoform 5b (TRACP-5b) after 4 months of dara monotherapy. Secondary endpoints included the change from baseline to 4 months of treatment in selected bone formation markers (e.g., bone-specific alkaline phosphatase [bALP], osteocalcin [OC], and procollagen type-I N-propeptide [PINP]), markers of osteoclast regulation (RANKL, osteoprotegerin, dicckopf-1 [DKK-1], sclerostin and C-C motif ligand-3 [CCL3]), progression-free survival (PFS), and overall survival (OS). Results: Among the 57 pts enrolled in the study, 33 (57.9%) pts had biomarker and other clinical data available on treatment initiation (baseline) and after 4 months of treatment; the following analyses refer to this pt subgroup. The pts' median (range) age was 73.0 (52.0-84.0) years, and most were female (18, 54.5%). Most (16, 48.5%) pts had >10 lytic bone lesions at baseline. Six (18.2%) pts received bisphosphonates along with dara monotherapy. The ORR (Partial Response or better [≥PR]) was 63.6% (CR: 3.0%, VGPR: 21.2%, PR: 39.4%). The respective 4-month ORR was 57.6% (VGPR: 24.2%, PR: 33.3%) The median CTX change from baseline to 4 months was 3.9%, with 13 (39.4%) and 11 (33.3%) pts exhibiting ≥20% and ≥30% reduction in CTX levels, respectively. The TRACP-5b levels decreased from baseline to 4 months by a median of 2.6%, with 10 (30.3%) and 6 (18.2%) pts showing ≥20% and ≥30% reduction in TRACP-5b levels, respectively. The median changes from baseline to 4 months in the CTX and TRACP-5b levels for pts with a ≥PR at 4 months were -1.3% and -2.6%, respectively; the respective changes for pts not achieving favorable response (i.e., pts with minimal response, stable disease, disease progression, or no response assessment) were 5.3% and -7.2%. The levels of the bone metabolism biomarkers bALP, OC, and PINP increased from baseline to 4 months, the median changes being 18.4%, 92.6% and 10.2%, respectively. For pts with ≥PR at 4 months, the median changes from baseline to 4 months in bALP, OC, and PINP levels were 25.3%, 146.0% and 15.7%, respectively; the respective changes for pts not achieving favourable response were 18.3%, 15.6% and -7.3% (Table 1). Other significant differences at 4 months included the decrease in DKK-1 by a median of 17.5%, the decrease in CCL3 by 16.0% (Table 1). The median (95% confidence interval) PFS and OS were 9.3 (6.7-15.3) and 21.2 (11.4-not reached) months, respectively; the respective results for all 57 patients were 4.7 (3.0-7.2) and 10.5 (8.4-18.1) months. Conclusions: In these highly pre-treated MM pt group, dara monotherapy showed a positive effect on bone metabolism which was more profound among responders; OC had a ~3-fold increase after 4 months of therapy. The reduction in TRACP-5b and in CCL-3 but not in CTX suggests a mild inhibitory effect on osteoclasts by dara, with a statistically non-significant trend to be greater in responders. Figure 1 Figure 1. Disclosures Terpos: Novartis: Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Genesis: Consultancy, Honoraria, Research Funding; GSK: Honoraria, Research Funding; Janssen-Cilag: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Kastritis: Janssen: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Genesis Pharma: Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Hatjiharissi: Gilead: Honoraria; Genesis: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Katodritou: GSK, Amgen, Karyopharm, Abbvie, Janssen-Cilag, Genesis Pharma, Sanofi: Honoraria, Research Funding. Verrou: Amgen: Honoraria; Genesis: Honoraria; Abbvie: Honoraria, Research Funding; Takeda: Honoraria; Roche: Honoraria; Janssen Cilag: Honoraria, Research Funding; Karyopharm: Research Funding. Gavriatopoulou: GSK: Honoraria; Karyopharm: Honoraria; Takeda: Honoraria; Janssen: Honoraria; Amgen: Honoraria; Genesis: Honoraria; Sanofi: Honoraria. Leonidakis: Health Data Specialists: Current Employment. Delimpasi: Janssen: Honoraria, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Kyrtsonis: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/Genesis Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria; Sanofi: Membership on an entity's Board of Directors or advisory committees. Symeonidis: AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi/Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Research Funding; MSD: Consultancy, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Demo: Research Funding; Astellas: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; WinMedica: Research Funding; GenesisPharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Dimopoulos: Beigene: Honoraria; Janssen: Honoraria; BMS: Honoraria; Amgen: Honoraria; Takeda: Honoraria.

Introduction: Lenalidomide-based regimens are currently among the standard-of-care treatment options for newly diagnosed patients with multiple myeloma NDMM). Second-line treatment of patients who are refractory to lenalidomide or have relapsed after prior use of lenalidomide is challenging. Daratumumab, an IgG1κ human monoclonal antibody that targets CD38, has shown durable responses and a favorable safety profile in heavily pretreated patients with relapsed/refractory MM (RRMM) as monotherapy, or in combination with other anti-myeloma treatments. Ixazomib, the first oral proteasome inhibitor, has shown deep responses and a positive safety profile in NDMM patients when it is combined with lenalidomide and dexamethasone. The aim of the DARIA study is to evaluate the effectiveness of daratumumab in combination with ixazomib and dexamethasone (DId) as second-line therapy in patients who have received prior treatment with lenalidomide-based regimens. Methods: DARIA is an ongoing prospective, open-label, multicenter, phase 2 study, which aims to enroll 43 patients who have received one prior line of therapy with a lenalidomide-based regimen. Patients must have a Karnofsky performance status (PS) ≥70. Exclusion criteria include previous daratumumab or anti-CD38, or ixazomib treatment exposure. The treatment phase consists of an induction therapy for 9 cycles, followed by a maintenance period. The induction phase included 28-day treatment cycles with 16 mg/kg intravenous daratumumab (weekly for cycles 1-2, every 2 weeks for cycles 3-6, and every 4 weeks thereafter), 4 mg oral ixazomib (Days 1, 8, and 15 of each cycle), and 40 mg oral dexamethasone for the first 9 cycles (weekly, each cycle). During the maintenance phase of the study daratumumab is administered every 4 weeks and ixazomib on the same schedule, until disease progression or unacceptable toxicity whereas dexamethasone is discontinued. The primary endpoint is the overall response rate (ORR). Secondary endpoints include evaluation of the toxicity profile of the study treatment, progression-free survival (PFS), overall survival, and levels of serum bone markers and angiogenic cytokines. All responses are based on investigators' assessment per International Myeloma Working Group criteria. This analysis presents results for patients who received the first dose of study treatment ≥5 months prior to the cut-off date (01/05/2020). Results: Fifteen patients, enrolled in 4 Sites, are included in the current analysis. Overall, 60% of the patients were refractory to lenalidomide. Median age was 70 years and the majority of patients were male (60%). The median time from diagnosis to first dose of study treatment was 1.3 years. Two patients (13%) had a prior autologous stem cell transplantation. Patients had a Karnofsky PS of 70 (27%), 80 (7%), 90 (53%), and 100 (13%) at baseline. At screening 47%, 26%, and 27% of the patients had an international staging system (ISS) of 1, 2, and 3, respectively, and a revised ISS of 1 (21%), 2 (71%), and 3 (7%). The median number of cycles reached until the cut-off date was 7. The median time from first dose of study treatment until first partial (PR) or very good partial response (VGPR) was 0.9 month. ORR was 60%, with 47% of the patients achieving VGPR and 13% PR. The 12-month PFS rate was 54%, and 8 patients (53%) were still on treatment by the cut-off date. From the patients who discontinued 71% was due to disease progression and 29% due to physician's decision. Overall, 10 patients (67%) had at least one adverse event (AE) grade 3 or 4, the most common being thrombocytopenia (6 patients, 40%). Four patients (27%) had a single SAE each: lower respiratory tract infection (fatal); peritonitis, gastroenteritis; nephrolithiasis. Conclusions: Rapid (median time to PR or better was 1 month) and deep responses (47% of the patients exhibited VGPR) were observed following treatment with daratumumab, ixazomib and dexamethasone as second-line therapy in patients who were previously treated with a lenalidomide-based regimen, more than half of whom were refractory to lenalidomide. The safety profile of DId combination is very good with one third of the patients not experiencing any grade 3 or 4 AEs, when at the same time the PFS rate at 12 months reached 54%. Disclosures Terpos: Janssen: Honoraria, Research Funding; Takeda: Honoraria, Other: travel expenses , Research Funding; Celgene: Honoraria; Amgen: Honoraria, Research Funding; Genesis pharma SA: Honoraria, Other: travel expenses , Research Funding; Sanofi: Honoraria; BMS: Honoraria. Gavriatopoulou:Amgen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Genesis Pharma: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Katodritou:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Theagenion Cancer Hospital: Current Employment; Takeda: Honoraria, Other: Expenses, Research Funding; Genesis Pharma: Honoraria, Other: Expenses, Research Funding. Hatjiharissi:Abbvie: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis pharma SA: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria. Verrou:Amgen: Honoraria; Abbvie: Honoraria; Janssen Cilag: Honoraria, Research Funding; Karyopharm: Research Funding; Takeda: Honoraria; Roche: Honoraria; Genesis: Honoraria. Leonidakis:Health Data Specialists S.A.: Current Employment. Delimpasi:Janssen: Honoraria; Takeda: Honoraria; Amgen: Honoraria; GENESIS: Honoraria. Symeonidis:Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GenesisPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Research Funding; WinMedica: Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck Sharp & Dohme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding. Kastritis:Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Genesis Pharma: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria. Dimopoulos:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees.

Introduction: Renal impairment (RI) is common in multiple myeloma (MM), with up to 40% of the patients (pts) experiencing this complication during the course of their disease. RI increases risk of early death and affects disease management in multiple ways, as it may complicate treatment options and dosing, and render pts more susceptible to infections and prolonged hospitalizations. Therefore, there is an urgent need to restore renal function in these pts in order to improve their quality of life and prognosis. Bortezomib-based therapies are the most commonly used in pts with RI, but eventually pts may become refractory to bortezomib and other drug classes such as IMIDs. Thus, new therapeutic options are needed in order to manage pts with MM and RI who fail these drugs. Daratumumab, an IgG1κ human monoclonal antibody that targets CD38, has shown durable responses and a favorable safety profile in heavily pretreated pts with relapsed/refractory MM (RRMM) as monotherapy. Pharmacokinetic analyses suggest that there are no clinically important differences in exposure to daratumumab in pts with normal or impaired renal function, but the available data on safety and efficacy of pts with RRMM and severe RI is scarce. Methods: DARE is a prospective, open-label, multicenter, phase 2 study, which completed the enrolment of 38 adult pts with documented RRMM and severe RI, defined as either eGFR Results: The current analysis includes 35 pts, enrolled in 7 Sites. Median age was 72 years, and 77.1% were male. The median time from diagnosis to first daratumumab dose was 4.2 years. Pts had a median of 3 prior systemic therapies, and 37.1% had a prior autologous stem cell transplantation. At study initiation 8.6% and 91.4% of pts had international staging system (ISS) 2 and 3 disease, respectively, while 51.4% and 48.6% were revised ISS 2 and 3. The median eGFR at baseline was 13 mL/min/1.73 m2.and 17 pts (48.6%) were on dialysis. The median number of cycles administered until the cut-off date was 5 and the median follow-up duration was 5.5 months. The 6-month progression-free survival rate, was 50% (figure). Overall, ORR was 45.7%, with 31.4% of all pts achieving a VGPR and 14.3% a PR. For pts on dialysis (n=17), ORR was 35.3%, equally divided between pts achieving VGPR and PR (17.6%). The median time from the first dose of study treatment until the first response (≥PR) was 0.9 months. RRR was 17.1%. By the cut-off date, 37.1% of the pts were still receiving protocol therapy, 17.1% discontinued treatment due to death, and 31.4% due to disease progression. Overall, 17 pts (48.6%) had at least 1 adverse event (AE) of grade 3 or 4, most frequent being anemia (17.1%) and hyperglycemia (8.6%). Nine (25.7%) pts had at least 1 SAE: Septic shock (fatal, 2 pts), and performance status decreased (fatal), lower respiratory tract infection (fatal), myocardial infarction (fatal), peritonitis (fatal, in a patient receiving peritoneal dialysis), cerebrovascular accident, pneumonia, acute kidney injury, and hyperkalemia (1 pt each). Conclusions: The administration of daratumumab with dexamethasone led to rapid hematologic responses in pts with RRMM and severe RI, including those in dialysis, and at the same time it resulted in a major renal response for 17.1% of the pts. Importantly, new safety signals were not observed, and daratumumab can be safely administered in pts with severe RI or those on dialysis. Figure 1 Disclosures Kastritis: Janssen: Consultancy, Honoraria, Research Funding; Genesis Pharma: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Terpos:Celgene: Honoraria; Takeda: Honoraria, Other: travel expenses , Research Funding; Janssen: Honoraria, Research Funding; Genesis pharma SA: Honoraria, Other: travel expenses , Research Funding; Amgen: Honoraria, Research Funding; Sanofi: Honoraria; BMS: Honoraria. Symeonidis:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Research Funding; WinMedica: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GenesisPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck Sharp & Dohme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Delimpasi:Genesis: Honoraria; Takeda: Honoraria; Janssen: Honoraria; Amgen: Honoraria. Cavo:Jannsen, BMS, Celgene, Sanofi, GlaxoSmithKline, Takeda, Amgen, Oncopeptides, AbbVie, Karyopharm, Adaptive: Consultancy, Honoraria. Zamagni:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Speakers Bureau; Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Katodritou:Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genesis Pharma: Honoraria, Other: Expenses, Research Funding; Abbvie: Research Funding; Karyopharm: Research Funding; Theagenion Cancer Hospital: Current Employment; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Other: Expenses, Research Funding. Rivolti:Celgene: Membership on an entity's Board of Directors or advisory committees. Kyrtsonis:Genesis pharma SA: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Gavriatopoulou:Amgen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Genesis Pharma: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Hatjiharissi:Abbvie: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria; Genesis pharma SA: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Dimopoulos:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees.

