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Therapeutic approaches in multiple myeloma (MM) are increasingly focused on restoring antitumor immunity. Immunomodulators have a variety of effects on the immune microenvironment, and are frequently incorporated into multidrug regimens. The monoclonal antibodies daratumumab and elotuzumab have been granted accelerated approval for use in the relapsed or refractory setting, and several other antibodies including immune checkpoint inhibitors are currently being evaluated for the treatment of MM. Antimyeloma vaccines have been developed, and may be useful in maintaining remission. The role of allogeneic stem cell transplantation continues to be an area of active research, as reduced intensity conditioning (RIC) regimens have significantly decreased treatment-related complications. Other immunotherapeutic approaches in development include marrow infiltrating lymphocytes, T cell receptor modified T cells (TCRts), and chimeric antigen receptor (CAR) T cells. Here we review the existing data on immunotherapy in MM, and discuss some promising areas of research which may impact the future of myeloma therapy.

Pomalidomide and low-dose dexamethasone (PomDex) is standard treatment of lenalidomide refractory myeloma patients who have received >2 prior therapies. We aimed to assess the safety and efficacy of the addition of oral weekly cyclophosphamide to standard PomDex. We first performed a dose escalation phase 1 study to determine the recommended phase 2 dose of cyclophosphamide in combination with PomDex (arm A). A randomized, multicenter phase 2 study followed, enrolling patients with lenalidomide refractory myeloma. Patients were randomized (1:1) to receive pomalidomide 4 mg on days 1 to 21 of a 28-day cycle in combination with weekly dexamethasone (arm B) or pomalidomide, dexamethasone, and cyclophosphamide (PomCyDex) 400 mg orally on days 1, 8, and 15 (arm C). The primary end point was overall response rate (ORR). Eighty patients were enrolled (10 in phase 1 and 70 randomized in phase 2: 36 to arm B and 34 to arm C). The ORR was 38.9% (95% confidence interval [CI], 23-54.8%) and 64.7% (95% CI, 48.6-80.8%) for arms B and C, respectively (P = .035). As of June 2015, 62 of the 70 randomized patients had progressed. The median progression-free survival (PFS) was 4.4 (95% CI, 2.3-5.7) and 9.5 months (95% CI, 4.6-14) for arms B and C, respectively (P = .106). Toxicity was predominantly hematologic in nature but was not statistically higher in arm C. The combination of PomCyDex results in a superior ORR and PFS compared with PomDex in patients with lenalidomide refractory multiple myeloma. The trial was registered at www.clinicaltrials.gov as #NCT01432600.

We describe baseline incidence and risk factors for new cancers in 4161 persons receiving autotransplants for multiple myeloma in the United States from 1990 to 2010. Observed incidence of invasive new cancers was compared with expected incidence relative to the US population. The cohort represented 13,387 person-years at-risk. In total, 163 new cancers were observed, for a crude incidence rate of 1.2 new cancers per 100 person-years and cumulative incidences of 2.6% (95% confidence interval [CI], 2.09 to 3.17), 4.2% (95% CI, 3.49 to 5.00), and 6.1% (95% CI, 5.08 to 7.24) at 3, 5, and 7 years, respectively. The incidence of new cancers in the autotransplantation cohort was similar to age-, race-, and gender-adjusted comparison subjects with an observed/expected (O/E) ratio of 1.00 (99% CI, .81 to 1.22). However, acute myeloid leukemia and melanoma were observed at higher than expected rates with O/E ratios of 5.19 (99% CI, 1.67 to 12.04; P = .0004), and 3.58 (99% CI, 1.82 to 6.29; P < .0001), respectively. Obesity, older age, and male gender were associated with increased risks of new cancers in multivariate analyses. This large data set provides a baseline for comparison and defines the histologic type specific risk for new cancers in patients with MM receiving postautotransplantation therapies, such as maintenance.

Background Therapeutic advancements following the introduction of autologous stem cell transplantation and ‘novel’ agents have significantly improved clinical outcomes for patients with multiple myeloma (MM). Increased life expectancy, however, has led to renewed concerns about the long-term risk of second primary malignancies (SPMs). This review outlines the most up-to-date knowledge of possible host-, disease-, and treatment-related risk factors for the development of SPMs in patients with MM, and provides practical recommendations to assist physicians. Design A Panel of International Myeloma Working Group members reviewed the most relevant data published in the literature as full papers, or presented at meetings of the American Society of Clinical Oncology, American Society of Hematology, European Hematology Association, or International Myeloma Workshops, up to June 2016. Here, we present the recommendations of the Panel, based on this literature review. Results Overall, the risk of SPMs in MM is low, multifactorial, and partially related to the length of patients’ survival and MM intrinsic susceptibility. Studies suggest a significantly increased incidence of SPMs when lenalidomide is administered either following, or concurrently with, oral melphalan. Increased SPM incidence has also been reported with lenalidomide maintenance following high-dose melphalan, albeit to a lesser degree. In both cases, the risk of death from MM was significantly higher than the risk of death from SPMs, with lenalidomide possibly providing a survival benefit. No increase in SPM incidence was reported with lenalidomide plus dexamethasone (without melphalan), or with bortezomib plus oral melphalan, dexamethasone, or thalidomide. Conclusion In general, the risk of SPMs should not alter the current therapeutic decision-making process in MM. However, regimens such as lenalidomide plus dexamethasone should be preferred to prolonged exposure to lenalidomide plus oral melphalan. SPM risk should be carefully discussed with the patient in the context of benefits and risks of different treatment options.

Increasing knowledge concerning the biology of hematologic malignancies as well as the role of the immune system in the control of these diseases has led to the development and approval of immunotherapies that are resulting in impressive clinical responses. Therefore, the Society for Immunotherapy of Cancer (SITC) convened a hematologic malignancy Cancer Immunotherapy Guidelines panel consisting of physicians, nurses, patient advocates, and patients to develop consensus recommendations for the clinical application of immunotherapy for patients with multiple myeloma, lymphoma, and acute leukemia. These recommendations were developed following the previously established process based on the Institute of Medicine’s clinical practice guidelines. In doing so, a systematic literature search was performed for high-impact studies from 2004 to 2014 and was supplemented with further literature as identified by the panel. The consensus panel met in December of 2014 with the goal to generate consensus recommendations for the clinical use of immunotherapy in patients with hematologic malignancies. During this meeting, consensus panel voting along with discussion were used to rate and review the strength of the supporting evidence from the literature search. These consensus recommendations focus on issues related to patient selection, toxicity management, clinical endpoints, and the sequencing or combination of therapies. Overall, immunotherapy is rapidly emerging as an effective therapeutic strategy for the management of hematologic malignances. Evidence-based consensus recommendations for its clinical application are provided and will be updated as the field evolves. Electronic supplementary material The online version of this article (doi:10.1186/s40425-016-0188-z) contains supplementary material, which is available to authorized users.

Effectively treating patients with multiple myeloma is challenging. The development of therapeutic regimens over the past decade that incorporate the proteasome inhibitor bortezomib and the immunomodulatory drugs thalidomide and lenalidomide has been the cornerstone of improving the outcome of patients with myeloma. Although these treatment regimens have improved patient survival, nearly all patients eventually relapse. Our improved understanding of the biology of the disease and the importance of the microenvironment has translated into ongoing work to help overcome the challenge of relapse. Several classes of agents including next-generation proteasome inhibitors, immunomodulatory agents, selective histone-deacetylase inhibitors, antibody and antitumor immunotherapy approaches are currently undergoing preclinical and clinical evaluation. This Review provides an update on the latest advances in the treatment of multiple myeloma. In particular, we focus on novel therapies including modulating protein homeostasis, kinases inhibitors, targeting accessory cells and cytokines, and immunomodulatory agents. A discussion of the challenges associated with these therapeutic approaches is also presented.

Elevated serum ferritin is associated with reduced survival following allogeneic transplantation and an increased risk of toxic and infectious complications after autologous hematopoietic stem cell transplantation (ASCT). We studied 315 patients who underwent ASCT for Hodgkin (HL) or non-Hodgkin lymphoma (NHL) at our institution in whom pretransplantation ferritin was available to determine its association with survival. On multivariate analysis, a pretransplantation ferritin >685 ng/mL was associated with significantly lower overall (OS; P = .002) and relapse-free survival (RFS; P = .021). Ferritin >685 ng/mL was associated with a higher incidence of relapse (P = .005) and relapse mortality (P < .001), but not of nonrelapse mortality (NRM; P = .23). Similar results were seen when pretransplantation ferritin was analyzed as a continuous variable and by quartiles. Our results indicate the need for studies designed to correlate an elevated ferritin with iron overload and to analyze the benefit of strategies to reduce the extent of iron overload.

Given the prevalence of osteolytic bone disease in multiple myeloma (MM), novel therapies targeting bone microenvironment are essential. Previous studies have identified activin A to be of critical importance in MM-induced osteolysis. Lenalidomide is a known and approved treatment strategy for relapsed MM. Our findings demonstrate that lenalidomide acts directly on bone marrow stromal cells via an Akt-mediated increase in Jun N-terminal kinase-dependent signaling resulting in activin A secretion, with consequent inhibition of osteoblastogenesis. Here, we attempted to augment the antitumor benefits of lenalidomide while overcoming its effects on osteoblastogenesis by combining it with a neutralizing antibody to activin A. Increased activin A secretion induced by lenalidomide was abrogated by the addition of activin A-neutralizing antibody, which effectively restored osteoblast function and inhibited MM-induced osteolysis without negating the cytotoxic effects of lenalidomide on malignant cells. This provides the rationale for an ongoing clinical trial (NCT01562405) combining lenalidomide with an anti-activin A strategy.

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by the aberrant expansion of plasma cells within the bone marrow, as well as at extramedullary sites. Decades of scientific research are now beginning to unravel the intricate biology that underlies the pathophysiology of MM. In particular, the roles of cellular differentiation, molecular pathogenesis, and oncogenes involved in the natural history of MM are becoming clearer. This has enabled the identification of specific cytokines, adhesion molecules, and stromal cells that affect MM cell development, disease progression, and treatment responses. This review describes our current understanding regarding the biology of MM, and how this has led to a robust pipeline of novel therapeutic agents with the potential to overcome resistance to existing MM therapies and, therefore, further improve outcomes in patients with MM.

The past decade has witnessed a dramatic improvement in the therapeutic options in multiple myeloma (MM). Several novel biologically targeted agents are in clinical use and have resulted in improved outcomes. However, the disease remains incurable, underscoring the need for continued efforts towards understanding MM biology, better risk stratification and exploitation of novel therapeutic approaches. Novel agents that target tumor and stromal compartments can be categorized as those that target protein dynamics (e.g., heat shock protein 90 and the ubiquitin–proteasome system), intracellular signaling kinases (e.g., JAK/STAT, PI3k/Akt/mTOR and MAPK pathways), cell cycle molecular machinery (e.g., cyclin-dependent kinase inhibitor and Aurora kinase inhibitors), membrane-bound receptors (e.g., IGF-1, VEGF and CD40), epigenetic modulators (e.g., DNA methyltransferase and histone deacetylase), tumor vasculature and microenvironment (e.g., angiogenesis and integrins) and agents modulating anti-MM immune responses. This article focuses on a series of new therapeutic targets that have shown promising preclinical results and early evidence of anti-MM activity in clinical studies, either alone or in combination with other conventional or novel anti-MM treatments.

