Citations

Abstract Plasma cell (PC) proliferative disorders vary in behavior ranging from MGUS, a usually indolent disorder, to multiple myeloma, an aggressive disease with poor long-term survival. Detecting PC immunoglobulin (Ig) light chain restriction is commonly used to demonstrate PC clonality and establish the diagnosis. Furthermore, greater degrees of peripheral blood (PB) and/or bone marrow (BM) involvement are associated with more aggressive disease types and, therefore, are adverse prognostic indicators. Flow cytometric immunophenotyping (FCIP) is often used for detecting PC Ig light chain restriction. Shortcomings of FCIP as traditionally performed include its relative insensitivity and its underestimation of PC number. For these reasons, the FCIP PC clonality assessment in our laboratory is currently supplemented with a slide-based immunofluorescence (IF) technique. Recent advances in flow cytometry have led to the development of powerful instruments and reagents, potentially enhancing the ability of FCIP to analyze PC clones. In this study a newly available single tube, 6-color FCIP approach for detecting and enumerating monotypic PCs was compared to the current combined FCIP/IF assay. 94 PB and 124 BM samples were analyzed by combined FCIP/IF and single tube 6-color FCIP assays. For PB, FCIP/IF used 2-color CD38-PE/ CD45-PerCP FCIP to enumerate PCs and slide based IF to detect Ig light chain restriction. For BM FCIP/IF used two 4-color FCIP tubes (Tube1: cytoplasmic (cy)-kappa Ig-FITC/CD20-PE/CD45-PerCP/CD38-APC; Tube 2: cy-lambda Ig-FITC/CD56-PE/CD45-PerCP/CD38 APC) and slide-based IF to confirm clonality. These FCIP assays were performed on a FACSCaliber. The single tube, 6-color FCIP assay used the following fluorochrome-antibody combination: cy-kappa Ig-FITC/cy-lambda Ig-PE/CD138-PerCP-Cy5.5/CD38-APC/CD19-PE-Cy7/CD45-APC-Cy7 with data acquired on a FACSCanto. The results are summarized in Tables 1 & 2, the concordance between the methods was high. All false-negative 6-color FCIP results were attributable to the use of isotonic lysis for cell isolation. When limiting the comparison to the ficolled cells alsoused for FCIP/IF, 6-color FCIP was consistently more sensitive. The 6-color assay also allowed for the discriminition between CD19-positive polytypic PCs and CD19-negative monotypic PCs and the detection of CD45-positive monotypic PC subsets which were often present and which were not identified in the combined FCIP/IF approach. Estimates of PB disease burden generated from the 6-color technique were similar to those determined from the FCIP/IF data which have previously been shown to be of prognositic significance. 6-color FCIP allows for the rapid, sensitive detection of monotypic PCs. The ability of 6-color FCIP to detect phenotypically distinct clonal PC subsets enhances the sensitivity and may provide prognositically significant data through both more complete cell enumeration and charactarization. Given these advantages, 6-color FCIP may eventually supplant traditional FCIP methods of PC analysis. Method Comparsion in BM Bone Marrow 6-color FCIP 6-color FCIP Positive negative total Positive = clone detected; negative = no clone detected. FCIP/IF Positive 81(65%) 3(2%) 84 FCIP/IF Negative 5(4%) 35(28%) 40 Total 86 38 124 Method Comparsion in PB Peripheral Blood 6-color FCIP 6-color FCIP Positive negative total Positive = clone detected; negative = no clone detected. FCIP/IF positive 24(26%) 7(7%) 31 FCIP/IF negative 9(10%) 54(57%) 63 total 33 61 94

Abstract Abstract 2943 Background: Inhibitor of apoptosis (IAP) proteins represents a conserved group of proteins that are important regulators of cell survival and apoptosis. X-linked IAP (XIAP) is the best studied IAP that inhibits pro-apoptotic caspases 3, 7 and 9. Multiple myeloma (MM) cell lines express high levels of XIAP. The levels of XIAP are further increased when stimulated by cytokines IL6 and IGF-1, both secreted in copious amounts in myeloma microenvironment. The other two main IAP proteins, namely cIAP1 and cIAP2 are not direct inhibitors of caspases. Instead, they modulate the levels of various signaling pathways by ubiquitinating proteins within the pathways. The NFKB pathway could be activated or inhibited by cIAP1 and 2. In MM, deletions of cIAP1 and cIAP2 have been shown to activate non-canonical NFKB pathway, which indicates a possible tumor suppressor role of these proteins. We wanted to investigate the role of the three IAPs by using a small molecule inhibitor. Our studies clearly indicate the importance of inhibiting all the three IAPs for the induction of apoptosis in MM cells. Methods: LCL161 was synthesized by Novartis Inc. (Basel, Switzerland). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry with Annexin V-FITC and propidium iodide (PI) for cell lines and patient cells. Immunoblotting was done on cell extracts at various time points following incubation with the drug in order to study the cell signaling pathways. siRNA to cIAP2 was purchased from Invitrogen and was electroporated into MM1S cells. Results: We first examined baseline levels of cIAP1, cIAP2 and XIAP in several MM cell lines and a few patient cells. We observed that the IAPs were constitutively expressed in MM cells. We then wanted to examine the functional significance of these IAP proteins in MM cells. For this, we used an IAP inhibitor LCL161. We observed that LCL161 was able to induce cytotoxicity and inhibit proliferation of MM cells, albeit with differences observed between cell lines. We then examined the factors contributing to resistance in the less sensitive cell lines. For this we chose H929, a sensitive cell line and MM1S, a less sensitive cell line to LCL161. Upon treatment with LCL161, cIAP1 and XIAP were down regulated accompanied by increase in levels of activated caspases 9, 8 and 3 in both H929 and MM1S cells. Using LCL161 in combination with a caspase 9 or a caspase 8 or a pan caspase inhibitor showed clearly that the extrinsic pathway is more involved in the LCL161 induced cell death process. LCL161, however, was unable to inhibit cIAP2 in the less sensitive cell line MM1S whereas cIAP2 was not found to be expressed in H929 cells. It has been shown that cIAP1 is required for ubiquitination and degradation of cIAP2. Therefore, cIAP1 down regulation by LCL161 could actually be contributing to the lack of down regulation of cIAP2 and the observed resistance to LCL161. In order to test this, we used a siRNA to cIAP2 and transfected it into MM1S cells by electroporation. We observed that the siRNA reduced cIAP2 levels and in combination with LCL161 led to marked increase in cells undergoing apoptosis. We also examined signaling pathways after treatment with LCL161 and observed upregulation of both canonical and non-canonical NFKB pathways and Jak/Stat pathway in MM1S cells and not in H929 cells. Combining LCL161 with a Jak2 specific inhibitor SD-1029 synergized in inducing cell death in MM1S and other cell lines less sensitive to LCL161. We are currently testing this combination in MM patient cells. Conclusion: These studies demonstrate the importance of inhibiting cIAP1, cIAP2 and XIAP together in MM cells. Furthermore, by this study we were able to identify resistance mechanisms that are upregulated due to inhibiting the IAP proteins and the importance of using agents that inhibit the IAPs along with inhibitors of these pathways in inducing apoptosis in MM cells. The findings from these studies form the basis of evaluation of IAP inhibitors in combination with a Jak/Stat pathway inhibitor in patients with MM. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Merck: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Research Funding; Novartis: Research Funding; Genzyme: Research Funding; Cephalon: Research Funding.

Abstract Background: Multiple myeloma cells are heterogenous in terms of the expression of CD45 and previous studies have suggested important clinical and biological implications of the CD45 expression. It has been shown that expression of CD45 on myeloma cells has prognostic value and that the CD45 positive cells contain the proliferative fraction of the myeloma cells. We have also demonstrated that the CD45+ population has a higher apoptotic rate compared to CD45− cells. Given the hypothesis that CD45+ cells represent myeloma cells earlier in their development and are more proliferative and more dependent on the marrow microenvironment we studied differences between the two populations in patients with different stages of monoclonal gammopathies and in normal plasma cells. Methods: Fresh bone marrow aspirates were subjected to ACK lysis for removal of RBCs subjected to multicolor flow cytometry to study the expression of different surface antigens on the plasma cells. Plasma cells were identified based on their characteristic CD38/45 expression. The surface expression of CD19 (B cell marker), CD56 (NCAM), CD87, (involved in cellular adhesion, cell motility and angiogenesis), CD126 (IL-6 receptor alpha chain), sVEGF (surface VEGF; surrogate measure of VEGF receptor expression), and CD71 (proliferation marker) were studied and compared between the CD45 + and CD45− cells by gating separately on these populations. Results: Patients with active myeloma (76), smoldering myeloma (32), MGUS/amyloid (44), and normal patients (23) were studied. Not all markers were performed on all patients. Among the patient samples, the expression of CD19, CD87, CD71, CD126, and sVEGF were higher among the CD45+ cells compared to CD45− cells (Table: % of cells with expression). There was no significant difference between the two in terms of the expression of CD56. Among the normal plasma cells, the expression of CD19, CD87, CD126 and sVEGF was greater on the CD45+ cells compared to CD45− plasma cells and CD56 was not different. When the patient cells were compared to normal cells, the expression of CD19 was higher among the normal CD45+ plasma cells and CD56, CD87 and CD126 were less. The expression sVEGF was comparable Conclusion: In this study we demonstrate differences between CD45+ and CD45− plasma cells in patients with monoclonal gammopathies. These findings are important for understanding the disease biology and provide an insight into the functional differences seen between these two populations. The increased expression of CD19 in the CD45+ cells supports the notion that these cells are likely to be at an earlier developmental stage. The comparable CD56 expression is not in accordance with previous studies and needs to be examined in a larger group of patients. Increased expression of sVEGF and CD126 as well as the adhesion molecules is in accordance with increased responsiveness and dependence of the CD45+ cells to VEGF and IL-6 and the increased proliferative rates. These findings indirectly support the hypothesis that CD45 negative cells are less dependent on the marrow microenvironment and the cytokine milieu that it provides.