Preliminary evidence for T cell receptor (TR) repertoire renewal and increased TR clonality has been reported by our group (Vlachonikola et al., ASH 2019) in multiple myeloma (MM) patients (pts) receiving daratumumab monotherapy within the context of the REBUILD study, an ongoing prospective, multicenter, non-comparative, open-label, phase II study in pts with relapsed and/or refractory MM (RRMM) who have had ≥2 prior lines of therapy, including lenalidomide and a proteasome inhibitor. Herein, we report the results from the longitudinal analysis of the TR repertoire employing next generation sequencing (NGS) and multi-color flow cytometry in 24 pts who completed 3 cycles (n=24) and 6 cycles (n=11/24) of daratumumab monotherapy, in order to assess the immunomodulatory effects of daratumumab. We assessed 59 peripheral blood samples collected at screening (SCR, n=24), on Day 1 of Cycle 4 (C4, n=24) and Day 1 of Cycle 7 (C7, n=11). Patients were grouped based on best responses at C4 into responders (i.e. pts with partial response [PR, n=7] and very good PR [VGPR, n=8]), and non-responders (i.e. pts with minimal response [MR, n=2], stable disease [SD, n=5], or progressive disease [PD, n=2]). TRBV-TRBD-TRBJ gene rearrangements were subjected to paired-end NGS and raw reads (n=13,886,646 | median 239,969/sample) were processed through a purpose-built bioinformatics pipeline. Productive TRBV-TRBD-TRBJ rearrangements were taken into consideration (n=6,324,986 | median 100,738/sample) for the computation of clonotypes (i.e. TRB rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 sequence). Overall, 325,789 distinct clonotypes (median 4,535 clonotypes/sample) were analyzed. The TR repertoire displayed clonal T cell expansions in both groups (responders/non-responders) in all pre/post-treatment timepoints. Clonality increased after treatment for both responders and non-responders in all assessed timepoints, with statistical significance at C4 in both groups (median cumulative frequency of the 10 most expanded T cell clonotypes/sample in responders: 31.6% pre-treatment vs 43% C4 post-treatment, p=0.009; and, in non-responders: 19.8% pre-treatment vs 39.6% C4 post-treatment, p=0.009). In both groups, the clonotype repertoire appeared to be renewed. Interestingly, in the responders' group a significant shift was noticed in the major clonotype repertoire at screening vs C4. In particular, the 10 most expanded clonotypes/sample at C4 represented expansions of clonotypes present at very low frequency at screening, whereas the most expanded clonotypes at screening decreased or even diminished post-treatment. Additionally, although the major post-treatment clonotype at C4 also dominated at C7 in most cases, certain lower frequency clonotypes at C4 emerged among the top-10 at C7. Thirteen shared clonotypes were identified amongst the post-treatment repertoires of different patients but not in other entities in public databases, raising the possibility that they may be "MM-specific" and selected by common MM-associated antigens. Finally, flow cytometry analysis revealed a significant increase post treatment in the percentage of CD3+ T cells (median frequency at SCR 63% vs 78.7% at C4 | p=0.0045 and 87.4% at C7 | p=0.0009), driven mostly by the expansion of the CD8+ T cell compartment (median frequency at SCR 31.4% vs 45.3% at C4 | p=0.0045 and 53.8% at C7 | p=0.001) in both groups. In conclusion, we document T cell clonal expansions and clonal drift after daratumumab treatment in MM. Our results suggest that daratumumab acts through renewing the greatest part of the pre-treatment TR clonotype repertoire, suggesting dynamic changes of the T cell compartment under treatment, a claim also supported by the significant increase in CD8+ cytotoxic T cell numbers overtime. The significant post-treatment expansion of certain low-frequency pre-treatment clones in responders raises the intriguing hypothesis that daratumumab treatment may have led to the outgrowth of anti-MM T cell clones, arguably contributing to clinical response. Disclosures Kastritis: Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Genesis Pharma: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Hatjiharissi:Abbvie: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis pharma SA: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria. Katodritou:Theagenion Cancer Hospital: Current Employment; Takeda: Honoraria, Other: Expenses, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genesis Pharma: Honoraria, Other: Expenses, Research Funding; Abbvie: Research Funding; Karyopharm: Research Funding; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Gavriatopoulou:Amgen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Genesis Pharma: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Delimpasi:GENESIS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Symeonidis:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Research Funding; Astellas: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GenesisPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck Sharp & Dohme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; WinMedica: Research Funding. Stamatopoulos:AstraZeneca: Honoraria; Janssen, Gilead, Abbvie: Honoraria, Research Funding. Dimopoulos:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau. Terpos:Amgen: Honoraria, Research Funding; Genesis pharma SA: Honoraria, Other: travel expenses , Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria, Other: travel expenses , Research Funding; Celgene: Honoraria; Sanofi: Honoraria; BMS: Honoraria. Chatzidimitriou:Janssen: Research Funding.

Introduction: Osteolytic lesions (OL) is a devastating characteristic of multiple myeloma (MM) that decreases patients (pts) quality of life. The interaction between MM plasma cells and the bone microenvironment leads to intracellular and intercellular pathways that adversely alter the delicate balance between bone formation and bone resorption. Pre-clinical studies have shown that anti-CD38 therapy inhibit osteoclast formation. Daratumumab (dara), an IgG1κ human monoclonal antibody that targets CD38, has shown deep hematological responses in heavily pre-treated pts with relapsed/refractory MM (RRMM) as monotherapy. The aim of the REBUILD study is to evaluate the effect of dara monotherapy on bone metabolism in advanced RRMM pts. Methods: REBUILD is a prospective, open-label, multicenter, phase 2 study which has completed the enrolment of 57 pts with documented RRMM and ≥2 prior lines of therapy, including lenalidomide and a proteasome inhibitor. Pts had a Karnofsky Performance Status score of ≥70, and a creatinine clearance of ≥30 mL/min. Exclusion criteria included previous treatment with dara or other anti-CD38 therapy. Pts receive dara at a weekly dose of 16 mg/kg for Cycles 1-2, every 2 weeks for Cycles 3-6 and every 4 weeks thereafter. The primary endpoint of this study was the change from baseline in the bone resorption markers C-terminal telopeptide of collagen type I (CTX) and tartrate-resistant acid phosphatase isoform 5b (TRACP-5b) after 4 months of dara monotherapy. Secondary endpoints include the change at 4 months from baseline in bone formation markers (bone-specific alkaline phosphatase [bALP], osteocalcin [OC], and procollagen type-I N-propeptide [PINP]); markers of osteoclast regulation (receptor activator of nuclear factor kappa-B ligand [RANKL], osteoprotegerin [OPG] and chemokine (C- C motif) ligand-3 [CCL-3]); markers of osteoblast control (sclerostin [SCL], dickkopf-1 [DKK-1]), and progression-free survival (PFS). This preliminary analysis included pts who received the first dose of study treatment at least 5 months before the cut-off date (01/05/2020). Results: Fifty-one pts were enrolled in 6 sites; among them 29 pts had bone markers and clinical data available on baseline and after 4 months of study treatment and are included in the present analysis. Median age was 73 years, and approximately half of them were female (15 pts, 52%). Median number of previous therapies was 3 (range: 2-5). Fifteen pts (52%) had >10 osteolytic lesions at study initiation, and only 4 pts (14%) received bisphosphonates together with dara monotherapy. The median changes in CTX and TRACP-5b levels for all pts (n=29) after 4 months in study treatment were 3.9% and -2.2%, respectively. Overall, 10 pts (35%) had ≥ 30% reduction in CTX and 5 pts (17%) in TRACP-5b levels. The median changes after 4 months of dara monotherapy for pts with a response [pts who achieved partial response or better (≥PR; n=18, 62%)] were 3% for CTX and -3% for TRACP-5b; among pts with no response [minimal response, stable disease, disease progression, or no response assessment prior to death (n=11; 38%)] 4% for CTX, and 9% for TRACP-5b. The median changes in bone formation markers for all pts after 4 months of dara were 24.5% for bALP, 116.8% for OC, and 15.7% for PINP. The differences for pts with a response versus pts without a response were respectively 26% versus 18% for bALP, 190% versus -61% (p=0.020) for OC, and 22% versus -3% for PINP. Other major differences in 4 months of dara monotherapy were the decrease in DKK-1 by 49% in pts with a response versus 2% increase in pts with no response, and the decrease in CCL3 by 15% in pts with response versus 101% increase in pts with no response (p=0.039). The median PFS for all 51 pts was 4.6 months (95% CI: 2.8-7.2). Conclusions: Monotherapy with dara has a positive effect on bone metabolism even in these highly pre-treated pts with MM. Reduction of TRACP-5b and of CCL-3 in responsive pts suggests an inhibitory effect on osteoclasts by dara. Interestingly, we found that there is a strong bone formation effect in all pts treated with dara monotherapy, especially in those who responded to therapy, i.e. OC had a 2-fold increase after 4 months of therapy. This is at least partially due to the reduction of DKK-1 (osteoblast inhibitor) in responding patients. Based on these positive results a preclinical study on the effect of dara on bone cells is currently being performed. Disclosures Terpos: Amgen: Honoraria, Research Funding; BMS: Honoraria; Sanofi: Honoraria; Celgene: Honoraria; Takeda: Honoraria, Other: travel expenses , Research Funding; Janssen: Honoraria, Research Funding; Genesis pharma SA: Honoraria, Other: travel expenses , Research Funding. Kastritis:Janssen: Consultancy, Honoraria, Research Funding; Genesis Pharma: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Hatjiharissi:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria; Abbvie: Honoraria; Genesis pharma SA: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Katodritou:Theagenion Cancer Hospital: Current Employment; Karyopharm: Research Funding; Abbvie: Research Funding; Genesis Pharma: Honoraria, Other: Expenses, Research Funding; Takeda: Honoraria, Other: Expenses, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Verrou:Abbvie: Honoraria; Amgen: Honoraria; Genesis: Honoraria; Roche: Honoraria; Takeda: Honoraria; Karyopharm: Research Funding; Janssen Cilag: Honoraria, Research Funding. Gavriatopoulou:Amgen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Genesis Pharma: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Leonidakis:Health Data Specialists S.A.: Current Employment. Delimpasi:Janssen: Honoraria; GENESIS: Honoraria; Takeda: Honoraria; Amgen: Honoraria. Kyrtsonis:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis pharma SA: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Papaioannou:Gilead: Honoraria; Genesis Pharma: Honoraria; Janssen-Cilag: Honoraria; Alexion: Membership on an entity's Board of Directors or advisory committees. Symeonidis:Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; WinMedica: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Research Funding; Astellas: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GenesisPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck Sharp & Dohme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Dimopoulos:Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees.