Elevated pretransplant serum ferritin levels have been associated with an increased incidence of morbidity and mortality after allogeneic haematopoietic stem cell transplantation (HCT). We studied 222 patients who underwent myeloablative allogeneic HCT in whom pretransplantation serum ferritin levels were available. Pretransplantation ferritin > 1910 microg/l was associated with lower overall survival (P = 0.003), lower relapse-free survival (P = 0.003), decreased chronic graft-versus-host disease (GVHD) (P = 0.019) and increased non-relapse mortality (NRM) (P = 0.042). Similar results were obtained when pretransplantation ferritin was analysed as a continuous variable and by quartiles. Our results indicate that an elevated pretransplant ferritin level adversely impacts transplantation outcomes. The adverse impact of elevated ferritin on NRM and survival was despite its association with lower incidences of acute and chronic GVHD, which are major causes of NRM. The association of ferritin with iron overload and its influence on HCT outcomes requires further prospective validation.

Iron overload is a well-established adverse prognostic factor for allogeneic hematopoietic SCT in thalassemia,1 and elevated pretransplant serum ferritin seems to adversely affect prognosis with myeloablative transplantation2, 3 for other diseases as well, primarily because of increased non-relapse mortality. We analyzed patients who underwent nonmyeloablative preparation for allogeneic transplantation to determine whether the presence of iron overload was also an adverse prognostic factor for patients undergoing nonmyeloablative transplants.

Background: Pomalidomide-dexamethasone results in an overall response rate of 33% and median PFS of 4.2 months in patients with prior lenalidomide and bortezomib (Richardson et al. Blood 2014). In this randomized phase II trial, we compared pomalidomide-dexamethasone (arm B) versus the addition of oral weekly cyclophosphamide to pomalidomide-dexamethasone (arm C) in patients with lenalidomide-refractory multiple myeloma (MM). We have previously reported that the recommended phase II dose of cyclophosphamide with standard-dose pomalidomide + dexamethasone was 400 mg PO D1, 8, 15. Patients and Methods: Eligible patients had relapsed and refractory MM after at least 2 prior therapies and were lenalidomide refractory. Patients had a platelet count ≥ 50,000/mm3 and ANC ≥ 1,000/mm3 (patients with ≥50% bone marrow plasmacytosis were allowed if platelet count was ≥ 30,000/mm3and ANC could be supported with GCSF during screening and therapy). Patients were randomized (1:1) to receive pomalidomide 4 mg PO D1-21 and dexamethasone 40 mg PO D1, 8, 15, 22 (20 mg if older than 75 years) (arm B) with or without oral cyclophosphamide 400 mg PO D1, 8, 15 of a 28-day cycle (arm C). Patients randomized to arm B were allowed to cross over to arm C in the event of disease progression. Thromboprophylaxis was mandated with aspirin, warfarin, or LMWH. The primary endpoint was overall response rate using IMWG criteria. Secondary endpoints included an evaluation of PFS, OS and safety of the two arms. Results: Between 7/2012 and 3/2014, 36 patients were randomized to arm B and 34 to arm C. Patients characteristics were not different between the 2 arms (table below). The median number of prior therapies was 4 (2-12). All patients were lenalidomide refractory and none received prior pomalidomide. After a median follow up of 15 months, the overall response rate (partial response or better) was 39% and 65% (p=0.03) for arm B and C, respectively. The clinical benefit rate (minimal response or better) was 64% and 79% (p=0.2) for arm B and C, respectively. The median PFS was 4.4 months (95% CI 2.3-5.9) for arm B and 9.2 months (95% CI 4.6-16) for arm C (log rank p=0.04). As of July 2014, 28 patients had died (16 arm B, 12 arm C) with median overall survival of 10.5 versus 16.4 months (p=0.08) for arm B and C, respectively. Hematologic grade 3/4 adverse events were more frequent in arm C, although this was not statistically significant (see table). Thirteen patients crossed over and oral weekly cyclophosphamide was added to their tolerated dose of pomalidomide dexamethasone. For those patients, the best response was as follows: 2 PR, 2 MR, and 6 SD, 3 PD. Conclusions: Pomalidomide-dexamethasone in combination with oral weekly cyclophosphamide resulted in a superior response rate and PFS compared to pomalidomide-dexamethasone alone in patients with relapsed and refractory MM. The increased hematologic toxicities, as a result of the addition of oral cyclophosphamide, were manageable. Table Arm B (N=36) Arm C (N=34) P value Age, years, median (range) 63 (50-78) 64 (47-80) 0.7 Male, n (%) 23 (64) 18 (53) 0.3 Number of prior therapies, median (range) 4 (2-12) 4 (2-9) 0.5 Bortezomib refractory, n (%) 28 (78) 24 (71) 0.3 Carfilzomib refractory, n (%) 16 (44) 13 (38) 0.5 Prior high-dose therapy, n (%) 27 (75) 28 (82) 0.6 Prior alkylating agent, n (%) 32 (89) 32 (94) 1 B2-microglobulin, median (range) 3.2 (1.6-10) 3.6 (1.5-13.9) 0.5 Serum creatinine, median (range) 1 (0.5-2.3) 0.9 (0.6-2.1) 0.6 High-risk cytogenetics, n (%) 5 (24) 6 (28) 0.8 Deletion 17p, n (%) 3 (14) 4 (20) 0.8 t(4;14), n (%) 3 (14) 3 (14) 0.9 Trisomy or tetrasomy 1q, n (%) 11 (55) 6 (33) 0.4 Best response (partial response or better), n (%) 14 (39) 22 (65) 0.03 Clinical benefit rate (MR or better), n (%) 23 (64) 27 (79) 0.2 Grade 3/4 neutropenia, n (%) 12 (33) 17 (50) 0.2 Grade 3/4 febrile neutropenia, n (%) 4 (11) 6 (18) 0.5 Grade 3/4 thrombocytopenia, n (%) 2 (5) 5 (15) 0.2 Grade 3/4 anemia, n (%) 3 (8) 7 (20) 0.2 Grade 3/4 pneumonia, n (%) 4 (11) 3 (9) 1 Grade 3/4 fatigue, n (%) 2 (5) 4 (12) 0.4 Number of serious adverse events 17 20 Disclosures Baz: Celgene: Research Funding; Millenium: Research Funding; Bristol-Myers Squibb: Research Funding; Karypharm: Research Funding; Sanofi: Research Funding. Off Label Use: Pomalidomide cyclophosphamide dexamethasone in relapsed refractory myeloma. Martin:Sanofi: Research Funding; Novartis: Speakers Bureau. Alsina:Triphase: Research Funding; Millenium: Research Funding. Shain:Onyx / Amgen: Research Funding; Treshold: Research Funding. Chari:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Array Biopharma: Membership on an entity's Board of Directors or advisory committees. Jagannath:Celgene: Honoraria; Millennium: Honoraria; Sanofi: Honoraria.

TPS8624 Background: The introduction of immunomodulatory agents and proteasome inhibitors, and advances in high dose therapy administration have made an impact on progression free survival (PFS) an...

Introduction Patients (pts) with MM often present with varying degrees of renal insufficiency (RI) which can cause significant morbidity. In younger pts with MM, high-dose chemotherapy with autologous stem cell transplantation (ASCT) has been shown to enhance remission and possibly improve survival. In pts with RI there has been concern about the safety and efficacy of proceeding to ASCT. At our center, ASCT has been routinely offered to pts with RI. Herein we report the results of MM pts with RI who received ASCT since 2000. Patients and Methods Outcomes of 50 MM pts with RI were compared to 100 matched ASCT recipients. The matched transplant pts studied were similar with respect to subject-, disease-, and transplant-related characteristics. All pts met standard eligibility criteria for ASCT and pts with RI had a serum creatinine (Cr) level of >2 mg/dL (average Cr = 6.07 with a range of 2 to 11.79 mg/dL), at the time of ASCT. 14 of the 50 pts were on dialysis at the time of ASCT. All patients had active MM requiring therapy and all pts had evidence of stable disease or treatment response prior to transplant. For pts with RI, the average age at transplant was 56 years (range 33 to 69 yrs) and the median time between diagnosis and ASCT was 302 days. The preparative therapy consisted of single agent Melphalan, given on Day (-3) or (-2) with a median administered dose of 140 mg/m2 (range 100-200 mg/m2) while the dose for the matched patients was 200mg/m2. All pts were hospitalized for ASCT and received standard supportive care; >2 x 10e6/Kg CD34(+) cells, G-CSF, transfusions as necessary and appropriate anti-infectives. Results The 30-day and 100-day treatment related mortality (TRM) rates for both the patients with RI and matches were 0%. Amongst the patients with RI, 4 patients required ICU care and the average duration of hospitalization was 23.36 days (range: 14 - 48 days) and duration of hospitalization for matches 19.4 days (range: 11-58 days). Time to ANC > 0.5 x10e9/L was 12 days (range 10-21 days) and 12 days (range 10-22 days) in the RI and matched cohort respectively. Time to platelets > 20 x 10e9/L was 11 days (range: 6-22 days) and 14 days (range 8-51days) in the RI and matched cohort respectively. 32% of pts with RI were in very good partial response (VGPR)+ complete remission (CR) of which 12% were in CR at ASCT. After ASCT, 50% of pts achieved a VGPR+CR of which 34% pts achieved a CR. The mean overall survival (OS) for patients with RI was 4.2 yrs and for controls was 6.5 yrs [Figure 1. HR 2.036, 95% CI (1.204-3.443) p=0.0084]. Conclusion ASCT is a safe and effective treatment option for MM pts with RI including those on dialysis with engraftment characteristics similar to pts without RI. Disclosures: Wolf: Onyx: Honoraria; Millenium: Honoraria; Celgene: Honoraria, Research Funding.

Abstract 328 In multiple myeloma (MM), deregulated osteoclast (OC)/osteoblast (OB) cross-talk induces osteolytic bone lesions. The HDAC6 selective inhibitor, rocilinostat (ACY-1215), in combination with bortezomib has shown potent anti myeloma activity in preclinical studies, which provided the rationale for a clinical trial that is currently recruiting relapsed/refractory MM patients (NCT01323751). However, while the beneficial role of bortezomib in tumor-related bone disease has been previously described, the effect of HDAC6 inhibition is not known. Evidence suggests a positive effect on bone turnover as pan HDAC inhibitors accelerate OB maturation and suppress OC maturation in vitro. Here, we evaluated effects of the selective HDAC6 inhibitor rocilinostat (Acetylon Pharmaceuticals, Inc), alone and in combination with bortezomib in MM bone disease. Rocilinostat (1 μM) alone and in combination with bortezomib (2.5 nM) inhibited OC differentiation, evidenced by a decreased number of TRAP positive multinucleated cells and bone-resorbing activity. In addition, rocilinostat (1 μM) significantly decreased cell growth of mature OC in co-culture with MM cell lines and in combination with bortezomib inhibited transcription factors implicated in OC differentiation including p-ERK, p-AKT, c-FOS, and NFATC1. Importantly, such an effect was cytokine (RANKL and M-CSF) dependent. Further, rocilinostat, alone and in combination, enhanced OB differentiation, evidenced by increased alkaline phosphatase (ALP) enzyme activity and alizarin red staining. In addition, we found increased mRNA expression of beta-catenin, osteocalcin, ALP, and RUNX2. Based on this promising in vitro data, we used the xenograft model of disseminated human MM in SCID mice to study the effect of rocilinostat, alone and in combination with bortezomib, on MM bone disease. MM.1S-GFP-Luc cells were injected intravenously, and MM disease progression was followed by bioluminescence imaging. A significant decrease in tumor burden was observed in mice following three weeks of treatment with rocilinostat, alone or in combination with bortezomib. Isolating serum from control and treated mice, we also observed a significant decrease of TRAPc5b levels, a marker of bone resorption, as well as a significant increase in osteocalcin levels, a marker of bone formation, in the serum of the combination treated cohort. Cells isolated from the calvaria from the combination treated group compared to the control group showed a significant increase in the mRNA expression of ALP, RUNX2, and osterix, as well as a significant decrease in the mRNA expression ratio of RANKL/OPG. To elucidate the role of HDAC6 inhibition on bone turnover, we used HDAC6 knockout mice. Cells were isolated from femurs, tibia, and spine of 2 month-old wild type (WT) and HDAC6 knockout (KO) mice and mRNA expression for osteocalcin, ALP, RUNX2 and osterix was assessed by qPCR. We observed a significant increase in osteocalcin mRNA expression without significant changes in the mRNA expression of ALP, RUNX2 and osterix. Bone marrow stromal cells (BMSCs) differentiated from WT and KO mice were co-cultured with MM murine cell lines and, notably, the proliferative advantage conferred by BMSC isolated from HDAC6 KO mice to MM cell lines was significantly decreased compared to WT BMSCs. These data suggest that a microenviroment lacking HDAC6 reduces MM cell proliferation. Moreover, treatment with rocilinostat (1mM) for 24 h inhibited proliferation of MM cells cocultured with WT BMSCs to levels observed in MM cells cultured with KO BMSC lacking endogenous HDAC6. Finally, the effect of co-treatment with rocilinostat (1μM) and bortezomib (2.5 nM) on proliferation of MM cells co-cultured with WT-BMSC was similar to that observed when bortezomib was added to MM cells in cocultures with KO BMSC. In conclusion, the in vitro data and the in vivo results from the xenograft models of human MM in SCID mice, as well as data in the HDAC6 KO mice, indicate a potential beneficial role of HDAC6 inhibition on MM-related bone disease. We are currently performing dynamic and static histomorphometric analysis to confirm this effect on bone remodeling at the tissue level. These effects on bone remodeling are an added benefit for MM patients and will be assessed prospectively in our ongoing clinical trial. Disclosures: Hideshima: Acetylon: Consultancy. Anderson:Onyx: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Jones:Acetylon Pharmaceuticals, Inc.: Employment. Raje:Onyx: Consultancy; Celgene: Consultancy; Millennium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding.