Abstract Background: Treatment of multiple myeloma has evolved considerably in the past few years with availability of several news drugs as well as increasing use of multidrug combinations. These changes have no doubt led to the improved survival seen among patients with MM. We have previously shown that outcomes of patients intolerant or refractory to one of the IMiDs and bortezomib had a poor outcome. Since that time, other drugs of the same class as well as new classes of drugs have been introduced for the treatment of MM. We designed this retrospective study to estimate the outcomes in patients with relapsed myeloma, who have become refractory to the current generation IMiDs and proteasome inhibitors. Patients and Methods: Patients with relapsed multiple myeloma who have received at least 3 prior lines of therapy, is refractory to both an IMiD (lenalidomide or pomalidomide) AND a proteasome inhibitor (bortezomib or carfilzomib), and has been exposed to an alkylating agent were identified from multiple centers. The time patients met the above criteria was defined as T0, and details of all treatment regimens before and after T0 were collected using electronic CRFs. The study was approved by the IRB at the respective centers. Results: 543 patients were enrolled in this study; median age was 62 years (31-87) and 61% were males. Patients were enrolled from centers in North America (n=181), Europe (n=318), and Asia Pacific (n=44). Patients were diagnosed between 2006 and 2014, the median duration between diagnosis of myeloma and study entry (T0) was 3.1 years (0.3 to 9). The median (95% CI) estimated follow up from diagnosis and from T0 were 61 (57, 66) months and 13 (11, 15) months respectively. The median number of lines of therapy prior to T0 was 4 (3-13), 48% had a prior transplant. The median OS from T0 for the entire cohort was 13 (11, 15) months. For these 462 patients, the median number of recorded regimens was 2 (1-9). The overall response and the depth of response to each line of treatment following T0 are as shown in the table. The median (95% CI) PFS and OS from T0 was 5 (4, 6), and 15.2 (13, 17), respectively. The overall survival for the 81 patients with no treatment post T0 was only 2.1 months. In a multivariate analysis, duration from diagnosis to T0, ISS stage III and number of lines of therapy were all associated with inferior PFS, as well as OS, and in addition, serum creatinine>2 mg/dL at T0 also predicted inferior OS. Conclusions: The study provides the expected outcome following development of myeloma that is refractory to a PI and an IMiD. The outcomes of these patients appear to be better than we had seen historically in patients refractory/ intolerant to bortezomib and IMiDs, highlighting the increased treatment options available for these patients. However, there is decreasing response rate to sequential regimens highlighting the development of drug resistance. The data provides a bench mark for comparison of new therapies that are being evaluated in this disease. Table Table. Disclosures Dimopoulos: Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Kastritis:Takeda: Consultancy, Honoraria; Genesis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Terpos:BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Celgene: Honoraria; Takeda: Consultancy, Honoraria; Genesis: Consultancy, Honoraria, Other: Travel expenses; Amgen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Novartis: Honoraria. Hillengass:Sanofi: Research Funding; Novartis: Research Funding; Amgen: Consultancy, Honoraria; BMS: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria. Leleu:TEVA: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; LeoPharma: Honoraria; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Pierre Fabre: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Takeda: Honoraria. Oriol:Janssen: Honoraria, Other: Expert board committee; Amgen: Honoraria, Other: Expert board committee. Cavo:Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Mateos:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Vij:Shire: Consultancy; Takeda: Consultancy, Research Funding; Jazz: Consultancy; Karyopharma: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Bristol-Myers Squibb: Consultancy; Amgen: Consultancy, Research Funding. Lokhorst:Genmab: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. van de Donk:Amgen: Research Funding; Janssen: Research Funding; BMS: Research Funding; Celgene: Research Funding. Mark:Onyx: Research Funding, Speakers Bureau; Millenium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ludwig:Amgen: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; BMS: Speakers Bureau; Janssen: Speakers Bureau. Jagannath:Novartis: Consultancy; Janssen: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Merck: Consultancy. Usmani:Array: Research Funding; Britsol-Myers Squibb: Consultancy, Research Funding; Skyline: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BioPharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Millenium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Speakers Bureau. Dytfeld:Janssen Poland: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees. Moreau:Novartis: Honoraria; Amgen: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda: Honoraria; Janssen: Honoraria, Speakers Bureau. Lee:Amgen: Membership on an entity's Board of Directors or advisory committees. Shustik:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. de la Rubia:Celgene: Consultancy; Bristol Myers: Consultancy; Amgen,: Consultancy; Janssen: Consultancy. Durie:Takeda: Consultancy; Amgen: Consultancy; Janssen: Consultancy.