A staging system was developed a decade ago for patients with Waldenström's macroglobulinemia (WM), however, since then WM treatments have changed. A revised staging system could better capture prognosis of WM patients in the chemoimmunotherapy era. We developed a revised system based on data from 492 symptomatic patients with at least 3 years and a median of 7 years of follow up while an independent validation cohort included 229 symptomatic patients. We identified age (≤65 vs 66-75 vs ≥76 years), b2-microglobulin ≥ 4 mg/L, serum albumin3.5 gr/dl, and LDH ≥ 250 IU/L (ULN 225) to stratify patients in five different prognostic groups and identify a very-low risk as well as a very-high risk group with a 3-year WM-related death rate of 0, 10, 14, 38, and 48% (p 0.001) and 10-year survival rate of 84, 59, 37, 19, and 9% (p 0.001). We evaluated this staging system separately in patients65 years and65 years, according to the reason for initiation of treatment, among patients receiving frontline rituximab or in patients treated primarily without rituximab. With further validation before clinical use, this revised IPSSWM could improve WM patient risk stratification, is easily available and may be used in the everyday practice to provide prognostic information.

Introduction: About 20-40% of patients (pts) with multiple myeloma (MM) present with moderate or severe renal impairment (RI) and about 25% of pts will also experience RI later during the disease course (Dimopoulos MA, et al; Leukemia 2008; 22:1485-1493). Moderate or severe RI is associated with poorer overall survival (OS) and higher risk of early death but also with challenges in the management and administration of the appropriate treatment. Daratumumab, an IgG1κ human monoclonal antibody that targets CD38, has shown efficacy and a favorable safety profile in pts with relapsed or refractory MM (RRMM) both as a monotherapy (Lonial S, et al; Lancet 2016; 387(10027):1551-1560) and in combination with other anti-myeloma agents (Dimopoulos MA, et al; N Engl J Med 2016; 375:1319-1331, and Palumbo A, et al; N Engl J Med 2016; 375:754-766). In population pharmacokinetic analyses, no clinically important differences in exposure to daratumumab were observed between pts with renal impairment and those with normal renal function. However, there are no prospective data on the safety and efficacy of daratumumab in pts with RRMM and severe renal dysfunction or those requiring dialysis. Methods: DARE is an ongoing multicenter, single arm, open-label, phase 2 study, aiming to enroll ~38 adult pts with documented RRMM and severe renal impairment (estimated glomerular filtration rate [eGFR]< 30 ml/min/1.73m2) or in need for hemodialysis. Pts must previously have had ≥ 2 lines of therapy with both bortezomib- and lenalidomide-based regimens and an Eastern Cooperative Oncology Group performance status (ECOG PS) score of ≤ 2. Exclusion criteria include previous treatment with daratumumab or other anti-CD38 therapy. All pts receive 28-day cycles of treatment with daratumumab given intravenously at 16 mg/kg weekly for Cycles 1-2, every 2 weeks for Cycles 3-6, followed by every 4 weeks thereafter, with dexamethasone 40 mg given weekly for each 4-week cycle. The primary endpoint of the study is the evaluation of progression-free survival (PFS). Key secondary endpoints include overall response rate (ORR; defined as the proportion of pts with partial response or better), renal response rate (RRR; defined as the proportion of pts with best response of renal partial response or better), and the assessment of daratumumab safety and tolerability. All responses were based on investigators' assessment per International Myeloma Working Group criteria. This interim analysis presents study results for pts who received the first dose of study treatment at least 3 months prior to the cut-off date (06/05/2019). Results: Eighteen pts, enrolled in 4 centers, were included in this prespecified analysis. The pts' median age was 74.0 years, and most were males (77.8%). The median time from MM diagnosis to first dose of daratumumab was 3.6 years. The number of pts with baseline ECOG PS 0, 1, and 2 were 4 (22.2%), 13 (72.2%), and 1 (5.6%), respectively. At the start of the study, 22.2% and 77.8% of patients had ISS Stage II and III disease, respectively. Moreover, 44.4%, and 55.6% of pts had a revised ISS stage II and III, respectively. Median number of prior lines of therapy was 3.5, and two (11.1%) pts had previous autologous stem cell transplantation; Median eGFR at baseline was 12 mL/min/1.73m2. The median number of therapy cycles received per patient was 4.5. The median time from first daratumumab dose to first partial response or better was 0.9 months. The median follow-up is 4.4 months and the Kaplan-Meier estimate of the 6-month PFS rate is 51.9% (Figure), ORR was 44.4% (8/18 pts) (including VGPR in 4, and PR in 4 pts), and RRR was 27.8% (5/18 pts). By the cut-off date, 10 (55.6%) pts were still on daratumumab; 7 (38.9%) pts discontinued treatment due to progressive disease and 1 (5.6%) due to a fatal serious adverse event (SAE). Overall, ten (55.6%) pts had ≥ 1 AE of grade 3 or 4. Of all grade 3 or 4 AEs, the most frequent were anemia (4, 25%), hyperglycemia (3, 18.8%), and hypocalcemia (2, 12.5%). Three (16.7%) pts suffered a single SAE each: lung infection, sepsis (fatal), and stroke. Conclusions: The combination of daratumumab with dexamethasone is efficacious and with a favorable safety profile and no new safety signals, in pts with RRMM with severe renal impairment. Importantly, hematologic responses are high in these heavily pretreated patients while about 28% achieved also a renal response. The study is ongoing and updated results will be presented at the meeting. Figure Disclosures Kastritis: Pfizer: Honoraria; Prothena: Honoraria; Genesis: Honoraria; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria. Terpos:Takeda: Honoraria, Other: Travel expenses, Research Funding; Genesis: Honoraria, Other: Travel expenses, Research Funding; Celgene: Honoraria; Medison: Honoraria; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding. Symeonidis:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding; Tekeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Delimpasi:Genesis: Honoraria, Other: Travel grant; Janssen: Honoraria; Takeda: Honoraria; Amgen: Honoraria. Cavo:Janssen, Celgene: Other: Travel Accommodations; Janssen, Celgene, Amgen, Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen, Celgene: Speakers Bureau; Celgene, Janssen, Amgen, BMS, Abbvie, Takeda: Honoraria. Zamagni:Celgene Corporation: Honoraria, Other: Advisory board, Speakers Bureau; Janssen: Honoraria, Other: Advisory board, Speakers Bureau; Amgen: Honoraria, Other: Advisory board, Speakers Bureau; BMS: Honoraria, Other: Advisory Board, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Sanofi: Honoraria, Other: Advisory Board, Speakers Bureau. Katodritou:Takeda: Honoraria; Janssen: Honoraria; Genesis: Honoraria; Amgen: Honoraria. Gamberi:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Gavriatopoulou:Genesis: Honoraria, Other: Travel expenses; Amgen: Honoraria; Janssen: Honoraria, Other: Travel expenses; Takeda: Honoraria, Other: Travel expenses. Hatjiharissi:Janssen: Honoraria. Dimopoulos:Sanofi Oncology: Research Funding.

Overexpression of the transcription factor MAF, as a result of its juxtaposition to the IgH enhancer [MAF-translocated t(14;16)], is a myeloma-initiating event in 3-5% of patients with multiple myeloma (MM) and confers a poor prognosis. MAF is also overexpressed in another 40% of cases, often in co-operation with the oncogene MMSET. The mechanisms by which MAF overexpression impacts on the regulatory genome to generate the MAF-driven oncogenic transcriptome and its direct targets are not known. To address this, we employed a multi-layer -omics approach using primary myeloma plasma cells (PC) as well as myeloma cell lines (MMCL). First, we determined the chromatin accessibility and transcriptome profiles of MAF-translocated myeloma by performing ATAC-seq and RNA-seq, respectively, in purified bone marrow CD138+ PC from two patients with t(14;16) and three healthy donors. We identified 6,640 differentially accessible regions, 87% of which displayed enhanced chromatin accessibility in MAF samples compared to normal PC. Secondary analysis comparing this with ATAC-seq data from a set of 28 other MM samples, including hyperdiploid, MMSET and CCND1-translocated MM, revealed 33% of those regions to be MAF subgroup specific (1,949 regions), with the rest shared between MAF and other cytogenetic groups. Gene annotation and pathway enrichment analysis using GREAT confirmed overrepresentation of the MF myeloma patient signature, as previously identified in microarray datasets. RNA-seq detected significant upregulation of approximately 900 genes in MAF samples compared to normal counterparts, including MAF itself (top 4th hit) as well as its presumed targets (CCND2, ITGB7 and NUAK1). Next, we obtained the MAF cistrome using ChIP-seq in the MAF-translocated MMCL MM1.S and integrated it with the primary PC ATAC-seq data. This revealed that 31% (618/1,949) of the differentially accessible regions in MAF-translocated MM PC are also MAF-bound. Additional overlay with ENCODE ChromHMM epigenome map showed that 47% of MAF binding sites are on active enhancers and 42% on active promoters signifying potential direct regulation of the corresponding genes. Next, we superimposed the accessible and MAF-bound loci on the epigenomic landscapes of normal PC and other B-cell types using their corresponding ChromHMM maps (Blueprint consortium data). Interestingly, 56% (345/618) of the MAF-specific regions were not active in any stage of B cell development. This suggests that aberrant MAF overexpression and chromatin binding in PC is associated with de novo activation of these chromatin regions, over half of which (200/345; 58%) are enhancers; we termed these 'neo-enhancers'. Upon de novo motif analysis of MAF ChIP-seq in MAF-translocated JJN3 and MM1.S MMCL, we confirmed MAF as the first and, interestingly, IRF4 as the second top hit, suggesting a possible MAF-IRF4 functional interaction in myelomagenesis. Indeed, overlay of the accessible MAF-bound loci with IRF4 ChIP-seq data in MM1.S revealed 63% co-occupancy (including 62% of "neo-enhancers"), proposing a novel and extensive co-operative chromatin-based network between the two transcription factors. Final integration of the accessible MAF-bound regions with the paired transcriptomes of primary myeloma PC revealed a set 206 candidate enhancer-gene pairs. Strikingly, we identified two IRF4-cobound "neo-enhancers" linked to overexpression of TLR4 and CCR1, two genes known for their roles in myeloma cell proliferation and migration. We confirmed significant downregulation of both genes upon shRNA-mediated knockdown of MAF in the two MAF-translocated MMCL, MM1.S and JJN3, as well as the lethality of MAF depletion. Further, MAF overexpression in MAF-negative myeloma backgrounds led to transcriptional upregulation of these genes, further validating them as MAF targets. While CRISPR/Cas9i experiments targeting TLR4 are ongoing, preliminary results validated the functional role of the "neo-enhancer" in CCR1 gene expression. In conclusion, we demonstrate for the first time an extensive re-organisation of the PC chromatin conferred by oncogenic MAF in MM; we reveal its extensive co-operation with IRF4 in this process; we validate the directly MAF-regulated genes and functionally characterise neo-enhancers of key MAF-dependent genes that in addition to MAF itself are also critical for myeloma biology. Disclosures Hatjiharissi: Janssen: Honoraria. Caputo:GSK: Research Funding. Karadimitris:GSK: Research Funding.