Abstract 3989 Bone Marrow Stromal Cells (BMSC), Osteoblasts (OB) and osteoclasts (OC) are a central part of the bone microenvironment and play a crucial role in multiple myeloma (MM) growth and survival. Their imbalance results in osteolytic lesions. Understanding the mechanisms underlying osteolytic lesions is important not only for the improvement of osteolytic bone disease but also for the treatment of MM. The osteocyte-secreted protein sclerostin, encoded by the SOST gene, is a potent inhibitor of osteoblastogenesis. However, the role of Sclerostin in MM remains to be elucidated. Our objective was to evaluate the role of sclerostin in MM bone disease and confirm that sclerostin directed strategies are an effective approach in MM. We observed higher levels of sclerostin in MM patients' plasma compared to leukemia patients, gastric cancer patients and healthy volunteers. Importantly, sclerostin levels were associated with an increase in tumor burden suggesting that MM cells are associated with the increase levels of sclerostin. Sclerostin concentrations similar to those detected in MM patients' plasma inhibited OB differentiation and an anti-sclerostin neutralizing antibody (R&D Systems) reversed this effect. Furthermore, sclerostin increased TRAP positive OC numbers differentiated from MM patients' peripheral blood mononuclear cell (PBMC) and their function as detected by pit formation assay. This was associated with stimulation of Ca2+/calmodulin-dependent protein kinases II (CaMKII) and c-Jun N-terminal kinase (JNK) signaling in preosteoclasts reversed by specific inhibitors with consequent inhibition of osteoclastogenesis. Moreover, sclerostin stimulated JNK and CaMKII phosphorylation, stimulated mRNA expression of RANKL and inhibited mRNA expression of OPG in MM patient derived BMSC. RANKL plays a crucial role in promoting OC differentiation and OPG, the decoy receptor for RANKL, inhibits OC differentiation; therefore our results indicate that sclerostin accelerates OC differentiation by JNK and CaMKII signaling stimulation in BMSC in addition to its direct affect against OC. We next examined OB derived from MM patients' BMSC cocultured with the MM cell line INA6 by using cell culture inserts to avoid cell-cell contact. INA6 inhibited OB differentiation and sclerostin neutralizing antibody reversed the INA6 effect as assessed by qPCR and alizarin red staining. Interestingly, co-culture with MM cells stimulated sclerostin mRNA expression and sclerostin protein expression in OB well as in OB cocultures with MM cells. Moreover recombinant CCL3 protein stimulated sclerostin mRNA expression in MM cells. Because CCL3 is secreted by MM cells, these data suggest in part the mechanism by which sclerostin is increased in MM-OB cocultures. These data suggest sclerostin is secreted by MM cells and OB and inhibits osteoblastogenesis and stimulates osteoclastogenesis directly and indirectly. Neutralizing sclerostin levels reverses these effects. Taken together, our data suggest that sclerostin is a good target to inhibit myeloma bone disease and help restore normal bone homeostasis. Disclosures: Raje: Onyx: Consultancy; Celgene: Consultancy; Millenium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding.

Abstract 4023 Background: mTOR kinase-targeted therapy is in the early phase of clinical evaluation in multiple myeloma (MM). Despite promising preclinical results with mTOR inhibitors, resistance to this class of drugs in MM patients may occur due to feedback Akt activation by mTORC1. This led to the development of mTORC1/2 inhibition strategies in the treatment for MM, predicated upon the rationale that mTORC2 inhibitors prevent inhibition of mTORC1 blockade-induced feedback AKT activation by mTORC1 inhibitors. Indeed, our previous studies using a novel dual mTORC1 and mTORC2 selective inhibitor AZD8055 show MM cell growth inhibition via apoptosis, associated with inhibition of mTORC1 and mTORC2 signaling, including rapamycin-resistant 4E-BP1 (downstream of mTORC1) and Akt as well as NDRG1 (an effector of mTORC2). Importantly, AZD8055 also inhibited PI3K/Akt signaling and related MM cell growth induced by cytokines (i.e., IL-6, IGF-1) or co-culture with bone marrow stromal cells (BMSCs). Recent studies, however, reveal that constitutively activated Akt signaling negatively regulates IGF-1 receptor (IGF-1R) at the transcriptional level, independent of mTOR activity. Moreover, AKT-induced IGF-1R down-regulation reduces sensitivity of IRS1 to IGF-1 stimulation. We have also shown that IGF-1R inhibitor triggers significant MM cell toxicity. Methods and Results: In this study, we therefore hypothesized that mTORC2 blockade may upregulate IGF-1R expression and/or activity via Akt modulation in MM cells, and that IGF-1R blockade may enhance the cytotoxic effects of mTOR kinase inhibition in MM cells. We first examined the tyrosine phosphorylation sites (Y1135/1136) in the activation loop of the IGF-1R kinase domain in three MM cell lines (MM.1S, OPM1 and RPMI8226) treated with AZD8055 or rapamycin. AZD8055 induced more pronounced upregulation of p-IGF-1R in MM.1S and OPM1 MM cells than rapamycin at earlier time periods. IGF-1 clearly upregulated Akt phosphorylation in MM cells; however, it had no effect on mTOR phosphorylation (Ser2481). Moreover, AZD8055-treated cells exposed to IGF-1 sustained p-Akt (Ser473) expression, while p-mTOR (Ser2481) remained fully inhibited. These results suggest that IGF-1/IGF-1R signaling may bypass mTORC2/Akt when mediating p-Akt (Ser473) upregulation. Moreover, reactivation of IGF-1 signaling in MM cells in the context of mTOR kinase inhibitors suggests that MM may survive in an IGF-1 R–dependent fashion. We therefore next treated MM.1S, OPM1 and RPMI8226 cells with AZD8055, in the presence or absence of IGF-1. MM.1S and OPM1 MM cells (with higher Akt baseline activity) partially escaped AZD8055 cytotoxicity, while RPMI8226 MM cells (with lower Akt activity) did not. Moreover, the addition of blocking IGF-1R antibody or of IGF-1R inhibitor enhanced AZD8055 cytotoxicity in MM.1S and OPM1 cells. Conclusions: Our study therefore shows interaction of mTOR/Akt and IGF-1R/Akt pathways in MM tumors with IGF-1-enabled Akt activation. Importantly, they suggest that combination treatment with AZD8055 and IGF-1R inhibitor is a promising strategy to mTOR kinase inhibition in MM with potential IGF-1R/Akt signaling mediated survival. Disclosures: Hideshima: Acetylon Pharmaceuticals, Inc.: Consultancy. Guichard:AstraZeneca: Employment, Shareholder Other. Anderson:Onyx: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Raje:Onyx: Consultancy; Celgene: Consultancy; Millenium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding.

Abstract 3990 Introduction: Multiple Myeloma (MM) is increasingly being recognized as a heterogeneous disease. Previous studies have reported various oncogenic mutations in myeloma. Recent genome sequencing studies have revealed activating mutations of BRAF kinase in 4% of patients, a potentially druggable target, underscoring the importance of identifying these and other potential oncogenic mutations. However, a validated methodology for clinical assessment of mutation profiles is lacking. Applying such technologies will be increasingly important to accelerate development of targeted therapies toward tumor-specific oncogenic pathways. Utilizing a high-throughput genotyping platform we evaluated the commonly described somatic mutations in bone marrow aspirates in patients diagnosed with MM. Methods: We developed a CLIA (Clinical Laboratory Improvement Amendments) validated, highly sensitive multiplexed PCR-based assay (SNaPshot) to simultaneously identify 70 genetic loci frequently mutated in 15 cancer genes. This assay has been used at our institution for over 3 years for tumor genotyping and to help guide therapeutic decisions for patients with solid malignancies. To test the value of this approach in patients with MM, we first validated the methodology on MM cell lines followed by testing patient derived bone marrow samples. Results: The detectable mutations in the 8 MM cell lines tested include: MMIR: KRAS 35G>C, G12A mutation; MMIS: KRAS 35G>C, G12A mutation; H929: NRAS 38G>A, G13D mutation; LR5: KRAS 35G>C, G12A mutation; RPMI8226: TP53 743G>A, R248Q mutation;U266: no mutations identified; OPM2: NRAS 182A>T, Q61L mutation; OPM1: TP53 524G>A, R175H mutation. Thirty six bone marrow aspirates from patients with MM have been analyzed to date. Tweny five percent of patients had a detectable mutation, with KRAS mutation in 17%, NRAS in 5% and TP53 in 3%. Additional data in a larger cohort is currently being analyzed and will be presented. Conclusion: Oncogenic driver mutations can be detected by SNaPshot from routinely-collected bone marrow aspirate samples. Experience from lung and breast cancer studies indicates that while the type and frequency of mutation varies somewhat by tumor phenotype, the common oncogenic mutations are often found in isolation. Broad tumor mutational analysis will be helpful to efficiently tailor personalized therapies based on individual tumor genetic profiles. Disclosures: Dias-Santagata: Bio-Reference Laboratories, Inc.: Consultancy. Borger:Bio-Reference Laboratories, Inc: Consultancy. Iafrate:Bio-Reference Laboratories, Inc: Consultancy. Raje:Eli-Lilly: Research Funding; Amgen: Research Funding; Acetylon: Research Funding; Millennium: Consultancy; Celgene: Consultancy; Onyx: Consultancy.