Abstract Background: Triplet regimens combining a proteasome inhibitor, an immunomodulatory drug, and a steroid have been shown to be active and well tolerated in previously untreated MM pts. Extended treatment represents an alternative paradigm to induction followed by ASCT in this setting; however, agents for continuous therapy need to be convenient and well tolerated. The investigational agent ixazomib is the first oral proteasome inhibitor to be studied clinically. Phase 1 studies have indicated single-agent activity in relapsed/refractory MM and shown ixazomib to be well tolerated with a manageable safety profile, including limited peripheral neuropathy (PN) (Richardson PG et al, Blood 2014; Kumar SK, et al, Blood 2014). Weekly ixazomib plus lenalidomide-dexamethasone has been investigated in a phase 1/2 study in previously untreated MM pts (NCT01217957); treatment comprised triplet induction therapy followed by single-agent ixazomib maintenance therapy. Results of induction therapy have been previously reported (Kumar SK, et al, ASH 2012). Here we report phase 2 efficacy and safety data in pts receiving ixazomib maintenance. Methods: In phase 2, pts aged ≥18 yrs with measurable disease and no grade ≥2 PN received oral ixazomib 4.0 mg on d 1, 8, 15, lenalidomide 25 mg on d 1–21, and dexamethasone 40 mg on d 1, 8, 15, 22, for up to twelve 28-d induction cycles, followed by maintenance ixazomib on d 1, 8, 15 every 28 d until progression at the dose last tolerated during induction. Transplant-eligible pts could discontinue from study for ASCT after 6 cycles. The primary objective was CR+VGPR rate. Responses were assessed by IMWG criteria. Adverse events (AEs) were graded by NCI-CTCAE v4.02. Results: Fifty pts were enrolled in phase 2; 29 discontinued during induction (cycles 1–12), primarily to undergo ASCT (n=14) or due to AEs (n=6) or pt withdrawal (n=4). Twenty-one pts received ixazomib maintenance therapy (cycle ≥13); 13 (62%) were male, and median age was 68 (range 34–77) years. At data cut-off (June 11, 2014), pts had received a median of 27 treatment cycles (range 15–32), including both induction and maintenance, with a median treatment duration of 26.6 mos (range 13.4–29.6). Mean ixazomib relative dose intensity was 95% and 89% in the induction and maintenance phases, respectively. The mean ixazomib dose administered during maintenance was 3.6 mg weekly. At data cut-off, 11 (52%) pts remained on ixazomib maintenance. Among 49 evaluable phase 2 pts, 44 (90%) achieved ≥PR, including 29 (59%) ≥VGPR. All 21 pts who received ixazomib maintenance had responded to induction therapy. Overall best confirmed/unconfirmed responses in the 21 pts included ≥CR in 11 (52%, of which 4 [19%] were stringent CRs [sCR]), ≥near-CR in 13 (62%), and ≥VGPR in 15 (71%), plus 6 (29%) PR. Responses were rapid and improved during treatment. Median time to first response (≥PR) was 0.99 mos (range 0.92–5.78) and to best response was 7.46 mos (range 1.02–24.74); 33% of pts improved their response during maintenance, including 2 VGPR to near-CR, 3 VGPR to CR, 1 VGPR to sCR, and 1 CR to sCR. At data cut-off, median duration of response was 26.5 mos (range 5.6–26.6+). Among 15 pts achieving ≥VGPR, 5 (33%) had progressed; median duration of ≥VGPR was 23.0 mos (range 3.7–26.1+). All pts were alive after follow-up of 24.6–30.3 mos, including a median follow-up from start of maintenance of 16.9 mos (range 12.9–18.9). Ixazomib maintenance was well tolerated; 15 (71%) pts had drug-related AEs, with only 2 (10%) pts with grade 3 drug-related AEs (hypokalemia, thrombocytopenia) and no grade 4 AEs. The most common (>1 pt) grade 1–2 drug-related AEs were diarrhea (n=8, 38%), nausea, pain in extremity (each n=3, 14%), anemia, and headache (each n=2, 10%). No PN was reported. Serious AEs were reported in 3 (14%) pts during maintenance, including grade 3 acute myocardial infarction, grade 3 pneumonia, and grade 3 orthostatic hypotension; all were considered not related to treatment. Only 1 pt required an ixazomib dose reduction due to an AE (neuralgia). Conclusions: These data indicate that single-agent ixazomib maintenance for up to 1.5 yrs was feasible and generally well tolerated, improved responses following ixazomib-lenalidomide-dexamethasone induction, and contributed to durable responses (median >2 yrs) in previously untreated MM pts not undergoing ASCT. The results support the ongoing phase 3 trial of ixazomib in the maintenance setting (NCT02181413). Disclosures Kumar: Celgene: Consultancy, Research Funding; Millennium: The Takeda Oncology Co.: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding; Sanofi-Aventis: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Skyline Diagnostics: Membership on an entity's Board of Directors or advisory committees. Off Label Use: Investigational proteasome inhibitor ixazomib in combination with lenalidomide and dexamethasone and as maintenance therapy for patients with previously untreated multiple