Recent evidence suggests immunomodulatory effects of daratumumab in heavily pre-treated Multiple Myeloma (MM) patients (pts); however, the precise effects remain under-characterized, particularly at the molecular level. REBUILD is an ongoing prospective, multicenter, non-comparative, open-label, phase II study that evaluates the effects of daratumumab monotherapy on bone metabolism of pts with relapsed and/or refractory MM (RRMM) who have had ≥2 prior lines of therapy, including lenalidomide and a proteasome inhibitor. Secondary endpoint of the study included the evaluation of T cell dynamics by comprehensive analysis of the T cell receptor (TR) repertoire employing next generation sequencing (NGS) and multi-color flow cytometry. Herein we report the results of this secondary endpoint for the first 14 pts who completed 3 cycles of daratumumab monotherapy. In total, we analyzed 28 paired samples collected at screening (n=14) and on Day 1 of Cycle 4 (C4D1, n=14) of treatment in order to assess potential changes in relation to treatment and clinical response. Patients were grouped based on best responses into responders (i.e. patients with partial response [PR, n=1] and very good PR [VGPR, n=6]), and non-responders (i.e. patients with minimal response [MR, n=2], stable disease [SD, n=4], or progressive disease [PD, n=1]). Starting material was peripheral blood mononuclear cells. TRBV-TRBD-TRBJ gene rearrangements were RT-PCR amplified and subjected to paired-end NGS. Raw NGS reads (n=6,715,406 | median 221,145/sample) were processed through a previously published, purpose-built bioinformatics pipeline. Only productive TRBV-TRBD-TRBJ rearrangements were taken into consideration (n=3,097,565 | median 101,670/sample) for the computation of clonotypes (i.e. TRB rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 sequence). Overall, 151,153 distinct clonotypes (median 5,084 clonotypes/sample) were assessed. Both groups (responders/non-responders) displayed clonal T cell expansions both pre- and post-treatment. Clonality was found to be increased after treatment for both responders and non-responders, with statistical significance in the former (median cumulative frequency of the 10 most expanded T cell clonotypes/sample: 31% pre-treatment versus 40% post-treatment, respectively | p=0.04). In both groups, the clonotype repertoire appeared to be renewed with only a small fraction of pre-treatment clonotypes remaining after treatment (1% for non-responders; 0.6% for responders). Interestingly, in the responders' group we noticed a significant shift in the major clonotype repertoire at screening vs C4D1. In particular, in the responders' group the 10 most expanded clonotypes/sample at C4D1 represented expansions of clonotypes present at very low frequency at screening, whereas the most expanded clonotypes at screening decreased or even diminished post-treatment, suggesting that daratumumab treatment led to the emergence of anti-myeloma T cell clones which contributed to clinical response. On the contrary, the 10 most expanded pre-treatment clonotypes in the non-responders' group tended to dominate also the post-treatment repertoire. Of note, 13 shared clonotypes were identified amongst the post-treatment repertoires of different patients (responders/non-responders); shared clonotypes were not found in other entities in public databases, raising the possibility that they may be "disease-specific" and selected by common tumor-associated antigens. With a single exception, shared clonotypes were detected in cases with relevant HLA restrictions, which is noteworthy given the random HLA background of our cohort. Finally, flow cytometry analysis revealed a significant increase post treatment in the percentage of CD3+ T cells (median frequency at screening 60% versus 83% at C4D1 | p=0.003), driven mostly by the expansion of the CD8+ T cell compartment (median frequency at screening 30.8% versus 48.9% at C4D1 | p=0.03) in both groups. In conclusion, TR clonality increases post-treatment through a renewal mechanism; however, pre-treatment clones significantly expanded post-treatment in responders, alluding to the existence of clonotypes with anti-MM properties that may be activated after treatment with daratumumab, arguably contributing to clinical response. Disclosures Kastritis: Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Prothena: Honoraria; Genesis: Honoraria; Amgen: Honoraria, Research Funding. Hatjiharissi:Janssen: Honoraria. Katodritou:Genesis: Honoraria; Janssen: Honoraria; Takeda: Honoraria; Amgen: Honoraria. Gavriatopoulou:Genesis: Honoraria, Other: Travel expenses; Janssen: Honoraria, Other: Travel expenses; Takeda: Honoraria, Other: Travel expenses; Amgen: Honoraria. Delimpasi:Takeda: Honoraria; Amgen: Honoraria; Janssen: Honoraria; Genesis: Honoraria, Other: Travel grant. Symeonidis:Sanofi: Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tekeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Dimopoulos:Sanofi Oncology: Research Funding. Terpos:Janssen: Honoraria, Other: Travel expenses, Research Funding; Medison: Honoraria; Genesis: Honoraria, Other: Travel expenses, Research Funding; Amgen: Honoraria, Research Funding; Celgene: Honoraria; Takeda: Honoraria, Other: Travel expenses, Research Funding. Chatzidimitriou:Janssen: Honoraria.

Introduction: A major clinical feature of multiple myeloma (MM) is osteolytic bone disease, with bone resorption prevailing bone formation. Daratumumab, an IgG1κ human monoclonal antibody that targets CD38, has been licensed for use in patients (pts) with relapsed or refractory MM (RRMM); however, its effect on bone disease has not yet been determined in a clinical setting. CD38 expression has been associated with osteoclast formation and bone resorption in in vitro studies and treatment with daratumumab has demonstrated an inhibitory effect on osteoclastogenesis. This study aims to evaluate the impact of daratumumab monotherapy on biochemical markers of bone metabolism in RRMM pts. Methods: REBUILD is an ongoing prospective, multicenter, non-comparative, open-label, phase II study aiming to enroll 57 adult pts with documented RRMM who have had ≥2 prior lines of therapy, including lenalidomide and a proteasome inhibitor. Pts should have a Karnofsky Performance Status score of ≥70, and a creatinine clearance of ≥30 mL/min. Exclusion criteria include previous treatment with daratumumab or other anti-CD38 therapy. Pts receive daratumumab at a weekly dose of 16 mg/kg for Cycles 1-2, every 2 weeks for Cycles 3-6 and every 4 weeks thereafter. The primary endpoint of this study is the change from baseline in the bone resorption markers C-terminal telopeptide of collagen type I (CTX) and tartrate-resistant acid phosphatase isoform 5b (TRACP-5b) after 4 months of daratumumab monotherapy. Secondary endpoints include the change at 4 months from baseline in bone formation markers (bone-specific alkaline phosphatase [bALP], osteocalcin [OC], and procollagen type-I N-propeptide [PINP]), in markers of osteoclast regulation (receptor activator of nuclear factor kappa-B ligand [RANKL], osteoprotegerin [OPG] and chemokine (C-C motif) ligand-3 [CCL-3]), osteoblast control (sclerostin, dickkopf-1 [DKK-1]), and progression-free survival (PFS). This interim analysis included pts who received the first dose of study treatment at least 6 months before the cut-off date (26/04/2019). Results: Twenty-seven pts had been enrolled in 5 study centres of the Greek Myeloma Study Group, and among them 15 had both biomarker and clinical data available after 4 months of therapy and they were included in the present analyses. The median number of previous therapies was 3. Nine (60%) pts had >10 lytic bone lesions at screening. Only 3/15 (20%) pts received bisphosphonates along with daratumumab monotherapy. Regarding the primary endpoint, the median change of CTX and TRACP-5b levels at 4 months from baseline was 13.4%, and -2.6%, respectively. The median change in CTX and TRACP-5b levels at 4 months from baseline for pts with partial response or better (≥PR) (n=7, 46.7%) was 13.4% and -9.4%, respectively; among pts with minimal response or stable disease (n=6, 40%) the change in CTX and TRACP-5b levels was 16.4% and 14.3%, respectively. The levels of all bone formation markers bALP, OC, and PINP showed a median increase at 4 months from baseline by 18.4%, 190.5%, and 19.3%, respectively. For pts with ≥PR, the median change at 4 months from baseline in bALP, OC, and PINP levels was even higher: 25.3%, 338.7%, and 19.3%, respectively. Interestingly, the median change at 4 months from baseline in osteoblast inhibitors sclerostin and DKK-1 levels was -20.3%, and -6.5%, respectively. The median PFS for all 27 pts enrolled was 7.1 months. Conclusions: Regarding the primary endpoint of the REBUILD study, results from this interim analysis indicate that daratumumab monotherapy decreases TRACP-5b levels, which was more pronounced in responders, but this is not accompanied by a reduction in CTX. An effect on CTX levels may become evident with additional follow-up. However, a clear increase in serum levels of all markers of bone formation tested (bALP, OC, PINP) was observed, especially among responders, which might at least partially be explained by a reduction of the osteoblast inhibitors sclerostin and DKK-1. Thus, daratumumab monotherapy in this RRMM population (heavily pre-treated pts with multiple lytic bone lesions and limited use of bisphosphonates) results in a decrease in TRACP-5b and osteoblast inhibitors and an increase in bone formation markers, suggesting an overall positive impact on bone remodeling. The study is ongoing and updated results will be presented at the meeting. Disclosures Terpos: Takeda: Honoraria, Other: Travel expenses, Research Funding; Genesis: Honoraria, Other: Travel expenses, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding; Amgen: Honoraria, Research Funding; Celgene: Honoraria; Medison: Honoraria. Kastritis:Amgen: Honoraria, Research Funding; Pfizer: Honoraria; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Prothena: Honoraria; Genesis: Honoraria. Hatjiharissi:Janssen: Honoraria. Katodritou:Takeda: Honoraria; Janssen: Honoraria; Genesis: Honoraria; Amgen: Honoraria. Gavriatopoulou:Takeda: Honoraria, Other: Travel expenses; Genesis: Honoraria, Other: Travel expenses; Janssen: Honoraria, Other: Travel expenses; Amgen: Honoraria. Leonidakis:HeaDS: Employment. Delimpasi:Janssen: Honoraria; Genesis: Honoraria, Other: Travel grant; Amgen: Honoraria; Takeda: Honoraria. Symeonidis:MSD: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding; Tekeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Dimopoulos:Sanofi Oncology: Research Funding.