Abstract 1822 The activation of Bruton's tyrosine kinase (Btk), a member of the Tec family of tyrosine kinases regulates B-cell development and activation, and plays a key role in antibody production. Interestingly, Btk and the other Tec kinase family, Tec, regulate OC differentiation via Receptor Activator of Nuclear Factor κ B (RANK) signaling. Moreover, patients with X-linked agammaglobulinemia (XLA) who harbor Btk null mutations have impaired OC function. Here we show that, a potent and specific inhibitor of Btk, AVL-292, inhibits OC function in MM patients. AVL-292 is a highly selective, covalent Btk inhibitor that potently silences Btk enzymatic activity (IC50 < 0.5nM) with high selectivity towards Btk with lack of significant inhibition against other kinases involved in BCR signaling (Syk, Lyn). We examined the mechanism of action of AVL-292 in the context of OCs function. OC derived from MM patient monocytes were assayed with or without AVL-292. Interestingly, OC function was inhibited in the presence AVL-292 as demonstrated by pit formation assay. However, mRNA expression of Cathepsin K and TRAP, markers of OC differentiation, were increased in the presence of AVL-292. These data suggest AVL-292 inhibits OC function without affecting the OC differentiation. It has been shown that BTK and Tec regulation of OC differentiation is related to calcium (Ca2+) signaling by increasing Ca2+ flux through direct phosphorylation of phospholipase C2 (PLCγ2). To delineate the mechanism of action of the BTK inhibitor against OC function, the RANK signaling proteins were detected by western blotting and Ca2+ concentration was measured by flourescence. AVL-292 inhibited phosphorylation of the BTK substrate, PLCγ2 in OCs. This was associated with an inhibition of intracellular Ca2+ release by AVL-292 which otherwise increased with RANKL stimulation in OCs. Given the effect of AVL-292 on RANK signaling we next evaluated its effect on Proline-rich tyrosine Kinase 2 (Pyk2) and c-Src. Pyk2 plays a role in OC activation and localizes to the sealing zone in OC. RANK signaling activates c-Src, which phosphorylates Pyk2. Moreover c-Src controls OC bone resorption by regulating actin organization via cortactin. Interestingly, AVL-292 inhibited c-Src and Pyk2 phosphorylation. Furthermore, AVL-292 inhibited cortactin protein and mRNA expression, and upregulated c-Cbl protein (E3 ubiquitin ligase for c-Src) expression in OCs derived from MM patient monocytes. These data demonstrate that the novel BTK inhibitor AVL-292 inhibits OC function through inhibition of OC sealing zone formation. These results suggest a potential novel mechanism of Btk inhibition with AVL-292 on osteoclast function and therefore bone resorption. Disclosures: Evans: Celgene: Employment. Singh:Celgene: Employment. Westlin:Celgene: Employment. Raje:Onyx: Consultancy; Celgene: Consultancy; Millenium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding.

Abstract 591 Background: Survival of patients with MM has improved over the past two decades, in part due to the use of AHCT. Increasingly, second primary malignancies (SPMs) are observed in MM survivors. Determining the baseline incidence and risk factors associated with SPMs after AHCT is important to assess risk and to evaluate the risk-benefit ratio of newer therapies. Methods: We analyzed the incidence of SPMs in 3784 MM patients receiving (“upfront”) AHCT for MM within 18 months of diagnosis between 1990 and 2010 and reported to the CIBMTR. Cumulative incidence rates of SPMs were estimated taking into account the competing risk of death. For each transplant recipient, the number of person-years at risk was calculated from the date of transplantation until date of last contact, death, or diagnosis of SPM, whichever occurred first. Incidence rates for all invasive cancers in the general population were obtained from the SEER database. Age-, sex-, and race- specific incidence rates for overall SPMs and particular anatomical sites were applied to the appropriate person-years at risk to compute the expected numbers of cancers. Observed–to –expected (O/E) ratios were calculated, and Poisson distribution 99% confidence intervals (CIs) were generated. Poisson regression model was used to analyze risk factors for overall SPMs and AML/MDS. Results: Pre-transplant therapy included novel agents in 56% including thalidomide (35%), lenalidomide (9%), bortezomib (16%) or their combinations (11%). Majority (80%) received high dose melphalan conditioning. Post-transplant maintenance therapy included thalidomide (16%), lenalidomide (8%), bortezomib (9%) and interferon (6%). Median follow-up of survivors was 52 months (range 3 to 192 months).With 12707 person years of follow up, 153 new malignancies were reported with a crude rate of 1.2 SPM per 100 person years of follow up. Observed/Expected [O/E] ratio for all SPMs was 0.99 (99% CI, 0.80–1.22). Cumulative incidence of SPM overall was 2.48% (95% CI, 1.96–3.05) at 3 years and 6.0% (95% CI, 4.96–7.10) at 7 years [Figure 1]. Individual SPMs observed significantly more frequently than expected are summarized in Table 1. The cumulative incidence of MDS/AML was 0.5% (95% CI, 0.28–0.78) at 3 years and 1.3 (95% CI, 0.85– 1.9%) at 7 years. Majority had MM progression prior to diagnosis of SPM (65 of 102 patients overall and 15 of 23 patients for MDS/AML). In multivariate analysis, significant risk factors for development of SPMs included: obesity [Hazard ratio = HR 1.89(95%CI, 1.21–2.93), p=0.0047 for BMI>30 vs. BMI The low number of MDS/AML (33 events out of 3784 cases) limited the power of multivariate analysis. Increasing age was significantly associated with development of MDS (HR10.77, (95%CI,92.09–55.51), p=0.004 for 70+ year old vs. 40–49 year olds). Conclusion: In this large cohort of AHCT recipients for MM, the incidence of MDS/AML, melanoma and other skin cancers was significantly higher compared to age and sex matched general population. However the overall risk of SPM was similar to that expected for age and sex matched population. It was also similar to the placebo arms of recent reports by McCarthy Pl et al and Attal M et al (N Engl J Med. 10; 366(19):1770–91). Lenalidomide (8%) or thalidomide maintenance (16%) used in a small subset of patients with comparatively short follow up, was not associated with risk of SPM in the analysis of the overall cohort. Disclosures: Gale: Celgene: Employment. Brandenburg:Celgene: Employment, Equity Ownership. Lonial:Millennium, Celgene, Novartis, BMS, Onyx, Merck all Consultancy. Krishnan:Celgene and Millennium: Consultancy, Speakers Bureau. Dispenzieri:Celgene and Millennium: Research Funding. Hari:Celgene: Consultancy, Honoraria.

Abstract 4217 Background: AHCT improves survival (OS) in newly diagnosed MM patients (pts) in large randomized trials. These trials have limited eligibility to younger, healthier pts. Selection of older pts and those with co-morbid illness for AHCT is problematic. HCT-CI, originally developed as predictor of post-allogeneic transplant outcomes, maybe valuable in stratifying risk of transplant related mortality (TRM) risk and OS in the AHCT setting. We investigated the relative impact of HCT CI along with other patient and MM related variables on outcomes after AHCT in a large cohort of transplant recipients. Methods: Outcomes of 1156 MM pts receiving AHCT after high dose Melphalan (MEL) between 2007 and 2010 reported to the CIBMTR (Center for International Blood and Marrow Transplant Research) were analyzed. HCTCI scores and individual comorbidities were prospectively reported at time of AHCT. Median follow up of survivors was 26 month. The impact of HCTCI and other potential prognostic factors including Karnofsky performance status (KPS) on OS were studied in multivariate Cox regression models. Results: HCTCI score was 0, 1, 2, 3, >3 in 42%, 18%, 13%, 13% and 14% respectively. Most common co-morbidities included pulmonary, diabetes, obesity, psychiatric, cardiac, renal and prior solid tumor. Using consolidated HCTCI scores, patients were stratified initially into 3 risk groups – HCTCI 0 (42%) vs. HCTCI 1–2 (32%) vs. HCTCI >2 (26%). Males and Caucasians were more likely to have greater HCTCI score. Higher HCTCI was associated with lower KPS 2). HCTCI score >2 was associated with MEL dose reduction to 140 mg/m2 (22% vs. 10% in score 0 cohort). Cytogenetic risk and MM related factors were not correlated with HCTCI. TRM at 12 month was 2%, 2%, and 3% for 3 risk groups. With extremely few TRM events, multivariate analysis did not suggest an impact of HCTCI. OS was 95%, 92%, 92% at 1 year and 87%, 81%, 80% at 2 year, respectively. OS was inferior for HCTCI >2 cohort (RR of death 1.48, p=0.02) and HCTCI cohort 1–2 (RR 1.37, p=0.04) compared with HCTCI 0 cohort. There was no significant difference in OS between HCTCI >2 vs. HCTCI 1–2 (p=0.64). Therefore the latter 2 groups were combined as the HCTCI >0 cohort [N=667] and compared with HCTCI=0 [N=489] in multivariate models. HCTCI >0 predicted inferior OS (RR of death= 1.41, p=0.01). Other significant predictors of inferior survival were KPS 1 pretransplant regimen (RR 1.47, p Conclusion: In clinical practice, higher HCTCI score was associated with MEL dose reduction. Mortality after AHCT is predominantly related to MM progression/relapse with low incidence of TRM. Higher HCTCI scores were independently associated with inferior OS. KPS remains an important tool for risk stratification. Disclosures: No relevant conflicts of interest to declare.

Abstract 1871 Multiple myeloma (MM) is characterized by the vast majority of patients developing osteolytic bone lesions consequent to biochemical imbalances in bone resorption and formation. As such, compound combinations that succeed in selectively inhibiting processes relevant to both osteolysis as well as MM cell viability would be of great therapeutic potential to the MM patient. Here we investigated the benefits of combining lenalidomide with an Activin A neutralizing antibody. Lenalidomide is an immunomodulatory drug which displays both direct anti tumor activity as well as indirect inhibition of osteoclastogenesis but lacks an anabolic bone effect. Conversely, Activin A, secreted predominantly by bone marrow stromal cells (BMSCs), is an extracellular protein complex with apparent paracrine functions critical in MM tumor-induced osteolysis whose sequestration has been recently demonstrated by us to restore bone architecture and reduce tumor burden in vivo. Given these properties, we hypothesized that an alleviation of the abnormal processes associated with the MM bone microenvironment via Activin A neutralizing antibody, combined with a selective targeting of MM cell growth and viability with lenalidomide could yield a positive outcome for MM patients with bone disease. Interestingly, our studies revealed that lenalidomide stimulated Activin A secretion in BMSCs, leading us to search for potential underlying mechanisms. Due to previous implications in MM induced Activin A secretion, as well as the presence of a highly conserved c-Jun-binding sequence in the promoter of the constituent subunit of the peptide, INHβA, the JNK-dependent pathway was of particular interest. Accordingly, BMSCs treated with lenalidomide displayed an early activation of JNK signaling evidenced by increases in both JNK phosphorylation and c-Jun mRNA expression, an effect which was found to be AKT dependent. We next investigated and accordingly observed that lenalidomide had a deleterious effect on osteoblastogenesis as evidenced via significant decreases in alkaline phosphatase activity associated with a downregulation of DLX5 mRNA expression and an increase in SMAD2 signaling. Expectantly, further studies revealed that osteoblasts differentiated both alone as well as in co-culture with MM cells exhibited variable levels of extracellular Activin A concentrations which were inversely correlated with ALP activity when treated with lenalidomide. Furthermore, the addition of lenalidomide was in fact noted to augment existing Activin A secretion as well as the resultant ALP dysfunction. This could be overcome by the addition of Activin A neutralizing antibody, which efficiently sequestered Activin A without compromising the cytotoxic MM activity of lenalidomide. Collectively, our results illustrate an Activin A mediated inhibition of osteoblastogenesis by lenalidomide, in which lenalidomide stimulates a JNK dependent secretion of Activin A by BMSCs. This however can be reversed via the addition of Activin A neutralizing antibody. Our work therefore indicates the validity of combining Activin A neutralizing antibody with lenalidomide, and provides the basis for a currently accruing Phase I clinical trial in MM (NCT01562405). Disclosures: Raje: Celgene: Consultancy; Onyx: Consultancy; Millennium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding.