myeloma.. Niesvizky:Onyx Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Millennium: The Takeda Oncology Company: Consultancy, Honoraria, Research Funding, Speakers Bureau. Lonial:Millennium: The Takeda Oncology Company: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding. Laubach:Novartis: Research Funding; Onyx Pharmaceuticals: Research Funding. Hamadani:Millennium: The Takeda Oncology Company: Research Funding. Stewart:Takeda Pharmaceuticals International Co.: Research Funding; Sanofi: Consultancy; Array BioPharma: Consultancy; Bristol Myers Squibb: Consultancy; Celgene: Consultancy; Novartis: Consultancy. Hari:Celgene: Consultancy, Honoraria, Research Funding; Onyx: Consultancy, Honoraria, Research Funding; Millennium: The Takeda Oncology Co.: Consultancy, Honoraria, Research Funding; Spectrum: Consultancy, Honoraria, Research Funding; Sanofi: Research Funding. Vescio:Celgene: Speakers Bureau; Millennium: The Takeda Oncology Co.: Speakers Bureau; Onyx Pharmaceuticals: Speakers Bureau. Kaufman:Millennium: The Takeda Oncology Co.: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Onyx: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Spectrum: Consultancy, Honoraria; Merck: Research Funding. Berg:Takeda Pharmaceuticals International Co.: Employment. Liao:Takeda Pharmaceuticals International Co.: Employment. Hui:Takeda Pharmaceuticals International Co.: Employment. Richardson:Millennium: The Takeda Oncology Co.: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Abstract Abstract 1848 Background: Aurora kinases play an important role in cell division by controlling chromatid segregation. Aberrant functions of aurora kinases result in genetic instability, a condition often seen in cancers. While Aurora A is important in the alignment of the sister chromatids, Aurora B, a spindle fiber associated kinase, heavily influences the equal division of sister chromatids and along with the Aurora C kinase also assists in a regulated cell division. Inhibition of either of the Aurora family kinases affects chromosomal alignment and segregation during the course of cell division resulting in polyploidy, cell growth arrest and ultimately cell death. VEGF has been implicated in the increased angiogenesis in MM patients. Increase in VEGF levels leads to upregulation in the Ras/Mek/Erk pathway and increased angiogenesis, cell proliferation and decreased apoptosis. Inhibiting this pathway has shown to induce apoptosis in MM cells. We therefore investigated the role of a small molecule inhibitor A-1014907 that inhibits both aurora kinases and VEGF stimulated Ras/Mek/Erk pathway. Methods: A-1014907 was synthesized and provided by Abbott Laboratories Ltd. Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI) for cell lines and patient cells. Immunoblotting was done on cell extracts at various time points following incubation with the drug in order to study the cell signaling pathways. Results: A-1014907 was able to induce cytotoxicity and inhibit proliferation in all MM cell lines tested with IC50 values between 50–100nM. Similar extents of inhibition of proliferation was also observed when MM cells were co-cultured with bone marrow stromal cells or HUVEC cells or tumor promoting cytokines IL6, IGF and VEGF. The increase in cytotoxicity was due to apoptotic cell death observed in both MM cell lines and patient cells. Cell cycle assays demonstrated that A-1014907 was able to induce cell cycle arrest at the G2/M stage of cell cycle followed by polyploidy indicative of Aurora B inhibition. We also observed an increase over time the proportion of cells in sub G0/G1 stage indicative of cell death. Western blots were performed to understand the mechanism of action of A-1014907. We observed that A-1014907 was able to significantly down regulate Aurora B activity as measured by pHistone H3 (Ser 10) down regulation. This was accompanied by up regulation of p21 and down regulation of CDK4 and cyclin E both indicative of G2/M arrest. We also observed the ability of A-1014907 to inhibit the Ras/Mek/Erk pathway by measuring levels of pErk post treatment with the drug. A-1014907 potently inhibited pErk levels and this down regulation was also observed when MM cells were co-cultured with either VEGF or HUVEC cells. Furthermore, A-1014907 at sub IC50 doses induced synergistic cell death of MM cell lines when combined with sub IC50 doses of dexamethasone Conclusion and current studies: A-1014907 clearly inhibits the Ras/Mek/Erk pathway and aurora B activity and induces apoptosis in MM cell lines and patient cells. We are currently using siRNA to aurora A in combination with A-1014907. We are also examining the mechanism of action of dexamethasone in combination with A-1014907. All this will help to design clinical trials with A-1014907 either alone or in combination with other anti-MM agents in MM patients. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Merck: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Research Funding; Novartis: Research Funding; Genzyme: Research Funding; Cephalon: Research Funding.