In multiple myeloma (MM), a malignancy of the bone marrow plasma cells (BMPC), hyperdiploidy (HY) and oncogene over-expression via chromosomal translocation [including CCND1- t(11;14), MAF- t(14;16), MMSET-t(4;14)] are the primary myeloma initiating events (MIE) that drive distinct transcriptional programs. These are further shaped by secondary SNV and CNV events. This genetic heterogeneity converges, in most cases, to a functionally dichotomous state of CCND1 or CCND2 overexpression. The molecular mechanisms underlying each of the distinct myelomagenic transcriptomes and the CCND1 vs CCND2 dichotomy have not been defined. To address these questions, we obtained highly purified BMPC from 3 healthy donors and 30 MM patients (HY: 15; CCND1: 4; MMSET: 5; MAF: 2; other: 4), either at diagnosis or relapse, and mapped their chromatin accessibility and transcriptome profiles by ATAC-seq and RNA-seq, respectively. In total, we obtained ~300K regions with accessible chromatin in either MM or normal PC. Overall chromatin accessibility increased in myeloma compared to normal PC, particularly in MAF- and MMSET-translocated subtypes. Analysis of combined ATAC-seq/RNA-seq by Multi-Omics Factor Analysis (MOFA) resulted in a clearer samples distinction than either ATAC-seq or RNA-seq alone, with altered chromatin accessibility accounting for more of the variance than expression. Of the top five identified factors, the top two (one transcriptome driven, one accessibility driven) distinguished normal from MM samples, whilst two more separated MMSET, MAF and CCND1 subgroups. Ninety seven, 157, 256 and 348 overexpressed genes in the CCND1, HY, MMSET and MAF subgroups, respectively, were predicted to be regulated by differentially accessible enhancers. Twenty percent (165/858) of these genes were overexpressed in >1 subgroup suggesting a process of chromatin accessibility-based convergence evolution. Enrichment analysis suggested direct or indirect involvement of Polycomb and chromatin remodellers; significant enrichment was also found for genes involved in neurogenesis. ATAC-seq footprinting predicted binding sites for 250 expressed transcription factors (TFs), 116 of which displayed higher binding frequency in myeloma than in normal PC and included both known (e.g., XBP1, RELA, IRF4, PRDM1) and potentially novel regulators of myeloma biology (e.g., CXXC1 and NFE2L1). The remaining 134 TF were predicted to be present in at least one MM subgroup, but absent in normal PC. Amongst them, as expected, MAF was active in the MMSET- and more so in the MAF-translocated subgroups. DepMap database analysis suggested myeloma cell dependency on 181/250 TF (CRISPR/Cas9 CERES score < -0.1 in >3/14 MMCL analysed). In dissecting the regulatory basis of CCND2 vs CCND1 dichotomy, one MOFA factor completely separated MAF from CCND1 samples, placing extreme opposite weights on the expression of CCND2 and CCND1 respectively. Interestingly, the same factor identified open-chromatin clusters upstream of CCND2 and linked them to its over-expression. These clusters were also open in the MMSET group and in CCND2-expressing HY samples. Conversely, no accessibility was detected in the CCND1 group, the CCND1-expressing HY samples or in normal PCs. Further, super-enhancer calling using the H3K27ac histone mark in MAF-translocated JJN3 cells identified the region of interest as a bona fide super-enhancer. Chromatin long range interactions, as assessed by Capture-HiC, demonstrated high frequency interactions of the CCND2 promoter with the constituent elements of the putative super-enhancer. Experimental validation using a CRISPR/Cas9i system confirmed the functional role of all 4 super-enhancer constituents tested in the regulation of CCND2 expression, while TF footprinting predicted MAF binding to the super-enhancer in MAF-translocated PC. In conclusion, we show that distinct oncogenic transcriptomes in MM are underpinned by extensive chromatin changes, accompanied by TF activity 're-wiring' that does not necessarily require transcriptional deregulation of the TF themselves. We identify novel, non-oncogene TF dependencies that suggest therapeutic opportunities in MM and we discover and characterise the critical super-enhancer that drives overexpression of the CCND2 oncogene in MM. Disclosures Auner: Amgen: Other: Consultancy and Research Funding; Takeda: Consultancy; Karyopharm: Consultancy. Hatjiharissi:Janssen: Honoraria. Caputo:GSK: Research Funding. Karadimitris:GSK: Research Funding.

We studied the role of histone deacetylase inhibitors in Waldenstrom's macroglobulinemia (WM). Gene expression profiling of bone marrow CD19+ cells from 30 patients and 10 healthy donors showed overexpression of HDAC4, HDAC9, and Sirt5, with validation of HDAC9 overexpression by q-PCR in primary and BCWM.1 cells. Suberoylanilide hydroxamic acid, trichostatin A, panobinostat, and sirtinol demonstrated dose-dependent killing of BCWM.1 cells. TSA showed the greatest potency with IC50 of 70 nM. Importantly, HDAC9 activity was decreased following TSA treatment suggesting an essential role for this HDAC in WM therapy. The combination of bortezomib plus HDAC inhibitors resulted in at least additive tumor cell killing in BCWM.1 cells. TSA and bortezomib-induced apoptosis depended on a similar set of caspase activation, whereas their effect on cell cycle regulators was distinctly different. These results provided a framework for examining HDAC inhibitors as monotherapy, as well as combination therapy with bortezomib in WM.

Waldenström's Macroglobulinemia (WM) is an indolent lymphoma with heterogeneous clinical presentation and prolonged course. In this context, determining the overall disease and therapy burden in terms of complications and associated comorbidities is necessary. Of special interest is the occurrence of a second malignancy and transformation to high grade lymphoma, due to their impact on survival and quality of life but also on health resource use and follow up strategies. Several factors including immune dysfunction, genetic predisposition, environmental factors and treatment effects have been suggested as possible underlying pathophysiologic mechanisms for development of second primary malignancies or transformation in patients with WM. While there have been data published from various series, it is crucial to have data after prolonged follow up, as the time for development of such complications may be long. The current study included all patients with symptomatic WM that have been registered in the prospectively maintained database of the Greek Myeloma Study Group, in which the development of a second malignancy and disease transformation is being recorded, including type of second cancer and date of diagnosis. All patients fulfilled criteria for symptomatic WM diagnosis and for treatment initiation according to the Consensus Recommendations. The analysis included 598 symptomatic newly diagnosed patients with WM with a median follow up of 114 months. The median age was 69 years and 284 (47%) have died. Primary therapy was rituximab-based in 65%, contained alkylating agents in 78%, a nucleoside analogue in 2% and an anthracycline in 4%. In 46 patients (7.7%) the diagnosis of a second tumor was made after the initiation of treatment for symptomatic WM. The most common malignancies were cancer of the prostate (18%), stomach (6%), colon (6%), lung (6%), pancreas (3%), non-melanoma skin (3%) and central nervous system (6%) and AML/MDS (6%). In addition, in 12 (2%) patients, a diagnosis of another malignancy preceded the diagnosis of WM. The median time from treatment initiation for symptomatic WM to the diagnosis of a second primary malignancy was 51 months [interquartile range (IQR): 16-79]. The incidence ratio (IR) of a second primary malignancy was 0.0095 per person-years and the cumulative incidence of a second primary malignancy, accounting for death due to WM or other causes as a competing event, at 5 and 10 years was 3.6% and 6.6%, while, the risk of death from WM or other causes was 22.5% and 46%, respectively (Figure). The IR of a second malignancy was higher in men [IR ratio (IRR): 0.5, 95% CI 0.2-1.1, p=0.067] while it was similar for younger vs older patients (>75 years) (IRR: 1.57, 95% CI 0.61-3.5, p=0.27). Importantly, the use of rituximab as primary treatment was not associated with any increased risk of a second malignancy (IRR: 0.99, 95%CI 0.5-2.1, p=0.97). The incidence of second malignancies has not changed significantly in the era before vs after 2000. Overall, 19 (3.1%) transformation events were identified; the median time from treatment initiation to transformation was 51 months (IQR: 28-106). The IR for transformation was 0.005 per person-years and the cumulative risk for transformation, accounting for death of any cause as a competing event, was 2.2% and 3.6% at 5 and 10 years respectively. The presence of anemia (hemoglobin We conclude that the incidence of a second malignancy among patients with symptomatic WM is 7.7%, corresponding to an incidence rate of about 1 case per 100 patients per year. Transformation occurred in 3.1% corresponding to an incidence rate of 0.5 cases per 100 patients per year; this risk was higher among patients presenting with anemia and has been reduced after 2000. This may be related to the extensive use of rituximab and less frequent use of alkylating agents and nucleoside analogues. Figure. Figure. Disclosures Kastritis: Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Prothena: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Terpos:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: member of steering committee, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grant, Research Funding; BMS: Consultancy; Novartis: Consultancy; Genesis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grant, Research Funding; Amgen Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grant, steering committee member, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: member of DMC, Research Funding. Dimopoulos:Takeda: Honoraria; Celgene: Honoraria; Janssen: Honoraria; Bristol-Myers Squibb: Honoraria; Amgen: Honoraria.

Waldenstrom Macroglobulinemia (WM) is a B-cell malignancy characterized by excess bone marrow (BM) lymphoplasmacytic cells (LPC). The accumulation of LPC in WM may represent a failure of B-cells to properly differentiate into plasma cells. The present study investigated transcriptional expression of genes involved in late B-cell differentiation, including PRDM1, PAX5, XBP1 transcripts and ERN1, in BM B-cells from 31 patients with WM and six healthy donors. Real time reverse transcription polymerase chain reaction (RT-PCR) determined that approximately 80% of the patients had high XBP1 spliced mRNA expression, 80% of whom had high mRNA ERN1alpha expression. XBP1, PRDM1 and PAX5 mRNA was present in all patients studied. Using relative quantitative RT-PCR we isolated two groups with low and high expression of XBP1, XBP1 spliced and ERN1alpha. Sequence analysis showed germline polymorphisms in all genes studied. These data depict for the first time a heterogeneous expression pattern of the genes involved in late differentiation process of plasma cells in patients with WM and propose a role of XBP1-ERN1alpha in WM pathogenesis.

Thalidomide enhances rituximab-mediated, antibody-dependent, cell-mediated cytotoxicity. We therefore conducted a phase 2 study using thalidomide and rituximab in symptomatic Waldenstrom macroglobulinemia (WM) patients naive to either agent. Intended therapy consisted of daily thalidomide (200 mg for 2 weeks, then 400 mg for 50 weeks) and rituximab (375 mg/m2 per week) dosed on weeks 2 to 5 and 13 to 16. Twenty-five patients were enrolled, 20 of whom were untreated. Responses were complete response (n = 1), partial response (n = 15), and major response (n = 2), for overall and major response rate of 72% and 64%, respectively, on an intent-to-treat basis. Median serum IgM decreased from 3670 to 1590 mg/dL (P < .001), whereas median hematocrit rose from 33.0% to 37.6% (P = .004) at best response. Median time to progression for responders was 38 months. Peripheral neuropathy to thalidomide was the most common adverse event. Among 11 patients experiencing grade 2 or greater neuropathy, 10 resolved to grade 1 or less at a median of 6.7 months. Thalidomide in combination with rituximab is active and produces long-term responses in WM. Lower doses of thalidomide (ie, ≤ 200 mg/day) should be considered given the high frequency of treatment-related neuropathy in this patient population. This trial is registered at www.clinicaltrials.gov as #NCT00142116.

Summary Waldenstrom macroglobulinaemia (WM) is an incurable lymphoplasmacytic lymphoma with secretion of serum monoclonal immunoglobulin M (IgM). We previously showed that patients receiving cholesterol-lowering statins, had the lowest IgM value in a large cohort of patients with WM. Simvastatin, a 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor, induced inhibition of proliferation, cytotoxic effect and apoptosis in IgM secreting cell lines as well as in primary CD19 + WM cells. Interestingly, those effects were reversed by addition of mevalonate and geranylgeranylpyrophosphate, demonstrating that simvastatin inhibited cell growth, survival and IgM secretion on BCWM.1 WM cells by inhibition of geranylgeranylated proteins. Furthermore, simvastatin overcame tumour cell growth induced by co-culture of WM cells with bone-marrow stromal cells. Simvastatin also decreased IgM secretion by BCWM.1 cells at an early time-point that had not affected cell survival. Simvastatin-induced cytotoxicity was preceded by a decrease in Akt (protein kinase B, PKB) and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathways at 18 h. In addition, simvastatin induced an increase in stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/ JNK) MAPK followed by caspase-8, -9, -3 and poly(ADP-ribose) polymerase (PARP) cleavages at 18 h, leading to apoptosis. Furthermore, simvastatin enhanced the cytotoxicity induced by bortezomib, fludarabine and dexamethasone. Our studies therefore support our earlier observation of statin-mediated anti-WM activity and provide the framework for future clinical trials testing simvastatin in WM.

Waldenström's macroglobulinemia is a B-cell disorder characterized by bone marrow infiltration with lymphoplasmacytic cells and demonstration of an immunoglobulin M monoclonal gammopathy. Despite advances in therapy, Waldenström's macroglobulinemia remains incurable. As such, novel therapeutic agents are needed for the treatment of Waldenström's macroglobulinemia. In ongoing efforts, we and others have sought to exploit advances made in the understanding of the biology of Waldenström's macroglobulinemia so as to better target therapeutics for this malignancy. Importantly, as part of these efforts, we have prioritized the development of stem cell-sparing drugs because autologous stem cell transplantation remains a viable salvage option in Waldenström's macroglobulinemia. These efforts have led to the development of several novel agents for treating Waldenström's macroglobulinemia, including bortezomib; monoclonal antibodies and/or blocking protein targeting CD40, CD52, or CD70, a proliferation-inducing ligand and B-lymphocyte stimulator; the immunomodulator thalidomide as an enhancer of rituximab activity, as well as agents interfering with stem cell factor, phosphatidylinositol 3-kinase/Akt, phosphodiesterase, cholesterol, and protein kinase C beta signaling. This report provides an update on biologic studies and clinical efforts for the development of these novel agents in the treatment of Waldenström's macroglobulinemia.