Abstract 3965 Background: Aminobisphosphonates (aBP) are standard of care for myeloma-related bone disease and are recommended every 3–4 weeks. Multiple myeloma patients may be susceptible to osteonecrosis of the jaw (ONJ) and stress fractures secondary to altered bone remodeling due to long-term aBP therapy. Monthly dosing of aBP is based on early studies that documented the suppression of N-telopeptide (NTX), a marker of bone resorption, for 4–8 weeks following a single dose of aBP. However, it is unclear whether NTX or other bone biomarkers are predictive of skeletal related events (SRE) or what the impact of cessation of aBP therapy has on bone remodeling. Methods: We studied markers of bone turnover after a single dose of zoledronic acid in myeloma patients in remission (NCT00577642). All patients had received 8–12 doses of aBP (pamidronate/zoledronic acid) prior to study inclusion. Patients were not receiving active anti-myeloma therapy besides maintenance therapy. Our primary objective was to determine the duration of time urinary NTX levels remain suppressed after a single dose of zoledronic acid on study. A secondary objective was the identification and correlation of other markers of bone remodeling with NTX changes. High throughput cytokine arrays of validated and potential surrogate markers of bone remodeling were performed. Bone marrow biopsies at start and end of protocol during which time aBPs were not administered were performed. Patients were removed from study if NTX crossed a threshold value (50 nMol), based on correlation of NTX levels with bone-related morbidity and mortality. Results: Twenty-nine patients were enrolled in a 6-month period. Cytokine array data is available in 28 patients. All patients had suppressed NTX levels, except one patient who had an increase in NTX levels associated with a SRE and disease progression. To identify other surrogate as yet unexplored cytokines that may serve as better markers in addition to NTX to monitor aBP activity, thirty cytokines were curated from the literature based on their possible role in bone remodeling and tested using custom designed cytokine arrays. Positive hits were confirmed by ELISA. GDF-15, which correlates with poor prognosis in myeloma and is thought to be produced by bone marrow mesenchymal stem cells, decreased in 24 of 28 patients (two-sided paired T test, p Conclusion: Our data suggest that suppressed NTX levels are predictive of freedom from SRE in this patient population. Furthermore, osteoblast suppression by aBP is reversible in myeloma. Importantly, the role of other surrogates such as decorin and GDF-15 need further validation and are the subject of ongoing studies. Taken together, this evidence may provide rationale for less frequent aBP dosing in multiple myeloma patients to help lower the incidence of long term toxicities such as ONJ and stress fractures, by allowing limited recovery of bone remodeling without adverse effects on multiple myeloma progression. Disclosures: Richardson: Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Ghobrial:Bristol Myers Squibb: Advisory Board, Advisory Board Other; Millennium: Advisory Board, Advisory Board Other; Onyx: Advisory Board, Advisory Board Other; Novartis: Advisory Board Other. Schlossman:Celgene: Membership on an entity's Board of Directors or advisory committees. Anderson:Onyx: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Raje:Onyx: Consultancy; Celgene: Consultancy; Millenium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding.

Abstract 3966 Background. Lenalidomide plus dexamethasone is approved for the treatment of relapsed and/or refractory multiple myeloma following prior therapy. Everolimus, an oral mTOR inhibitor, has been studied as a single agent in multiple myeloma but does not have significant activity. Based on previous preclinical studies showing synergistic anti-myeloma activity of mTOR inhibitors when combined with lenalidomide, we studied this combination as a non-steroid containing oral regimen in advanced multiple myeloma. In order to help determine the molecular mechanisms of response, we comprehensively assessed patient samples using gene expression analysis, western blotting, and immunohistochemistry of the mTOR pathway as well as cytokine analysis and measurement of T cell subsets. Methods. Patients with relapsed and refractory multiple myeloma were assigned to lenalidomide and everolimus for 21 days out of a 28 day cycle until disease progression or unacceptable toxicity (NCT00729638). We measured levels of p70S6K phosphorylated protein in peripheral blood mononuclear cells by western blotting before and during treatment. Immunohistochemical analysis for target proteins of the mTOR/AKT pathway was performed on bone marrow (BM) aspirates. Gene expression of CD138+ selected BM aspirates was analyzed on Affymetrix expression arrays prior to treatment. IGF-1 and IL-6 cytokine levels were determined by ELISA. T cell subsets were defined immunophenotypically over the course of treatment. Results. Twenty-six patients were evaluable for toxicity. The MTD was lenalidomide 15 mg and everolimus 5 mg for 21 days with a 7 day rest period. Nineteen patients finished at least two cycles of treatment and were evaluable for response. The overall response (OR) was 58% (1 CR + 3 PR + 7 MR). Three patients had SD and 5 patients had PD. The median progression free survival was 6.3 month at a median follow-up of 8.2 months. Biomarker data demonstrated that treatment with everolimus and lenalidomide consistently downregulated protein expression of phosphorylated p70S6K, a downstream target of mTOR. Microarray gene expression data was available for 12 patients, including 9 responders (MR, PR, SD) and 3 non-responders (PD). Gene set enrichment analysis showed enrichment for genes in the mTOR pathway gene set among the responders (p = 0.033, FDR 0.026). At the individual gene level, expression of members of the mTOR pathway, e.g. IGF1 (p = 0.034, FDR = 0.20) and RICTOR (p = 0.016, FDR = 0.15) was significantly higher in responding patients than non-responding patients. IHC for p-Akt (Ser 473) was evaluable in 10 patients, and 7 patients scored positively for pAkt expression. Given the small numbers, we were unable to correlate pAkt expression with response. DEPTOR was evaluable in 12 cases and strongly expressed in the majority of tumors. Quantification of T cell subsets was available for 25 patients. Treatment with everolimus and lenalidomide did not affect populations of CD4+ or CD8+ T cells or NK cells. Of the patients where IGF-1 levels were available, IGF-1 levels rose in the non-responding patients (N = 2) over the course of treatment; conversely, IGF-1 levels had an initial peak followed by decrease at time of best response in 4 of 5 responders. There was no difference in IL-6 levels between responders and non-responders. Conclusions. The combination of lenalidomide and everolimus showed durable responses in a heavily pretreated population. The doses of lenalidomide and everolimus used in this phase I study inhibited the downstream target of mTOR, p70S6K; was active in cases of constitutive mTOR activation; did not alter T cell subsets; and modulated IGF-1, but not IL-6 levels. With confirmation in larger patient numbers, this analysis may serve as a framework for guiding patient selection for future clinical trials investigating the role of mTOR inhibition in multiple myeloma. Disclosures: Off Label Use: The combination of everolimus and lenalidmoide is an off-label use for multiple myeloma. Richardson:Millennium Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Johnson & Johnson: Consultancy. Hideshima:Acetylon: Consultancy. Ghobrial:Noxxon: Research Funding; Bristol-Myers Squibb: Research Funding; Millennium: Research Funding; Noxxon: ; Millennium: ; Celegene: ; Novartis:. Munshi:Celgene: Consultancy; Millenium: Consultancy; Novartis: Consultancy; Onyx: Consultancy. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding.

Abstract 2916 Inhibition of the PI3K/mTOR pathway is a promising therapeutic strategy in targeting multiple myeloma (MM) cells in the bone marrow (BM) microenvironment, which abnormally activates PI3K/mTOR signaling cascade mediating proliferation, anti-apoptosis and drug resistance. Exploring the targeting of PI3K/mTOR pathway has led to the development of different therapeutic approaches; however, mTORC1 inhibitors (i.e., temsirolimus and everolimus) have demonstrated only modest activity as single agents. In this regard, several mechanisms underlying rapamycin resistance, including mTOR/S6K1-mediated feedback loops resulting in activation of PI3K/Akt and ERK signaling, have been proposed. Importantly, recent studies have identified mTOR kinase and the mTOR-DEPTOR counter-regulatory cascade as key mediators of mTORC1 and mTORC2 multi-protein complexes, with differential sensitivity to rapamycin. Indeed, targeting DEPTOR/mTORC1/mTORC2 signaling by inhibition of mTOR kinase proved an effective strategy to overcome some of the limitations of TORC1 inhibition in MM cells, evidenced in our studies of the novel dual mTORC1 and mTORC2 selective inhibitor AZD8055. Unlike rapamycin, AZD8055 induced apoptosis and inhibited MM cell growth even when co-cultured with cytokines (i.e., IL-6, IGF1) or BMSCs, presumably through simultaneous suppression of mTORC1 and mTORC2 signaling including the rapamycin-resistant 4E-BP1 (downstream of mTORC1) and Akt as well as NDRG1 (effectors of mTORC2). We examined mRNA and protein level of DEPTOR in MM cell lines treated with AZD8055 versus rapamycin and observed no significant changes. To examine the functional significance of DEPTOR in response to mTOR inhibitors, we utilized lentiviral shRNA to knockdown DEPTOR in OPM1 MM cells. DEPTOR-knockdown cells acquired resistance to AZD8055 treatment, suggesting that DEPTOR is a key modulator of mTORC1/2 signaling. Moreover, DEPTOR knockdown triggered decrease in Akt phosphorylation (Ser473), associated with suppression of Rictor phosphorylation (Thr1135). DEPTOR co-immunoprecipitation with Rictor was also abrogated by both AZD8055 and rapamycin treatment. Taken together, our results indicate the role of DEPTOR, either alone or as an mTOR/Rictor interacting molecule, in mediating the anti-MM activity induced by mTOR kinase inhibitors in MM cells. These data therefore both provide insights into the molecular profiles that may predict sensitivity/resistance to second generation of mTOR inhibitors in MM, and may be useful to select MM patients for mTOR inhibitor therapy. Disclosures: Hideshima: Acetylon: Consultancy. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees. Guichard:AstraZeneca, UK: Employment, Shares from AstraZeneca, UK. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding.

Abstract 1590 Background: The occurrence of a secondary lymphoma in patients with a prior history of B-cell lymphomas has been reported.1, 2 There are few reported occurrences of Multiple Myeloma (MM) in patients (pts) with a prior history of lymphoma and the biologic relationship between the two neoplasms in such cases is unknown. Methods: We queried our IRB approved clinicopathologic database of hematologic malignancies for patients with lymphoma and MM. Of the 4165 pts with B-cell lymphoma and 804 pts with MM, 6 pts with a history of B-cell lymphoma developed MM and 1 patient with a prior history of MM developed a B-cell lymphoma. We describe the morphology, immunophenotype, and clinical features of the 7 pts. The clonal relationship of the 2 components was analyzed using sequencing analysis of immunoglobulin heavy chain variable region (IgVH) genes and by light chain restriction. Results: There were 5 men and 2 women (median age of diagnosis of lymphoma, 65 years; median age of diagnosis of MM, 71 years). The pts with lymphoma included 2 pts with diffuse large B cell lymphoma, 2 pts with small lymphocytic lymphoma, 2 pts with follicular lymphoma and 1 patient with lymphoplasmacytic lymphoma. The development of MM was metachronous in 5 cases, following B-cell lymphoma by 3 years to 23 years and synchronous in 1 case. In 1 patient, the B-cell lymphoma developed 6 years after the diagnosis of MM. 6 pts achieved complete remission after treatment for lymphoma and 1 patient is ongoing treatment. 6 of the 7 pts required treatment for MM soon after diagnosis. 1 patient has smoldering MM and continues to be observed 57 months after diagnosis. FISH analysis indicated IgH rearrangement in 3 pts with MM; 1 patient with 17p deletion and monosomy 13; 3 pts had normal FISH and metaphase cytogenetics. In 3 pts, both neoplasms were kappa light chain restricted; in 1 patient both were lambda restricted; in 1 patient, the lymphoma was lambda light chain restricted while the MM was kappa light chain restricted and the reverse in another pt; in 1 patient the B-cell lymphoma was light chain negative and the MM was kappa restricted. IgVH rearrangement studies in 4 patients in whom tissue samples were available indicated that the two were clonally unrelated in 3 patients and related in only 1 patient. Conclusion: Clonality analysis of rearranged immunoglobulin genes from patients with both B-cell lymphoma and MM provide evidence of separate clonal origins of the two tumors in the majority of cases, thus excluding secondary transformation of the original B-cell clone. The presence or absence of a genetic predisposition to the development of multiple B cell malignancies requires further study.3 Disclosures: Off Label Use: The combination of lenalidomide and everolimue is an off label use in multiple myeloma. Abramson:Genentech: Consultancy; Novartis: Consultancy. Raje:Amgen: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Millenium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding.