Abstract Abstract 1859 Genome wide RNA interference studies identified Aurora Kinase (AURK) A and B as lethal targets in Multiple Myeloma (MM) while suppression of these genes also sensitized MM to bortezomib (BTZ). MLN8237 is an oral inhibitor of AURKA. We therefore conducted a Phase I clinical trial of MLN8237 in combination with BTZ. The study enrolled a total of 19 patients at 5 institutions. 9 patients are still receiving active treatment as of the date of this report. Study Design: The phase I portion of this study uses a standard 3+3 design to determine the maximum tolerated dose (MTD) of MLN8237 and BTZ in patients with relapsed/refractory MM. Eligibility required a minimum of 1 and maximum of 4 lines of prior therapy. Patients who have received prior BTZ therapy were allowed on trial as long as they did not progress during prior BTZ or ≤ 60 days of therapy discontinuation. The following laboratory values were required £7 days prior to registration. ANC3 1000/mL, Hgb ≥9 g/dl, PLT3 100,000/mL, Total bilirubin £1.5 × upper limit of normal (ULN), Creatinine £ 2.5 × ULN, a baseline LVEF ≥45%. Patients were required to be able to take oral medication and to maintain a fast as required for 2 hours before and 1 hour after MLN8237 administration. Treatment Overview: The first 3 patients received MLN8237 at 25 mg po days 1–14 and BTZ at 1.3 mg/m2/dose iv. days 1, 4, 8, 11 on a 28 day schedule. Based on data from other concurrent trials an amendment changed dosing of MLN8237 to 20, 30, 40 or 50 mg po twice daily on days 1–7 and BTZ was given at 1.5mg/m2 iv weekly on a 28 day schedule. Results: Median age of patients was 64, 63% were male, 31% had high risk genetics, 84% had prior stem cell transplant, 53% of patients were relapsed and 47% were relapsed and refractory to therapy. No DLTs were observed even at the highest dose level tested. However, one patient at the highest dose level required a platelet transfusion in order to initiate treatment on time in Cycle 2. Thus, a further 3 patients accrued at the highest dose level before declaring the MTD and proceeding to phase II. The highest dose level (Dose Level 3: MLN8237 50 mg po twice daily on days 1–7; BTZ 1.5 mg/m2 iv. given on days 1, 8, 15, 22) was the final dose level tested (the MTD of single agent MLN8237 is 50mg as defined in other Phase I trials). The ORR was 26% (1 CR, 4 PR); when minor responses are included the ORR was 52%. Median follow up was 4.3 months (range 0.9–23.4) and PFS was 5.5 months. At last follow up 12 patients showed no progression and 7 had progressed. Toxicity: 63% of patients experienced a grade 3AE and 5% a grade 4 AE. Grade 3 or 4 toxicity seen in more than one patient was all hematologic with thrombocytopenia and neutropenia being common. Other toxicity of any grade regardless of attribution occurring in more than 20% of patients included neuropathy 63%, fatigue 63%, diarrhea 53%, nausea 47%,vomiting 26%, infection 32%, alopecia 21%, Conclusions: The MTD of the combination is MLN8237 50 mg po twice daily on days 1–7 and BTZ at 1.5mg/m2 iv weekly. Phase II testing is underway and updated results will be presented. Disclosures: Stewart: Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy. Vij:Millennium: Speakers Bureau. Hofmeister:Celgene: Advisory board Other, Honoraria.