WM is a disease of the elderly with a protracted course in many patients and a median survival of 7 to 10 years,[1][1] however, in many patients, other factors rather than WM or its treatment may be the cause of death.[2][2] Non-WM-related mortality mainly affects outcomes of elderly patients and is

Background Alemtuzumab is a monoclonal antibody used in the treatment of CD52-expressing B-cell malignancies, including Waldenstrom's macroglobulinemia (WM). Recent studies demonstrate high levels of alemtuzumab activity in relapsed/refractory disease. One potential target of alemtuzumab is bone marrow mast cells (BMMCs), which provide growth and survival signaling for WM lymphoplasmacytic cells. Patients and Methods We therefore examined BMMCs (FcɛRI + , CD117 + ) from WM and other mast cell (MC) disorders for expression of CD52. Results We identified cell surface antigen expression by multicolor flow cytometric analysis and found CD52 expressed on human mast-derived cell line–1 (HMC-1) and LAD2 MC lines, on BMMC from 13 of 15 patients with WM, and on BMMCs from 4 of 4 patients with systemic mastocytosis (SM). None of 4 healthy donors expressed CD52. Reverse-transcriptase polymerase chain reaction analysis confirmed CD52 expression in the HMC-1 and LAD2 MC lines, in BMMCs from 14 of 15 patients with WM, and 3 of 3 patients with SM. CD52 transcripts were also detected in BMMCs from 6 of 6 healthy donors, despite the absence of CD52 cell surface expression. Importantly, we observed high levels of alemtuzumab-mediated, antibody-dependent, cell-mediated cytotoxicity against LAD2 MCs and BMMCs from patients with WM and SM. Conclusion These studies demonstrate that CD52 is widely expressed on human MCs and WM bone marrow lymphoplasmacytic cells and provide the preclinical rationale for the use of alemtuzumab in the treatment of WM and possibly other MC-related disorders.

Familial clustering of B-cell disorders among Waldenström's macroglobulinemia (WM) patients has been reported, though the frequency and any differences in disease manifestation for familial patients remain to be defined.We therefore analyzed clinicopathological data from 257 consecutive and unrelated WM patients. Forty-eight (18.7%) patients had at least one first-degree relative with either WM (n = 13, 5.1%), or another B-cell disorder including non-Hodgkin's lymphoma (n = 9, 3.5%), myeloma (n = 8, 3.1%), chronic lymphocytic leukemia (n = 7, 2.7%), monoclonal gammopathy of unknown significance (n = 5, 1.9%), acute lymphocytic leukemia (n = 3, 1.2%) and Hodgkin's disease (n = 3, 1.2%). Patients with a familial history of WM or a plasma cell disorder (PCD) were diagnosed at a younger age and with greater bone marrow involvement.Deletions in 6q represented the only recurrent structural chromosomal abnormality and were found in 13% of patients, all non-familial cases. Interphase FISH analysis demonstrated deletions in 6q21-22.1 in nearly half of patients, irrespective of familial background.The above results suggest a high degree of clustering for B-cell disorders among first-degree relatives of patients with WM, along with distinct clinical features at presentation based on familial disease cluster patterns. Genomic studies to delineate genetic predisposition to WM are underway.

The preclinical evaluation of investigational agents for Waldenström macroglobulinemia (WM) has been limited by the lack of in vivo models that enable the use of explanted patient cells. We describe here a novel in vivo model of human WM in severe combined immunodeficient (SCID) mice implanted with human fetal bone chips (SCID-hu mice) into which WM cells from patient bone marrow are engrafted directly into the human bone marrow (huBM) microenvironment. WM cells in SCID-hu mice produced human monoclonal paraprotein (immunoglobulin M [IgM] and/or κ or λ chain) detectable in mice sera. Immunohistochemical analysis of human bone retrieved from SCID-hu mice showed infiltration with CD20+, IgM+, and monotypic light chain+ lymphoplasmacytic cells. Mast cells were observed to be associated with the infiltrate in these sections. Treatment of SCID-hu mice bearing WM with rituximab induced tumor regression, associated with a decrease in serum paraprotein. This model, therefore, recapitulates the in vivo biology of WM and allows the study of novel investigational drugs targeting WM cells in the huBM milieu. (Blood. 2005;106:1341-1345)

We report two patients with severe thrombocytopenia and life-threatening bleeding that were successfully managed with recombinant activated factor VII (rFVIIa). The first was a 75-year-old male with Waldenström's macroglobulinemia. During a therapeutic course with fludarabine, he developed severe autoimmune thrombocytopenia resistant to conventional treatment, followed by persistent uncontrollable nasal bleeding. Platelet transfusions failed to increase the platelet count and control the hemorrhage. When hemoglobin levels fell below 8.5 g/dL and the patient's clinical condition got much worse, a single dose of 4.8 mg rFVIIa (90 microg/kg) was given as an i.v. bolus. Ten minutes after the rFVIIa injection, nasal bleeding stopped, the patient's clinical condition progressively improved, and splenectomy could be carried out uneventfully 2 days later. The second patient, a 52-year-old female, was under treatment for pre-B lymphoblastic leukemia. She developed severe thrombocytopenia, secondary to chemotherapy, complicated by massive gastrointestinal bleeding. Despite intensive treatment with platelet transfusions, hemorrhage continued and her condition deteriorated rapidly. She was then given an i.v. bolus injection of 4.8 mg rFVIIa, which resulted in cessation of hemorrhage and dramatic improvement of her clinical status. No adverse effects from the treatment with rFVIIa were observed. In conclusion, rFVIIa appears to be an attractive alternative for controlling hemorrhage in patients with severe thrombocytopenia, especially when platelet transfusions are unavailable or ineffective.

Abstract 2909 Unlike the established role of acquired genetic events and bone marrow microenviroment in the pathogenesis of multiple myeloma (MM), the phenotype and function of cells enriched in tumor-propagating activity and their relationship to the phenotypic architecture in MM are controversial. Because the immunoglobulin heavy (IgH) chain class-switched myeloma plasma cell (PC) is the final event of a linear, B cell lineage developmental process, it was suggested that myeloma cell growth is sustained by a minority of cells more immature than the PC. We combined multicolour flow-cytometry and sorting with patient-specific quantitative PCR (qPCR) to dissect the myeloma clonal organisation in the bone marrow (BM) and peripheral blood (PB). In a cohort of 30 patients we show that MM comprises at least four hierarchically organised, clonally-related sub-populations which, although phenotypically distinct, share the same oncogenic chromosomal abnormalities as well as IgH chain complementarity region 3 area sequence. We found rare CD19+ clonotypic cells with phenotype either of resting memory cell (CD19+CD38-IgD-CD27+/−) or plasmablasts (CD19+CD38++CD319+CD138-) and CD19-CD38hiCD319+CD200+CD56+ clonotypic cells, comprising CD138- (∼3% of the clone, termed Pre-PC), CD138low and CD138+ PC. The clonal populations resemble their normal counterparts and exist in nearly logarithmic incremental frequencies in the BM. Using dynamic mathematical models and a Bayesian approach a Pre-PC->PC transition was predicted, outside the linear developmental process. Following intravenous injection of purified clonotypic populations into sub-lethally irradiated NSG mice, we found that both PC (in 9/12 of the injected mice, 75%) and Pre-PC (4/16, 25%) can engraft, but not the CD19+ clonotypic cells (0/10). Of note, upon engraftment both PC and Pre-PC regenerate the original CD19- hierarchy of the human BM, as predicted by mathematical modelling. In addition, Pre-PC are more quiescent and unlike PC, preferentially localize at extramedullary niches, such as the liver and spleen of the engrafted animals. Therefore, the myeloma-propagating activity is the exclusive property of a population characterized by its ability for bi-directional transition between the dominant PC and the low frequency Pre-PC. To gain insights into the molecular mechanisms underpinning this reversible phenotypic transition we used gene expression profiling of highly purified BM Pre-PC and PC (n=9). Differential expression analysis and principal component analysis clearly separate Pre-PC from PC, with 7 of 9 samples following the same expression pattern in hierarchical clustering. Functional annotation analysis using DAVID shows that Pre-PC are enriched in epigenetic regulators, including histone methyl-transferases (belonging to the Polycomb repressive complex 2 or Trithorax MLL activating complex) and de-methylases, histone acetyl-transferases and de-acetylases as well as several members of SWI/SNF chromatin remodeling complex, suggesting that epigenetic plasticity underpins the phenotypic diversification of myeloma-propagating cells. Finally, to study the clinical importance of the myeloma clonal organisation, with emphasis to clinical drug resistance, we prospectively assessed the size of the different phenotypes in paired, pre- and post-treatment BM samples (n=8). We show that in all cases a higher proportion of Pre-PC than PC persisted after treatment suggesting that Pre-PC are clinically more drug-resistant than PC (median 10.3-fold, range 4.4–332, p=0.008). Thus, clinical drug resistance in MM is linked to reversible, bi-directional phenotypic transition of myeloma-propagating cells, likely under the orchestration of epigenetic regulators. These novel biological insights have important clinical implications in relation to assessment of minimal residual disease and development of alternative therapeutic strategies in MM. Disclosures: No relevant conflicts of interest to declare.

Waldenström's Macroglobulinemia (WM) is a low grade lymphoma with a prolonged course and a median survival exceeding 7 years. However, there are patients who die of WM early during the course of the disease while a significant proportion of WM patients can survive ≥10 years. The characteristics of these two groups of patients may differ and their identification may augment the choice of risk adapted treatment strategies. The aim of our study was to identify and characterize patients with short survival due to WM as well as those with survival exceeding 10 years, and to compare their characteristics in order to evaluate clinical factors associated with poor or with good outcomes, based on data from a large database with long follow up. The analysis included 492 patients that have been entered in the prospectively maintained database of the Greek Myeloma Study Group, who fulfill the criteria form symptomatic WM requiring therapy. The median follow up of all the patients in the database is 10 years. For the first part of the analysis we included 292 patients who have at least 10 years of follow up (thus, they started therapy at least 10 years ago, before 2006). Among them, we identified 101 (34.5%) patients who survived ≥10 years, and 13% who died due to WM within As a validation of the previous results, we evaluated the presence of the above clinical characteristics in patients with survival 65 in 63%, hgb4 mg/L were the two most important predictors of early WM-related death. When age >65 years was also included as a predictor, patients with 0, 1, 2 or 3 of the above factors had 3-year WM-related death rate of 3%, 4%, 16% and 25% (p In conclusion, 34.5% of patients with WM survive ≥10 years, but 10%-13% die of WM within < 3 years from initiation of treatment. These patients with high risk disease are older with higher tumor bulk and increased LDH. However, by applying only the presence of serum albumin < 4 gr/dl, b2 microglobulin ≥4 mg/L and age >65 years we can identify patients at very low risk of early death as well as patients with significant risk of early death and short survival. This simple staging system may also outperform IPSSWM. Figure 1 Figure 1. Disclosures Kastritis: Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Genesis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Kyrtsonis:Genesis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Katodritou:Genesis: Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Delimpasi:Amgen: Honoraria; Janssen: Honoraria; Genesis: Honoraria. Terpos:Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Dimopoulos:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