Abstract 3716 Background: Rituximab monotherapy as initial treatment for low-grade B-cell lymphomas produces responses in approximately 70% of patients with one third achieving a complete response, and progression-free survival (PFS) of approximately 2 years. Maintenance rituximab appears to prolong initial remissions after rituximab alone. Current dosing for rituximab is largely empiric, so we sought to investigate whether increased doses of rituximab induction would increase the complete response rate (CRR) over that expected from standard dose rituximab. We also sought to assess whether high-dose induction followed by standard maintenance would produce a PFS comparable with that of combination chemoimmunotherapy strategies. Methods: We conducted a phase II trial of increased-dose rituximab monotherapy induction, followed by a standard maintenance schedule. Eligible patients were adults with previously untreated low-grade B-cell lymphomas with measurable disease >2cm and were not candidates for potentially curative radiotherapy to localized disease. Subjects were treated with induction rituximab at a dose of 750 mg/m2 on days 1,8,15, and 22. Patients without progressive disease were then treated with maintenance rituximab at 375mg/m2 every 3 months for 8 doses or until disease progression. The primary end point was CRR as defined by the International Workshop Response Criteria (1999). Secondary endpoints included overall response rate (ORR), PFS, and toxicity. The study was designed with 90% power to show a 50% CRR, with a 30% CRR considered unworthy of further study. Results: Between August 2009 and August 2010, 40 eligible subjects were enrolled (31 grade 1–2 follicular lymphomas, 4 marginal zone lymphomas, 3 small lymphocytic lymphomas, and 2 indolent B cell lymphoma not otherwise specified). The median age was 60 (range 36–88) All subjects had advanced Ann Arbor stage disease. Twenty-two subjects (55%) had involvement of >4 nodal sites, 6 (15%) had a Hgb Conclusions: Increased dose rituximab monotherapy is well tolerated, but does not improve the CRR compared to what would be expected from rituximab at standard doses. Significant improvement in the CRR occurred with ongoing maintenance therapy, and the vast majority of patients remain in remission after 1 year. Ongoing follow-up will determine whether this approach produces a PFS comparable to a chemoimmunotherapy treatment program. Disclosures: Hochberg: Genentech: Consultancy. Fisher:Genentech: Consultancy. Abramson:Genentech: Consultancy.

Abstract 791 Targeting PI3K/Akt/mTOR signaling is among one of the promising therapeutic strategies in multiple myeloma (MM), since it facilitates MM cell survival and development of drug resistance in the context of the bone marrow microenvironment. Specifically, regulation of PI3K activity, which mediates MM cell growth and drug resistance, by mTOR complex 1 (mTORC1) provides the rationale for use of rapamycin analogs for MM treatment. However, rapamycin alone fails to overcome bone marrow-induced proliferation of MM cells, at least in part, because of the mTORC1-dependent feedback loops which activate PI3K/Akt. More recently, extensive studies of the mTOR network have identified mTORC2 as a “rapamycin-insensitive” complex. Sharing mTOR kinase as a common catalytic subunit, mTORC1 and mTORC2 mediate two distinct pathways: mTORC1 controls cell growth by phosphorylating key regulators of protein synthesis S6 kinase 1 (P70S6K) and the eIF-4E-binding protein 1 (4E-BP1); mTORC2 modulates cell survival and drug resistance by phosphorylating target proteins including Akt and serum/glucocorticoid regulated kinase 1(SGK1)/N-myc downstream regulated 1 (NDRG1). Moreover, studies have also revealed overexpression of a novel mTOR-interacting protein DEP domain containing 6 (DEPTOR), which can modulate mTOR activity and promote PI3K/mTORC2 signaling in primary MM tumor cells and in MM cell lines while mTORC1 remains silenced. We therefore hypothesized that targeting mTOR may disrupt DEPTOR/mTOR interaction and silence mTORC1/mTORC2 signaling, thereby overcoming mTOR resistance in MM cells. To confirm this idea, we used AZD8055, an orally bioavailable selective ATP-competitive mTOR kinase inhibitor, in our MM preclinical models. AZD8055- treatment of MM.1S inhibited phosphorylation of both mTORC1 and mTORC2 substrates: P70S6K; 4E-BP1 including the rapamycin-resistant T37/46 – downstream targets of mTORC1; as well as Akt and NDRG1 – effectors of mTORC2 refractory to rapamycin. Interestingly, AZD8055-mediated mTORC1/mTORC2 downregulation was associated with DEPTOR upregulation, which is consistent with the finding that DEPTOR expression is negatively regulated by mTORC1 and mTORC2. Moreover, inhibition of mTORC1 alone by rapamycin resulted in reduction of DEPTOR, associated with Akt activation. Furthermore, we observed that DEPTOR expression was decreased in MM.1S cells cultured with IL-6, IGF-1 or bone marrow stromal cells (BMSCs), which stimulate PI3K/Akt/mTOR signaling, evidenced by enhanced P70S6K and Akt phosphorylation. Unlike rapamycin, AZD8055 reversed those effects and inhibited MM.1S proliferation, even in the presence of these cytokines or BMSCs. AZD8055-induced growth inhibition was associated with apoptosis, evidenced by caspase-9, -3 and PARP cleavage in a time-dependent fashion (80% apoptotic cells at 72 hour culture as detected by Annexin V/PI staining). Moreover, AZD8055 induced cytotoxicity even in rapamycin resistant MM cell lines and primary patient MM cells. Finally, AZD8055 demonstrated significant anti-MM activity in an in vivo human MM cell xenograft SCID mouse model. Taken together, our data show that disruption of DEPTOR/mTORC1/mTORC2 cascade in MM cells results in significant anti-tumor effects, providing the framework for future clinical trials of AZD8055 to improve patient outcome in MM. Disclosures: Guichard: AstraZeneca: Employment, Shareholder AstraZeneca. Anderson:Millenium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; BMS: Consultancy; Acetylon: Membership on an entity's Board of Directors or advisory committees, Ownership interest (inc stock options) in a Start up company. Raje:AstraZeneca: Research Funding; Acetylon: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Abstract 4042 Plasmacytomas are rare clonal proliferations of plasma cells that though cytologically identical to plasma cell myeloma, present with osseous or extraosseous growth pattern. Understanding their molecular characteristics can provide crucial insights into their pathogenesis and risk of progression to multiple myeloma (MM). To investigate the differences between extramedullary (EMP) and medullary plasmacytomas (MP) and MM without plasmacytomas, we sought to molecularly profile these tumors by tissue microarrays, gene expression, microRNA, and FISH. We identified 85 patients from our data base with a pathological diagnosis of plasmacytoma. Of the 85 patients, 13 patients presented with EMP, and 72 had MP. Among the patients with EMP (n=13), 2 patients presented with multiple lesions. Three of 13 (23%) patients progressed to develop MM at a median of 12 months. 72 patients presented with MP, of which 21 had solitary lesions and 27 (37%) progressed to MM at a median of 20.5months. There was a male preponderance (67% vs 33%) and the median age at diagnosis was 60.5 years (range 27.7–87.6). The mean overall survival for patients with EMP was 121 months (95% confidence interval[CI] 97–144 months) and for patients with MP was 102 months (95% CI 93–128 months) {p=0.025}. MicroRNA (miRNAs) profiling was performed on MP (n=19), EMP (n=7) and MM samples (n=66). Data was normalized using U6 endogenous control. Gene expression profiling was performed and correlated with the miRNA data to identify genes and transcripts of interest. miRNA 127, which regulates SET D8, was upregulated four fold in both MP and EMP compared to MM. miRNA 493, which regulates cadherin 11 and PTCH 1, both of which have been associated with metastatic potential in solid tumors, was similarly downregulated four fold in both MP and EMP compared to MM. A tissue microarray was created on 52 patients (8: EMP, 44: MP,) in whom paraffin-embedded tissue was available. Additional evaluation using SET 8, cadherin 11 antibodies and validation of additional functional targets is ongoing and will be reported. Differential expression patterns of factors involved in proliferation, survival, adhesion, and stroma-tumor cell interactions may help explain plasmacytoma biology and identify factors responsible for progression to MM. These insights may help identify new therapeutic approaches and targets in the treatment of these plasma cell disorders. Disclosures: No relevant conflicts of interest to declare.

Abstract 3051 Background: Lenalidomide (Revlimid®, Len) plus dexamethasone (Dex) is approved for the treatment of relapsed or refractory multiple myeloma (MM) patients following ≥1 prior therapy. RAD001, an mTOR inhibitor has been studied as a single agent in MM, and although well tolerated, did not have single agent activity. Informed by our previous studies demonstrating synergistic anti-MM activity of mTOR inhibitors when combined with Len, we studied this combination to evaluate its safety and activity as a non-steroid containing oral regimen in advanced MM. Methods: Patients with relapsed and refractory MM were assigned to Len and RAD001 to be taken for 21 days of a 28 day cycle (NCT00729638) in a standard 3+3 design. Patients were allowed to continue therapy until disease progression or unacceptable toxicity and were required to receive concomitant anti-thrombotic (aspirin 81 or 325 mg/day) therapy. Peripheral blood and bone marrow samples were collected before and after treatment for pharmacokinetic and pharmacodynamic studies. Results: Twenty-eight patients were registered on the trial between December 2008-December 2009. Two patients were inevaluable because of either rapidly progressive disease or failure to meet eligibility criteria on day 1. Data on 26 patients are therefore available. Pts had received a median of 4 prior lines of treatment.14 pts had received Len previously of which 11 pts had relapsed disease and 3 pts had relapsed/refractory disease. Both cohort 1 (Len: 10mg/day and RAD001: 5mg/every other day ×21 days of 28 day cycle) and 2 (Len: 15mg/day and RAD001: 5mg daily ×21 days of 28 day cycle) required expansion because of grade 3 neutropenia and grade 4 thrombocytopenia. Dose limiting toxicities included grade 4 neutropenia and thrombocytopenia in 2/3 patients in cohort 3 (Len: 20mg and RAD001: 5 mg × 21 days). The maximum tolerated dose (MTD) was therefore declared at 15 mg of len and 5mg of RAD001 for 21 days with a 7 day rest period. Most common (≥10%) Grade 1/2 events included nausea, fatigue, dyspnea, diarrhea, constipation, neuropathy and muscle cramps, all of which proved manageable with supportive care. One patient developed RAD related non-infectious pneumonitis requiring therapy discontinuation. Grade 3/4 adverse events (≥ 5%) included thrombocytopenia (11%) and neutropenia (22%). Nineteen patients completed 2 cycles and were evaluable for response. Median follow up is currently 8.7 months and median PFS is 4.3 months, with 12 patients receiving treatment at MTD. Overall response rate was 68% (13/19) (90% CI: [30-76%]) including CR(1) PR(2), MR(8) and SD(2). Four patients continue on the combination at 13, 14, 15 and 17 months respectively. Pharmacokinetic and pharmacodynamic data including immunohistochemistry for phosphorylated AKT, cytokine profiles, T cell subsets and transcription profile on MM cell pre and post treatment as well as correlation of response with pharmacodynamic studies will be presented. Conclusions: The combination of Len plus RAD001 is a well tolerated regimen with predictable hematological toxicities. Promising responses were noted in this heavily pretreated patient population with advanced MM. This combination thus provides an oral steroid free combination treatment strategy which warrants future evaluation in phase II studies. Disclosures: Richardson: Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees. Adams:Novartis: Employment. Makrides:Celgene: Employment. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Raje:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Astra Zeneca: Research Funding; Acetylon: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees. Off Label Use: Lenalidomide and RAD 001 for treatment of relapsed and refractory multiple myeloma.