Abstract Abstract 2965 Background: Alkylating agents have been the mainstay of multiple myeloma (MM) therapy for decades and despite introduction of several new therapies, it continues to play a significant role in its management as part of various drug combinations. While melphalan has been the most commonly used alkylator in MM, recent studies have suggested significant activity for bendamustine, a bifunctional alkylator. The combination of lenalidomide and melphalan has been associated with high response rates in relapse and newly diagnosed MM. Based on these promising results we designed a trial to evaluate the maximally tolerated dose of lenalidomide and bendamustine when used in combination as well as the efficacy of the combination in relapsed disease. Patients and Methods: Patients with relapsed MM and measurable disease were enrolled on this phase 1/2 trial provided they had not more than 4 prior lines of therapy for MM, had adequate performance status and organ and hematological function. Patients refractory to lenalidomide were allowed to enroll. The primary objectives were to (i) to determine the MTD of bendamustine and lenalidomide in combination with dexamethasone in subjects with relapsed MM (phase 1) and (ii) to evaluate the confirmed response rate of bendamustine in combination with lenalidomide and dexamethasone in patients with relapsed MM (phase 2). Bendamustine (B) was administered on days 1 and 2 of a 28-day cycle at doses of 50–100 mg/m2. Lenalidomide (R) was given days 1–21 at doses of 15–25 mg daily. Dexamethasone (D) was administered at 40 mg weekly. Dose escalation was done using a 3+3 design and MTD was defined as one dose level below that resulted in >=2 DLTS among 6 patients. The primary end point for this trial was the proportion of patients with confirmed hematologic response (sCR, CR, VGPR, or PR) over the first 6 cycles of treatment. Results: A total of 72 patients were accrued to this study from March 2010 to May 2012: 21 patients in phase 1 and 51 in phase 2. The 6 patients from the MTD dose level of phase 1 were also included in phase 2. The median age of all 72 patients was 62.1 (range, 40–86) and 57% were male. Majority (75%) of patients had previously been exposed to lenalidomide and 69% had prior exposure to bortezomib. Median # of prior therapies was 3 (range, 1–5) and 74% of patients had a prior autologous stem cell transplant. Patients have received a median of 4 cycles (range, 1–25), with 27 patients still continuing on active treatment. Disease progression led to study discontinuation in 22 (49%) and adverse events were the reason for discontinuation in 14 (31%). In phase I, two DLTs (Grade (Gr) 2 neuropathy and Gr 4 neutropenia) were seen at the highest dose level (100 mg/m2 B, 25 mg R), and the MTD was determined as 75 mg/m2 of B given days 1 and 2 and 25 mg of R days 1–21, along with D 40 mg weekly. Overall patients, 12/21 (57%) had a PR or better. In phase 2, 17 (40%) confirmed responses (>=PR) were seen among the 43 patients evaluable for response (received at least 6 cycles of treatment or have gone off study prior to 6 cycles); including 9 (21%) VGPR and 8 (19%) PR. An additional 5 patients had a minor response. Over all dose levels, a gr 3 or higher adverse event at least possibly attributed to the study was seen in 75% of patients. The most common toxicities were all hematological (thrombocytopenia and leukopenia), and most common non-hematological toxicity was infection. Prolonged time to recovery of blood counts was seen in a few patients, but majority of patients were able to tolerate the regimen with adequate dose reductions. Conclusion: The recommended dose of the combination for further studies is bendamustine at 75 mg/m2 days 1 and 2, lenalidomide 25 mg daily on days 1–21 and dexamethasone days 1, 8, 15, 22; with cycles repeated every 28 days. The regimen is well tolerated with hematological toxicity being the most common and manageable with dose reductions. The regimen is effective with high response rates and durable responses seen. Updated results with response rates and time to event analyses will be available for the entire cohort at the time of meeting. Disclosures: Kumar: Merck: Consultancy, Honoraria; Celgene: Research Funding; Millennium: Research Funding; Novartis: Research Funding; Cephalon: Research Funding; Genzyme: Research Funding. Krishnan:celgene: Consultancy, Speakers Bureau. Zimmerman:Celgene: Honoraria; Millennium: Honoraria; Novartis: Expert Testimony, Expert Testimony Other. Vij:Teva: Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau; Millennium: Speakers Bureau; Onyx: Honoraria, Research Funding.