BACKGROUND - AIM Ruxolitinib can be effectively used for the treatment of splenomegaly or constitutional symptoms in patients with myelofibrosis. Its main side effect is myelosuppression which leads to dose modifications. Real-life data from the use of ruxolitinib in every-day practice were analysed to assess efficacy, safety, dose modifications or reasons for treatment discontinuation. PATIENTS - METHODS A retrospective observational study was conducted in patients with primary or secondary myelofibrosis, treated in 13 sites. Statistical analysis was done with non-parametric tests using SPSSv17 software. A total of 116 patients, diagnosed between 1993 and 2016 were included in the analysis. The male:female ratio was 2:1, median age at diagnosis was 63 years (range 27 to 82) and median duration of ruxolitinib therapy was 15.3 months. The study expands over 664.2 patient-years of observation, including 176.7 patient-years while on ruxolitinib. RESULTS The diagnosis, revisited according to the WHO 2008 criteria, was primary myelofibrosis in 56%, post-essential thrombocythemia myelofibrosis in 22% and post-polycythemia vera myelofibrosis in 22% of patients. At the beginning of ruxolitinib treatment, the international prognostic scoring system (IPSS) risk category was low (6.2%), intermediate-1 (26.5%), intermediate-2 (39.8%), high (23%). Constitutional symptoms were present in 48% of patients at diagnosis and in 73% of them at the onset of ruxolitinib therapy. Median spleen size was 5cm (centimeters) below costal margin at diagnosis and 12cm at onset of ruxolitinib. Ruxolitinib starting dose was 5mg (16%), 10mg (18%), 15mg (31%), 20mg (35%) twice daily (BID), while the final dose was Ruxolitinib was permanently discontinued in 29% of patients, due to progressive disease in 6.4%, transformation to acute leukemia in 2.7% and death in 8.2% of patients. Side effects (including anemia, thrombocytopenia, investigator decision, patient preference) was the reason for treatment discontinuation in 5.5% and lack of response in 2.7% of patients. Efficacy (reduction in constitutional symptoms and spleen size) and hematological toxicity (values of platelets, white blood cell counts, hemoglobin) of ruxolitinib was assessed after 1, 6 and 12 months of treatment (table 1). Thrombocytopenia (PLT Constitutional symptoms after 1 month were significantly less frequent with increased doses of ruxolitinib, whereas there was no statistically significant difference among the various doses at 6 and 12 months of treatment. Platelet counts were significantly lower in patients taking 5mg BID at 6 months of treatment (p=0.02), and they also tended to be lower in 1 and 12 months (p=0.07, p=0.08 respectively). White blood cells and hemoglobin values did not differ significantly among the various doses of ruxolitinib in either time point. Table 1. Results at 1, 6, 12 months of treatment, according to different doses. CONCLUSIONS Ruxolitinib can effectively improve splenomegaly and alleviate constitutional symptoms in patients with myelofibrosis. The symptoms are relieved slower with the low dosage, whereas the main side effect is thrombocytopenia. It can however be maintained within safety limits (grade < 3), with dose modifications without hampering efficacy. Table Table. Disclosures No relevant conflicts of interest to declare.

Immunogenetic analysis of MM has proven instrumental in elucidating disease ontogeny e.g. by revealing the clonal relationship between switch variants expressed by the bone marrow plasma cells and myeloma progenitors in the marrow and blood; demonstrating the marked under-representation of the inherently autoreactive IGHV4-34 gene; and, identifying patterns of somatic hypermutation (SHM) indicative of post-germinal center derivation. Yet, limited information exists about the composition of the immunoglobulin (IG) gene repertoire in MM cases expressing different heavy chain isotype, in particular A versus G. This is relevant in light of studies showing an overall higher SHM impact in CD27+IgA+ compared to CD27+IgG+ normal memory B cells, perhaps reflecting a distinct location of the immune response, especially considering that IgA class switching mostly occurs in mucosa-associated lymphoid tissues. From a clinical perspective, it is also relevant to note that IgA patients exhibit a higher incidence of the t(4;14) translocation, shorter progression-free survival and worse median overall survival compared to IgG patients. Here, we explored potential differences in the immunoprofiles of IgA versus IgG MM focusing on IG gene repertoire and SHM characteristics. In total, 428 patients with a diagnosis of MM following the IMWG criteria from collaborating institutions in Greece, Italy and Spain (n=355) or retrieved from the LIGM-DB (n=73) were included in the study. Of these, 135 and 293 belonged to IgA and IgG MM groups, respectively. Amongst the evaluated productive IG rearrangements, IGHV3 subgroup genes predominated in both groups (IgA: 58.5%; IgG: 52.2%). However, at the individual gene level, major asymmetries were noted, since only 7 IGHV genes accounted for 41.6% of the IgA and 46.7% of the IgG cases, respectively. Of these, 3 genes were shared between IgA and IgG MM cases: IGHV3-30 (IgA: 11.9% - IgG: 13.3%), IGHV3-23 (IgA: 5.2% - IgG: 6.8%) and IGHV3-9 (IgA: 6.7% - IgG: 4.4%), whereas the remaining 4 of the 7 most frequent genes were specific for each group with significant (p Disclosures Terpos: Celgene: Honoraria; Novartis: Honoraria; Genesis: Consultancy, Honoraria, Other: Travel expenses; BMS: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel expenses, Research Funding. Stamatopoulos:Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding.

Backgound: The assessment of disease burden is often difficult in patients with Waldenstrom’s macroglobulinemia (WM) who receive rituximab due to the induction of an IgM flare, and following the removal of serum IgM by plasmapheresis. Soluble CD27 (sCD27) is a tumor necrosis factor family member secreted by WM cells which is strongly correlated with serum IgM levels and clinical responses in patients with WM. As such, we attempted to delineate its potential role in WM patients experiencing a rituximab-induced IgM flare and following plasmapheresis. Patients and Methods: sCD27 levels were serially measured by serum-based ELISA in 8 patients who ultimately demonstrated a response to therapy, and in whom a rituximab-mediated IgM flare was observed, as well as in 3 WM patients undergoing plasmapheresis. Results: Among the 8 patients who experienced a rituximab-mediated IgM flare, IgM levels rose from 3515 to a peak of 5270 mg/dL (P = .008), while sCD27 levels decreased from 174.1 to 155.9 U/mL (P = .012), with a decline observed in all patients. Among 3 patients undergoing plasmapheresis, IgM levels declined from a median of 6940 to 4770 mg/dL (P = .031), while median sCD27 levels remained without significant change (P = .317). Conclusion: sCD27 is a faithful marker of disease burden and is unaffected by the rituximab-induced IgM flare, as well as plasmapheresis in WM. The use of this marker may aid in correctly predicting clinical outcome in patients undergoing treatment with rituximab and/or plasmapheresis in WM.

Waldenström macroglobulinemia (WM) is a B-cell malignancy characterized by an IgM monoclonal gammopathy and bone marrow (BM) infiltration with lymphoplasmacytic cells (LPCs). Excess mast cells (MCs) are commonly present in WM, and provide growth and survival signals to LPCs through several TNF family ligands (CD40L, a proliferation-inducing ligand [APRIL], and B-lymphocyte stimulator factor [BLYS]). As part of these studies, we demonstrated that WM LPCs secrete soluble CD27 (sCD27), which is elevated in patients with WM (P < .001 vs healthy donors), and serves as a faithful marker of disease. Importantly, sCD27 stimulated expression of CD40L on 10 of 10 BM MC samples and APRIL on 4 of 10 BM MC samples obtained from patients with WM as well as on LAD2 MCs. Moreover, the SGN-70 humanized monoclonal antibody, which binds to CD70 (the receptor-ligand partner of CD27), abrogated sCD27 mediated up-regulation of CD40L and APRIL on WM MCs. Last, treatment of severe combined immunodeficiency–human (SCID-hu) mice with established WM using the SGN-70 antibody blocked disease progression in 12 of 12 mice, whereas disease progressed in all 5 untreated mice. The results of these studies demonstrate a functional role for sCD27 in WM pathogenesis, along with its utility as a surrogate marker of disease and a target in the treatment of WM.

Waldenstrom macroglobulinemia (WM) is an incurable low-grade lymphoma characterized by bone marrow (BM) involvement of IgM secreting lymphoplasmacytic cells. The induction of unfolded protein response (UPR) genes (“physiologic” UPR) enables cells to differentiate into professional secretory cells capable of production of high amounts of endoplasmic reticulum (ER)–processed proteins, such as immunoglobulins. Ultimately, the initially cytoprotective UPR triggers an apoptotic cascade if ER stress is not corrected, called proapoptotic/terminal UPR. We show that WM cells inherently express the physiologic UPR machinery compared with normal BM cells, and that increased ER stress leads to proapoptotic/terminal UPR in WM cells. We therefore examined tunicamycin, ER stress inducer, for potential antitumor effects in WM. Tunicamycin induced significant cytotoxicity, apoptosis and cell-cycle arrest, and inhibited DNA synthesis in WM cell lines and primary BM CD19+ cells from patients with WM with an inhibitory concentration (IC50) of 0.5 μg/mL to 1 μg/mL, but not in healthy donor cells. Importantly, coculture of WM cells in the context of the BM microenvironment did not inhibit tunicamycin-induced cytotoxicity. Finally, we demonstrate that ER stress inducer synergizes with other agents used in the treatment of WM. These preclinical studies provide a framework for further evaluation of ER stress inducing agents as therapeutic agents in WM.

The serum IgM level has been utilised as a marker of tumor progression and to assess response to therapy in patients with Waldenstrom macroglobulinemia (WM). However, there are many limitations to the IgM protein level. The objective of this study was to evaluate the association of known tumor burden markers and prognostic factors with serum free light chain (sFLC) in 98 patients with WM. We demonstrated that sFLC measurement accurately differentiated IgM-MGUS compared with WM reflecting a measurement of tumor burden. In univariate and multivariate analysis, median sFLC at the cut-off at 60 mg/L was higher for WM patients with low hemoglobin and high beta2M, when we applied the WM-IPSS cut-offs, but showed no association with IgM level. This study demonstrates that sFLC is a new marker in WM disease. Further analysis is required to prospectively study the role of sFLC in monitoring response to therapy and as a prognostic marker in WM patients.

The nuclear factor-κB (NF-κB) path-way has been implicated in tumor B-cell survival, growth, and resistance to therapy. Because tumor cells overcome single-agent antitumor activity, we hypothesized that combination of agents that target differentially NF-κB pathway will induce significant cytotoxicity. Therapeutic agents that target proteasome and Akt pathways should induce significant activity in B-cell malignancies as both pathways impact NF-κB activity. We demonstrated that perifosine and bortezomib both targeted NF-κB through its recruitment to the promoter of its target gene IκB using chromatin immunoprecipitation assay. This combination led to synergistic cytotoxicity in Waldenstrom macroglobulinemia (WM) cells that was mediated through a combined reduction of the PI3K/Akt and ERK signaling pathways, found to be critical for survival of WM cells. Moreover, a combination of these drugs with the CD20 monoclonal antibody rituximab further increased their cytotoxic activity. Thus, effective WM therapy may require combination regimens targeting the NF-κB pathway.

Waldenstrom macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma. We demonstrate up-regulated Akt activity in WM, and that Akt down-regulation by Akt knockdown and the inhibitor perifosine leads to significant inhibition of proliferation and induction of apoptosis in WM cells in vitro, but not in normal donor peripheral blood and hematopoietic progenitors. Importantly, down-regulation of Akt induced cytotoxicity of WM cells in the bone marrow microenvironment (BMM) context. Perifosine induced significant reduction in WM tumor growth in vivo in a subcutaneous xenograft model through inhibition of Akt phosphorylation and downstream targets. We also demonstrated that Akt pathway down-regulation inhibited migration and adhesion in vitro and homing of WM tumor cells to the BMM in vivo. Proteomic analysis identified other signaling pathways modulated by perifosine, such as activation of ERK MAPK pathway, which induces survival of tumor cells. Interestingly, MEK inhibitor significantly enhanced perifosine-induced cytotoxicity in WM cells. Using Akt knockdown experiments and specific Akt and PI3K inhibitors, we demonstrated that ERK activation is through a direct effect, rather than feedback activation, of perifosine upstream ERK pathway. These results provide understanding of biological effects of Akt pathway in WM and provide the framework for clinical evaluation of perifosine in WM patients.