Abstract 4075 The introduction of novel treatment strategies targeting tumor cells within their microenvironment have resulted in prolonged survival for Multiple Myeloma (MM) patients. Lenalidomide belongs to this category of agents working via tumoricidal, anti-osteoclast and immunomodulatory activities. However, lenalidomide lacks bone anabolic effects. We have recently reported that activin A mediates osteoblast inhibition in MM and neutralizing activin A via a soluble receptor, RAP-011 (murinized form of ACE-011) (Acceleron Pharma, Cambridge, MA), restores bone architecture and reduces tumor burden in vivo. We therefore hypothesized that the combination with RAP-011 may potentiate lenalidomide effects and vice versa as they act via complementary mechanisms. Our previous data demonstrates that 50% of MM patients have increased bone marrow (BM) levels of activin A that correlate with osteolytic burden. Here, we observed that 2 and 10 μ M lenalidomide (Selleck Chemicals, Houston, TX) upregulated activin A in 3 out 6 bone marrow stromal cell (BMSC) samples by 1.9 and 2.8 fold (average ± st.dev. 1052 ± 190 and 1667 ± 732 pg/ml respectively, compared to 734 ± 553 in control, p=0.2). There was no time-dependent upregulation of activin A. Of note, no augmentation of activin A was noted in BMSC which already expressed high baseline levels of the cytokine (average ± st.dev 3638 ± 3755 pg/ml in control vs 3074 ± 2997 after lenalidomide 10 μ M). Previous data suggest that high concentrations of activin A induce growth arrest and apoptosis in myeloma and breast cancer cells and may therefore mediate lenalidomide cytotoxicity. To ensure that inhibition of activin would not antagonize lenalidomide anti-tumor effects, we investigated whether activin A inhibition affected the cytotoxic and anti-proliferative effects of lenalidomide on MM cells alone and in co-culture with BMSC. As previously demonstrated, RAP-011 did not exert any direct anti-tumor effects. Lenalidomide 10 μ M induced between 20 and 40% of apoptosis in several myeloma cell lines, such as MM1.S, LR5, DOX40 and RPMI, independent of activin A inhibition. Similarly, lenalidomide almost completely reversed the proliferative advantage conferred by BMSC to tumor cells. Combining lenalidomide and RAP-011 was not antagonistic to the inhibition of the proliferative advantage conferred by BMSC to myeloma cells, suggesting that lenalidomide's direct anti-tumor activity is not mediated through activin A. Finally we assessed OB differentiation in the presence of both RAP-011 and lenalidomide. We have previously reported that activin A inhibitory effects on OB differentiation are reversed by RAP-011 treatment. Here, we noted diminished alkaline phosphatase (ALP, a marker of osteoblast activity) expression during osteoblastogenesis in the presence of increasing concentrations of lenalidomide (17% ± 3 decrease by 2 μ M and 26% ± 11 by 10 μ M compared to control, p=0.01 and 0.06 respectively). In contrast, combination with RAP-011 restored the osteogenic potential by increasing ALP expression close to control levels in healthy donor-derived BMSC and above control levels in MM-derived BMSC. These preliminary data suggest that lenalidomide results in upregulation of activin A expression in MM BMSCs which have low baseline levels. Combining lenalidomide with RAP-011 results in restored osteogenesis presumably by inhibiting activin signaling. Importantly, we observed no antagonistic effect of RAP-011 on lenalidomide's anti-tumor activity, confirming that activin A does not mediate the anti-tumor activity of lenalidomide. Ongoing in vivo studies using a murine model of myeloma will confirm the efficacy of this promising combination. Our results provide a preclinical rationale for combining lenalidomide with ACE-011 to target myeloma by manipulating the microenvironmental compartment, specifically the bone compartment. Disclosures: Seehra: Acceleron Pharma: Employment. Scadden:Fate Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Raje:Celgene, Novartis: Consultancy; Astrazeneca, Acetylon: Research Funding; Celgene, Amgen: Membership on an entity's Board of Directors or advisory committees.

Abstract 2264 Poster Board II-241 Prior studies have demonstrated that high-dose etoposide followed by granulocyte colony-stimulating factor (VP+G) significantly improves peripheral-blood progenitor cell (PBPC) mobilization for autologous stem cell transplantation (ASCT) compared to granulocyte colony-stimulating factor (G-CSF) alone. We compared the safety and effectiveness of VP+G to G-CSF alone for PBPC mobilization in patients (pts) with non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL) who underwent ASCT at the Cleveland Clinic and Ohio State University. Primary end points were CD 34+ cell collection, post-priming hospitalization for neutropenic fever and incidence of secondary hematologic malignancies. Secondary endpoints included relapse-free and overall survival. From 02/1992 through 06/2006, 640 pts who received etoposide (2g/m2) followed by G-CSF (10mcg/kg/day) and 318 who received G-CSF (10mcg/kg/day) alone underwent ASCT. Patients who had inadequate cell collection ( Priming regimen was not a significant variable for relapse-free or overall survival. The present study shows that even when restricted to patients who collect adequate numbers of PBPCs, the addition of etoposide significantly improves the effectiveness of mobilization. This occurs at the cost of an increased incidence of neutropenic fever but with no mortalities. Following transplantation, there is no evidence of an increased incidence of secondary malignancies in pts receiving VP+G. Thus VP+G remains a safe and effective regimen for mobilization in pts with lymphoma. Disclosures: No relevant conflicts of interest to declare.

532 Primary Plasma cell leukemia (PCL) is a rare plasma cell disorder characterized by an aggressive clinical course. The efficacy of autologous and allogeneic HCT in PCL is uncertain due to the small number of patients that have been reported in the literature. To determine the potential for long-term disease control using HCT in PCL, we performed a retrospective review of 160 patients with primary PCL who received autologous (n=107), allogeneic (n=52) or syngeneic (n=1) HCT and were reported to the CIBMTR between 1995 and 2006. Kaplan–Meier analysis was used for overall survival (OS) and progression free survival (PFS). Cumulative incidence estimates of non-relapse mortality (NRM), relapse, acute and chronic GVHD were calculated. Median age at the time of autologous HCT was 57 years (range, 32-74 years) and allogeneic HCT was 48 years (range, 24-62 years). Median time from initial treatment to autologous HCT was 7 months and allogeneic HCT was 6 months. At the time of transplant, in the autologous HCT group, 19% patients were in complete remission, 56% in partial remission and 1% had relapsed/progressive disease; in the allogeneic HCT group, 17% were in complete remission, 46% in partial remission and 10% had relapsed/progressive disease. In the allogeneic HCT group, 83% of the patients received myeloablative conditioning regimens and 17% received nonmyeloablative/reduced-intensity conditioning. The rate of acute GVHD was 35% (95% confidence interval [CI], 22%-49%) and the rate of chronic GVHD at 3 years was 27% (95% CI, 13%-40%). After a median follow-up of 38 months, 20 of the 52 patients (38%) are alive in the allogeneic HCT group and 68 of the 107 pts (64%) are alive in the autologous HCT group ([Figure 1][1]). Progressive disease accounted for 38% of the deaths in the allogeneic HCT group and 86% of the deaths in the autologous HCT group. The outcomes are summarized in [table 1][2]. The remarkable overall survival of patients after autologous HCT in this analysis is noteworthy and indicates that autologous HCT is a safe and acceptable treatment option for patients with primary PCL. Although relapse rates are lower, allogeneic HCT carries a much higher risk of NRM and should be considered primarily in the context of a clinical trial. | Outcomes | Allogeneic Probability (95% CI) | Autologous Probability (95% CI) | |:-----------------:| ------------------------------- | ------------------------------- | | TRM @ 1 year | 26 (15 - 39) | 2 (0 - 5) | | TRM @ 3 years | 40 (27 - 55) | 5 (1 - 10) | | Relapse @ 3 years | 40 (26 - 54) | 61 (49 - 72) | | PFS @ 3 years | 20 (10 - 33) | 34 (24 - 46) | | OS @ 3 years | 39 (25 - 53) | 62 (51 - 73) | Table 1. ![Figure 1:][3] Figure 1: Disclosures: Lonial: Celgene: Consultancy; Millennium: Consultancy, Research Funding; BMS: Consultancy; Novartis: Consultancy; Gloucester: Research Funding. [1]: #F1 [2]: #T1 [3]: pending:yes

Inpatient length of stay (LOS) has been studied with respect to its influence on treatment costs in patients (pts) undergoing high dose chemotherapy (HDC) followed by autologous stem cell transplantation (ASCT). We conducted a retrospective study to identify associations between pretransplantation patient characteristics and LOS, and to assess the prognostic effect of LOS in pts undergoing HDC followed by ASCT for lymphoma. 630 pts who received a busulfan-based HDC preparative regimen followed by ASCT at the Cleveland Clinic between 1997 and 2006 form the study cohort. 510 pts (81%) had non- Hodgkin’s lymphoma and 120 pts (19%) had Hodgkin’s lymphoma. Pts were admitted to the hospital for administration of the preparative regimen followed by infusion of autologous stem cells and were discharged after granulocyte recovery (neutrophils >500/ μL) unless complications prolonged hospitalization. The median age at transplant was 50 years (range, 18–77) and median time from diagnosis to transplant was 17 months (range, 2–372). The median length of stay was 21 days (range, 16–77). Recursive portioning analysis with a log rank splitting method identified three LOS groups predictive of overall survival: 22 days (170 patients). LOS was analyzed with this categorization and also as a continuous variable. Longer LOS was associated with older age (p 370 pts (59%) are alive with a median follow up of 51 months (range, 3–137). The median relapse-free survival was 2.2 years for pts with LOS>22 days, 3.6 years for pts with LOS 21–22 days and 6.1 years for pts with LOS 22 days, 8.5 years for LOS 21–22 days and the median has not been observed in pts with LOS less than 21 days ( p On multivariate analysis, the significant adverse prognostic factors for overall survival were longer length of stay (p These data indicate that we can segregate pts into distinct prognostic groups based on LOS. Pts with longer LOS should be targeted for more intensive follow-up. Figure: Length of stay and overall survival Figure:. Length of stay and overall survival