Abstract Abstract 1858 Systemic light chain amyloidosis (AL amyloidosis) is a condition where clonal plasma cells produce misfolded insoluble immunoglobulin light chains that deposit in various organs causing progressive organ dysfunction. Chemotherapy and autologous stem cell transplant (ASCT) when eligible is the standard treatment options for patients with AL amyloidosis. There are several studies who report long term outcomes of patient post ASCT. However, there is a paucity of literature describing the outcomes of patients who have received ASCT but have relapsed. We performed a retrospective study to assess the outcomes and treatment regimens employed following relapse after ASCT. Between 1996 and 2009, 410 patients received ASCT at the Mayo Clinic as first line therapy. Of those 410 patients 42 patients died within 3 months of transplant, 64 patients died without documented relapse, 158 patients were alive without documented progression, and 146 patients had documented progression. Those 146 patients are the subject of our study. The median time to hematologic relapse was 2 years (range: 0.2–15.5 years). At relapse, 59 patients were treated with IMiD based therapy, 36 with alkylator based therapy, 24 with bortezomib, 15 with steroids, and 5 with second ASCT. The respective hematologic response rates were 58%, 33%, 50%, 53%, and 60%. The remaining six patients were not evaluable for response for one other following reasons: organ transplants; no further therapy; inevaluable disease. With a median post relapse follow up of 3.6 years, the median overall survival (OS) from the first post ASCT relapse was 4.6 years. The median post transplant follow up was 6.1 years, the median OS for these patients was 7.3 years from the time of transplant. These data provide novel information about outcomes after SCT relapse, which should be useful not only for patients and doctors but also for investigators designing studies for salvage therapies post-transplant. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Myeloma remains incurable due to a stem cell/proliferative component that responds poorly to standard treatments. We have shown that aberrant IL-1beta stimulates the generation of paracrine IL-6, a central myeloma growth factor. A Phase II trial was completed using IL-1 receptor antagonist (IL-1Ra/Anakinra), which inhibits paracrine IL-6 production, and low dose Dexamethasone (Dex), which decreases IL-1 levels through myeloma cell apoptosis, in patients with SMM/IMM. SMM/IMM patients are most likely to benefit from anti-cytokine therapy in order to delay/prevent the development of active myeloma. Methods: Patients that had ≥ 10% bone marrow plasma cells and/or an IgG or IgA M-spike ≥ 3 g/dL and did not require immediate chemotherapy were eligible. All patients received 100 mg of IL-1Ra SQ qd for 6 months unless clinical progression occurred. Non-progressors were allowed to continue on therapy with IL-1Ra alone. Low dose Dex (20 mg qweek) was added after 6 months of IL-1Ra or for evidence of clinical progression; the dose was adjusted based on response/toxicity. Results: To investigate the effects of IL-1Ra and Dex alone and in combination in vitro, we co-cultured IL-1beta transduced +/− myeloma cells with stromal cells +/− dexamethasone, IL-1Ra, or both for 48 hours and quantitated the percent apoptotic cells by flow cytometry and IL-6 production by ELISA. The results showed that: 1) IL-1Ra was superior to Dex at inhibition of IL-6 but caused no myeloma apoptosis; 2) Dex induced apoptosis was inhibited by IL-6 3) Dex and IL-1Ra combined induced maximal IL-6 inhibition and apoptosis of myeloma cells. In the clinical trial, data were available on 47 patients; smoldering (79%) / indolent (21%). All patients received IL-1Ra initially and 20/47 subsequently received IL-1Ra/Dex. Seven patients had a decrease in the plasma cell labeling index (PCLI), a marker of cell proliferation, on IL-1Ra alone which paralleled a decrease in the high sensitivity C-reactive protein (CRP). Interestingly, in one patient treated with Dex alone for 6 months, Dex induced a decrease in the M-protein and IL-1 levels, however, the PCLI and CRP values increased. The IL-1Ra/Dex combination has resulted in stability of disease in the majority of the 47 patients; 28 continue on therapy with a median overall progression-free survival (PFS) of 37.5 months. Four of the 47 pts achieved a minor response (MR) with IL-1Ra alone, and an additional 6 pts achieved a MR/PR after addition of dex. The continuous measure of % reduction in CRP was significantly associated with time to progression (p=0.02). Similarly, those patients who had a >5% decrease in serum M protein also had a longer PFS (p<0.001). Of note, 2 patients with high PCLI values of 6.4 and 3.0, characteristic of poor prognosis, responded with a reduction in the PCLI values and continue on combination therapy for 3 and 1.5 years, respectively with stable disease. Conclusion: Agents such as IL-1Ra that specifically inhibit paracrine IL-6 production are more effective at targeting the proliferative component than apoptosis inducing agents such as Dex; the combination may be especially useful in patients that have an elevated PCLI at presentation.