The presence of valine (V) at position 158 of FcgammaRllla (CD16) is known to improve clinical response to rituximab in indolent non-Hodgkin lymphoma (NHL). Little is known about the basic mechanisms for this observation. We examined natural killer (NK) cells from healthy donors representing the FcgammaRIIIa-158 polymorphic subgroups (V/V, V/F, and F/F) for gene transcript and cell surface CD16 expression, rituximab binding, and rituximab-dependent NK cell-mediated cytotoxicity. We observed higher levels of FcgammaRIIIa transcripts among individuals with the FcgammaRIIIa-158 V/V versus V/F or F/F genotype (P < .001); increased cell surface CD16 expression by quantitative flow cytometry on NK cells from individuals expressing at least one valine at FcgammaRIIIa-158 versus F/F (P = .029); as well as augmented rituximab binding and rituximab-mediated, antibody-dependent cellular cytotoxicity (ADCC). These results suggest that individuals expressing at least one valine at FcgammaRIIIa-158 might, in part, have better clinical outcomes due to increased CD16 expression, rituximab binding, and rituximab-mediated ADCC.

A significant impairment in understanding the biology and advancing therapeutics for Waldenstrom's macroglobulinemia (WM) has been the lack of a representative cell line and animal model. We, therefore, report on the establishment of the BCWM.1 cell line, which was derived from the long-term culture of CD19(+) selected bone marrow lymphoplasmacytic cells isolated from an untreated patient with WM. BCWM.1 cells morphologically resemble lymphoplasmacytic cells (LPC) and propagate in RPMI-1640 medium supplemented with 10% fetal bovine serum. Phenotypic characterization by flow cytometric analysis demonstrated typical WM LPC characteristics: CD5(-), CD10(-), CD19(+), CD20(+), CD23(+), CD27(-), CD38(+), CD138(+), CD40(+), CD52(+), CD70(+), CD117(+), cIgM(+), cIgG(-), cIgA(-), ckappa(-), clambda(+), as well as the survival proteins APRIL and BLYS, and their receptors TACI, BCMA and BAFF-R. Enzyme-linked immunosorbent assay studies demonstrated secretion of IgMlambda and soluble CD27. Karyotypic and multicolor fluorescence in situ hybridization studies did not demonstrate cytogenetic abnormalities. Molecular analysis of BCWM.1 cells confirmed clonality by determination of IgH rearrangements. Inoculation of BCWM.1 cells in human bone marrow chips implanted in severe combined immunodeficient-hu mice led to rapid engraftment of tumor cells and serum detection of human IgM, lambda, and soluble CD27. These studies support the use of BCWM.1 cells as an appropriate model for the study of WM, which in conjunction with the severe combined immunodeficient-hu mouse model may be used as a convenient model for studies focused on both WM pathogenesis and development of targeted therapies for WM.

Background: Polymorphisms in FcγRIIa and FcγRIIIa receptors are associated with responses to the CD20-directed immunoglobulin G1 (IgG1) monoclonal antibody rituximab among patients with indolent lymphoma. At odds with the aforementioned clinical observations has been the finding that IgG1 binding is impacted by polymorphisms in FcγRIIIa but not FcγRIIa. One possibility for this discrepancy might involve linkage of polymorphisms between FcγRIIa and FcγRIIIa. Materials and Methods: As such, we performed allelespecific polymerase chain reaction and directed sequencing of the genomic DNA coding region of FcγRIIA and FcγRIIIA for 52 healthy individuals. Results: Two common polymorphisms were observed for FcγRIIA (at positions 27 and 131) and FcγRIIIA (at positions 48 and 158). Importantly, we observed linkage among polymorphisms within and between FcγRIIa and FcγRIIIa, including the expression of histidine at FcγRIIa-131 and valine at FcγRIIIa, both of which are associated with enhanced responses to rituximab. The results of these studies demonstrate that there is wide linkage within and between polymorphisms in FcγRIIa and FcγRIIIa and might provide an explanation for why polymorphisms at FcγRIIa are associated with rituximab responses despite a lack of impact on IgG1 binding. Conclusion: Knowledge of such linkages could facilitate the development of diagnostic tests aimed at identifying patients who might be more suitable for treatment with rituximab and possibly other therapeutic antibodies.

Waldenström macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Protein kinase Cβ (PKCβ) regulates cell survival and growth in many B-cell malignancies. In this study, we demonstrate up-regulation of PKCβ protein in WM using protein array techniques and immunohistochemistry. Enzastaurin, a PKCβ inhibitor, blocked PKCβ activity and induced a significant decrease of proliferation at 48 hours in WM cell lines (IC50, 2.5-10 μM). Similar effects were demonstrated in primary CD19+ WM cells, without cytotoxicity on peripheral blood mononuclear cells. In addition, enzastaurin overcame tumor cell growth induced by coculture of WM cells with bone marrow stromal cells. Enzastaurin induced dose-dependent apoptosis at 48 hours mediated via induction of caspase-3, caspase-8, caspase-9, and PARP cleavage. Enzastaurin inhibited Akt phosphorylation and Akt kinase activity, as well as downstream p-MARCKS and ribosomal p-S6. Furthermore, enzastaurin demonstrated additive cytotoxicity in combination with bortezomib, and synergistic cytotoxicity in combination with fludarabine. Finally, in an in vivo xenograft model of human WM, significant inhibition of tumor growth was observed in the enzastaurin-treated mice (P = .028). Our studies therefore show that enzastaurin has significant antitumor activity in WM both in vitro and in vivo, providing the framework for clinical trials to improve patient outcome in WM.

Interaction between receptor activator of nuclear factor κB ligand (RANKL) and RANK/osteoprotegerin (OPG) plays a dominant role in osteoclast activation and possibly in plasma cell survival in multiple myeloma (MM). We measured soluble RANKL (sRANKL), OPG, and bone remodeling markers in 121 patients with newly diagnosed MM to evaluate their role in bone disease and survival. Serum levels of sRANKL were elevated in patients with MM and correlated with bone disease. The sRANKL/OPG ratio was also increased and correlated with markers of bone resorption, osteolytic lesions, and markers of disease activity. The sRANKL/OPG ratio, C-reactive protein (CRP), and β2-microglobulin were the only independent prognostic factors predicting survival in multivariate analysis. We generated a prognostic index based on these factors that divided our patients into 3 risk groups. The low-risk group had a 96% probability of survival at 5 years, whereas the intermediate-risk and the high-risk groups had probabilities of survival of 52% and 0%, respectively. Not only do these results confirm for the first time in humans the importance of sRANKL/OPG in the development of bone disease, they also highlight the role of this pathway in the biology of plasma cell growth as reflected by its influence on survival.

While a familial predisposition may exist in up to 20% of patients with Waldenström's Macroglobulinemia (WM), the precipitating cause of this B-cell malignancy remains unknown in most patients. In previous studies, an association between hepatitis C virus (HCV) infection and WM has been suggested as etiological. This relationship has been the subject of debate, however, with some studies demonstrating increased incidence of HCV infection among WM patients and other studies showing no such association exists. This discordance might be attributable to the analytical method used, HCV antibody detection, which might be ineffective in patients with immunosuppression. We therefore analyzed the prevalence of HCV in a large population of WM patients utilizing both an HCV antibody detection immunoassay as well as qualitative polymerase chain reaction assay to directly detect HCV presence in serum samples. None of 100 randomly tested WM patients in this study tested positive for HCV by either analytical method. Our results therefore demonstrate a lack of association between HCV and WM.

WM is a disease of the elderly with a protracted course in many patients. There are limited data which indicate that several WM patients die due to causes which are not directly related to their underlying malignancy. However, the realization and the estimation of the contribution of unrelated mortality in WM are important for the design of treatment strategy in patients of advanced age. To our knowledge there are no such data published for WM patients. Thus, we analyzed the outcomes of 408 patients with symptomatic WM who received therapy within the centers of the Greek Myeloma Study Group in order to assess disease related survival. In this analysis unrelated death was considered to be a competing risk event. Causes of death other than WM, treatment toxicity or myelodysplasia/transformation were considered as unrelated deaths. Median age of patients was 68 (28-92) years; 21% were >75 years and 9% were ≤50 years of age. Patients who started therapy after 2000 were older (median age 70 vs. 65 years before 2000, p75 years (vs. 13% before 2000). In terms of ISSWM stage, more patients had high and intermediate risk disease after 2000 (41% & 42% vs. 25.5% & 38% before 2000, p Median follow up for all patients was 5.5 years (9 years in the pre-2000 and 4.5 years in the post-2000 group) and 52% of patients have died (77% in the group before 2000 and 40% in the group after 2000). However, 23% of deaths were considered unrelated to WM. Thus, 5-year and 8-year overall survival (OS) was 70% and 54% respectively, with a median OS of all patients of 8.8 years. When we performed survival analysis with unrelated deaths as competing risk, then 5-year risk of WM-related death was 21.4% (95% CI 17-26%) and of unrelated death was 7.6% (95% CI 5-10.5%), while 8-year WM-related death rate was 32% (95% CI 27-37%) and unrelated death 11.5% (95% CI 8-15%). Because older patients are at higher risk of unrelated deaths we performed an age-specific analysis. The median survival of patients >75 years was 5.3 vs. 9.7 years for patients ≤75 years (p75 years, the 5-year death rate due to WM was 22% (95% CI 13-32%) vs. 21% (95% CI 16-26%) for patients ≤75 years (p=0.193), while the 5-year unrelated death rate was 17% (95% CI 10-27%) and 5.1% (3-8%), respectively (p75 years) >40% of deaths are unrelated to WM, while WM-specific death rates were similar for patients >75 or ≤75 years. In patients ≤50 years there were no WM-unrelated deaths. We then evaluated the prognostic significance of IPSSWM, which discriminated 3 groups with 5-year overall survival of 86%, 68% and 51% for low, intermediate and high risk groups, respectively (p In conclusion, this is the first analysis in a large cohort of patients with symptomatic WM in which WM-unrelated death is treated as a competing risk. Many patients of advanced age die of causes unrelated to WM and this fact should be taken into account in the evaluation of long term outcomes and the design of clinical trials in patients with WM, especially since more patients of advanced age are diagnosed and treated for WM. Disclosures: No relevant conflicts of interest to declare.

Abstract 3032 The treatment of Waldenström's Macroglobulinemia (WM) has changed over the last decades, mainly since the introduction of nucleoside analogues and of rituximab in the management of this disease. Furthermore, novel agents such as bortezomib have been recently introduced. Several analyses in multiple myeloma indicated that the outcome of these patients has significantly improved over the last decade as a result of the introduction of novel agents. However, such data are not available for patients with WM. Thus, we performed an analysis in order to investigate whether the outcome of patients with WM has improved over the last years, compared to that of patients who started treatment before new drugs, such as rituximab became widely available, especially as part of the frontline treatment. We analyzed the database of the Greek Myeloma Study Group which includes 345 patients, who started treatment after January 1985: 130 patients initiated treatment before 1/1/2000 (Group A) and 215 patients started treatment after 1/1/2000 (Group B). More patients were males in both groups (54% in group A vs. 63% in group B, p=0.084), but patients in group B were older (median age 70 years vs. 65 years in group A, p=0.001). Patients in both groups started treatment mainly due to anemia (40% and 43% in groups A and B, respectively). Similar percentages of patients in groups A and B had hemoglobin ≤11.5 g/dl (74% vs. 77%, p=0.57), platelets ≤100,000/ml (12% vs. 14%, p=0.643), albumin ≤3.5 g/dl (44% vs. 50%, p=0.306) and elevated LDH (≥250 IU/L) (21% vs. 17%, p=0.422). The median serum M-peak levels were also similar (3.95 g/dl vs. 3.75 g/dl, p=0.485) while 5% and 7% of patients in groups A and B had a serum M-peak >7 g/dl (p=0.491). However, more patients in group B had serum beta2-microglobulin above 3 mg/dl (62% vs. 42%, p=0.004). Thus, according to the International Prognostic Scoring System (IPSS) for WM, 30%, 48%, and 22% had low, intermediate and high risk disease in group A and 15%, 42% and 43% in group B, respectively (p Disclosures: No relevant conflicts of interest to declare.