Iron overload is a well-established adverse prognostic factor for allogeneic hematopoietic stem-cell transplantation in thalassemia and appears to adversely affect prognosis with myeloablative transplantation for other diseases as well. In thalassemia, modification of the preparative regimen improves prognosis in patients who are at high risk due to iron overload. We analyzed a large group of patients who received a nonablative preparative regimen for allogeneic transplantation to determine whether the adverse impact of iron overload was circumvented by nonablative transplantation. Sixty-four consecutive patients undergoing nonablative allogeneic transplantation between 12/00 and 12/06 had pretransplant ferritin levels within 100 days preceding transplantation. Median age was 57 (range, 24–67) years; 24 patients were female. Median interval from diagnosis to transplant was 14.5 months (range, 2.4 to 151.6). Diseases included AML (n=16), NHL (15), MDS (10), myelofibrosis (6), CLL (6), CML (5), MM (3), Hodgkin (2), and ALL (1). Six had undergone prior transplantation. Median ferritin was 961 ng/mL (range, 302–6251). All but two received Flu/TBI preparation. Thirty-nine were sibling and 25 unrelated donors. Recursive partitioning identified significantly better survival for patients with ferritin ≤ 1615 ng/mL (n=45) compared to those with ferritin >1615 ng/mL (n=19; P=.012) as shown: Figure Figure Among age, gender, diagnosis, interval from diagnosis to transplant, extent of therapy prior to transplant, ferritin level, albumin, AST, alkaline phosphatase, bilirubin, LDH, risk group, donor source, degree of HLA match, donor to recipient gender, and CD34+ dose, only ferritin >1615ng/ml (P=.010, HR= 2.74), older age (P=.049, HR= 1.63), and prior transplantation (p=.002, HR=5.78) were adverse prognostic factors for survival in multivariable analysis. The poorer survival was related to both a higher incidence of treatment-related mortality and a higher rate of relapse mortality, neither of which reached statistical significance. The addition of albumin, a negative acute phase reactant, did not change the prognostic impact of ferritin. Conclusion: Similar to patients who undergo myeloablative transplantation, survival of patients who undergo nonablative allogeneic transplantation is adversely affected by elevated ferritin. It is important to assess the influence of specific factors on comparative results of myeloablative and nonablative transplantation. These results should stimulate study of iron chelation prior to allogeneic transplantations.

Iron overload is an adverse prognostic factor in patients who undergo allogeneic hematopoietic stem-cell transplantation (HSCT) for thalassemia and apparently in patients with acute leukemia and myelodysplastic syndrome as well. Iron overload has also been associated with susceptibility to infection following autotransplantation and with impaired immunity in other settings. We report here the results of a large study of consecutive patients undergoing autologous hematopoietic stem-cell transplantation (ASCT) for various hematologic malignancies to assess the influence of pretransplantation ferritin on outcomes following transplant. Pretransplantation ferritin, obtained within 100 days preceding transplant, was available on 397 patients undergoing autologous HSCT for various disorders (NHL=257, MM= 61, HD=58, AML=21) from 11/2/2000 to 12/28/2006.The median ferritin was 529.3 ng/ml (range, 12.8–4330). The median patient age was 52 (range, 19–77). Recursive partitioning analysis identified baseline ferritin >685 ng/ml as an adverse prognostic factor for survival as shown: ESTIMATED SURVIVAL BY PRETRANSPLANT FERRITIN LEVEL Figure Figure Age, gender, disease, disease status at transplant, interval from diagnosis to transplant, number of prior chemotherapy regimens, prior radiation therapy, ferritin, albumin, AST, Alkaline phosphatase, LDH, preparative regimen and CD 34+ dose were analyzed in a multivariable analysis. Elevated ferritin was an independent, adverse, significant prognostic factor for survival (p Figure Figure Conclusions: Elevated ferritin adversely influences survival, relapse-free survival, and relapse mortality following autologous transplantation. Whether the increased number of deaths due to relapse is related to the established immunosuppressive affect of iron overload is unknown. Iron chelators have been shown to inhibit the growth of tumor cells in vitro and in vivo. Trials designed to analyze the benefit of iron chelation therapy prior to ASCT in patients with iron overload are warranted.

Skin toxicity is a known but understudied complication of autologous stem cell transplantation (ASCT). Its assessment is complicated by the development of graft-vs-host disease following allogeneic transplantation. We chose therefore to study skin toxicity following autologous transplantation. We retrospectively reviewed the records of 392 patients undergoing ASCT to analyze risk factors for skin toxicity, its association with mucositis and its effect on survival. Skin toxicity was assessed three times a week during the transplant admission and was classified as none, mild, moderate, severe or life threatening. The most severe skin toxicity is used in this analysis. Mucositis was assessed using the modified oral mucositis assessment scale (OMAS). Median patient age was 53 years; 63% of patients had non-Hodgkin s lymphoma (NHL), 17.6% multiple myeloma, 13.5% Hodgkin disease (HD) and 5.9% acute leukemia. Peripheral blood progenitor cells were mobilized with G-CSF alone (36%) or the combination of etoposide plus G-CSF (64%). The preparative regimen was busulfan (Bu)/cyclophosphamide (Cy) /etoposide in 82% and Bu/Cy alone in 18%. Two hundred and sixty patients (67%) developed skin toxicity of which 143 patients (36.5%) had mild, 105 (26.8%) had moderate and 16 (3.9%) had severe skin toxicity. Factors associated with the development of any skin toxicity in univariable analysis were male gender (p=0.028), diagnosis of NHL (p Patients with severe skin toxicity appeared to have worse overall survival (p=0.054) and relapse-free survival (p=0.08), but these findings did not reach statistical significance. Conclusion: A substantial proportion of patients develop skin toxicity following autotransplantation. The inclusion of etoposide in the preparative regimen is associated with a significant increased frequency in multivariable analysis. The severity of skin toxicity correlates closely with the severity of mucositis. Figure Figure

We compared the safety and effectiveness of high-dose etoposide followed by granulocyte colony-stimulating factor (G-CSF) to G-CSF alone for peripheral-blood progenitor cell (PBPC) mobilization regimen in patients with non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL) who underwent autotransplantation (ASCT). Primary end points were CD 34+ cell collection, post-priming hospitalization for neutropenic fever and incidence of secondary malignancy. Prior studies have demonstrated that high dose etoposide significantly improves the proportion of patients who collect sufficient PBPC’s for transplantation. From 1/1993 through 12/2005, 352 patients received etoposide (2g/m2) followed by G-CSF (10mcg/kg/day) and 318 who received G-CSF (10mcg/kg/day) alone underwent ASCT. Patients who had inadequate cell collection were excluded. Mean age of patients receiving etoposide and G-CSF was 49 years and G-CSF alone was 45 years (p Figure Figure There was no significant difference in the incidence of secondary malignancies (p=0.73) or in the incidence of relapse (p=0.5) or survival (p=0.15) between the two groups. Conclusion: The present study shows that even when restricted to patients who collect adequate numbers of PBPCs, the addition of etoposide significantly improves the effectiveness of mobilization. This occurs at the cost of an increased incidence of neutropenic fever but with no mortalities. Following transplantation, there is no evidence of increased adverse effects due to addition of etoposide for priming, including on the incidence of secondary malignancies.

Iron overload is a well-established adverse prognostic factor in patients who undergo hematopoietic stem-cell transplantation (HSCT) for thalassemia and appears to play a similar role in patients with acute leukemia and myelodysplastic syndrome. We report the results of a large study of consecutive patients undergoing myeloablative allogeneic transplantation to assess the influence of pretransplantation ferritin on treatment-related mortality (TRM), graft-versus-host disease (GVHD) and survival. Pretransplantation ferritin, obtained within 100 days preceding transplant, was available on 222 consecutive patients ≥18 years of age undergoing allogeneic HSCT from 10/20/2000 to 12/04/2006. Transplantation was performed for various disorders (AML n= 105, ALL= 44, CML= 25, MDS= 19, NHL=18, and other= 11) and mainly with busulfan-based preparation (n=153). One hundred twenty four donors were siblings and 98 unrelated. The median patient age was 43 years. Median ferritin level was 1433 ng/ml (range 7–17029). Seventy-five patients had ferritin levels >1910 ng/ml. Median follow-up for surviving patients was 2.5 years. Recursive partitioning analysis identified significantly lower survival for patients with serum ferritin >1910 ng/ml as shown: ESTIMATED SURVIVAL BY PRETRANSPLANTATION FERRITIN LEVEL Figure Figure Age, gender, disease, disease status at transplant, interval from diagnosis to transplant, number of prior chemotherapy regimens, prior radiation therapy, ferritin, albumin, AST, Alkaline phosphatase, LDH, preparative regimen and CD 34+ dose elevated were assessed. In a multivariable model for survival, elevated ferritin was a significant,independent,adverse prognostic factor for survival (p=0.011, HR=1.68). The addition of albumin, a negative acute phase reactant, did not change the prognostic impact of elevated ferritin. Patients with ferritin >1910 ng/ml had a higher incidence of TRM(p=0.046), as illustrated below: INCIDENCE OF TRM BY FERRITIN LEVELS Figure Figure This siginificantly higher incidence of TRM occured despite a significantly lower incidence of chronic GVHD (p=0.019) and a trend towards a lower incidence of acute GVHD (p=0.10) in patients with elevated ferritin levels. The incidences of hepatic venoocclusive disease and relapse mortality were not different between the two groups. Conclusions: Elevated ferritin is an independent adverse risk factor in a large group of patients undergoing myeloablative transplantation for various diseases. Higher TRM occured despite the association of elevated ferritin with a significantly decreased incidence of chronic GVHD, consistent with an immunosuppressive effect demonstrated in other settings. Trials should be undertaken to analyze the benefit of iron chelation therapy prior to BMT in patients with iron overload.

Abstract 1806 Poster Board I-832 Plasmacytomas are rare clonal proliferations of plasma cells that though cytologically identical to plasma cell myeloma, present with osseous or extraosseous growth pattern. Understanding their molecular characteristics can provide crucial insights into their pathogenesis and risk of progression to multiple myeloma (MM). To investigate the differences between extramedullary (EMP) and medullary plasmacytomas (MP) and MM without plasmacytomas, we sought to molecularly profile these tumors by tissue microarrays, gene expression, microRNA, and FISH. We identified 85 patients from our data base with a pathological diagnosis of plasmacytoma. Of the 85 patients, 13 patients presented with EMP, and 72 had MP. Among the patients with EMP (n=13), 2 patients presented with multiple lesions. Three of 13 (23%) patients progressed to develop MM at a median of 12 months. 72 patients presented with MP, of which 21 had solitary lesions and 27 (37%) progressed to MM at a median of 20.5months. There was a male preponderance (67% vs 33%) and the median age at diagnosis was 60.5 years (range 27.7-87.6). The mean overall survival for patients with EMP was 121 months (95% confidence interval[CI] 97-144 months) and for patients with MP was 102 months (95% CI 93-128 months) { p=0.025} MicroRNA (miRNAs) profiling was performed on MP (n=19) and MM samples (n=66). Data was normalized using U6 endogenous control. Three hundred and one miRNAs out of a total 665 were significantly differentially expressed between MP vs MM samples. Gene expression profiling performed on MP will be correlated with the miRNA data to identify genes and transcripts of interest which will be functionally validated. Tissue microarrays were performed on 52 patients (8: EMP, 44: MP,) in whom paraffin-embedded tissue was available. Of samples analyzed, CD56 positivity was observed in 55% MP and 71% EMP samples (p=0.67). Additional staining for cyclin D1, Bcl 2 and FISH analysis will be reported. Differential expression patterns of factors involved in proliferation, survival, adhesion, and stroma-tumor cell interactions may help explain plasmacytoma biology and identify factors responsible for progression to MM. These insights may help identify new therapeutic approaches and targets in the treatment of these plasma cell disorders. Disclosures Hochberg: Enzon: Consultancy, Speakers Bureau; Biogen-Idec: Speakers Bureau; Genentech: Speakers Bureau; Amgen: Speakers Bureau. Anderson:Millennium: Research Funding. Raje:Celgene, Norvartis, Astrazeneca: Research Funding.