Ghobrial, Irene M.

Abstract The objective of this study was to investigate the underlying molecular alteration in multiple myeloma at the protein level in order to identify regulators of pathogenesis, discover novel targets of therapy, and compare genetic and proteomic alterations. We employed antibody protein microarrays (BD Clonetech, CA) to measure changes in the patterns of protein expression between MM and normal plasma cells. The antibody array is a new technique enabling protein differences to be assayed directly by hybridizing fluorescently labeled protein mixtures from cell extracts onto glass slides spotted with 512 different monoclonal antibodies. CD138+purified plasma cells were obtained from cryopreserved bone marrow samples of 12 newly diagnosed patients with MM. The labeling index was high (1% cutoff) in 7 samples and low in the other 5. Control plasma cells were obtained from 9 pooled CD138+ purified normal donor bone marrow plasma cells. Interphase FISH analysis for 17p deletion, 13q deletion, and t(11:14) were performed. To assess differential expression, the mean of the ratios of Cy5/Cy3 for each sample were analyzed using the Clontech software to calculate an internally normalized ratio. The normalized data were analyzed by the Genespring software. Unsupervised clustering identified 4 groups of MM. Changes of protein expression ≥2 fold in 70% of the samples as compared to control were identified. There were 6 proteins differentially expressed between all MM samples and control cells including proteins in the ras signaling pathway (KSR-1), the ubiquitin pathway (Ubc-H6), cyclin-dependent kinases (CDK4), cytokines (IL-6), DNA toposisomerase II, and the rho-interacting serine-threonine kinase CRIK. Proteins differentially expressed in MM groups 1 and 2 compared to normal control included cell cycle regulators (cul-2, MCM6, PCNA, TGFb1), kinases (p70S6K, PKC), and chromatin regulators (Ran, AKAP450, Rad50). Protiens differentially expressed in MM group 3 included cell cycle regulators (CDK2, CLK1, MENA), apoptosis regulators (XIAP, caspase 4, perforin) kinases (IKKa and RAC1 in the Wnt signaling pathway) and P53 regulators, while proteins identified in MM group 4 included NFkB/ubiquitin proteins (IKKa and Ubch6), cell cycle regulators (c-myc, CDK4), p53 pathway proteins (53bp2), ras-signaling proteins (KSR1), and the kinase CRIK. There were no differences in protein expression between the high and low labeling index groups. 13q was identified in 5 (42%), 17 p in 1(8%) and t(11:14) in 1(8%) patients. 80% of the 13 q deletion cases clustered in MM group 1 and 2 patients. Cyclin D-1 was upregulated in 5 (42%) patients including the patient with (11:14) translocation. This is the first proteomic study of patients with MM. The results are consistent with previously identified genetic alterations in MM indicating that this novel technique could be used in identifying molecular changes in MM. It identifies novel proteins dysregulated in MM that differ between the 4 MM groups. These results may be used in the future to individualize therapy based on the proteins dysregulated in each group. For example, IKK inhibitors may be useful in group 3 MM patients, while mTOR inhibitors (upstream of p70S6K) could be used in groups 1 and 2 patients. Future correlations with gene expression arrays and prognosis in a larger cohort of patients is warranted.

Abstract Angiogenesis plays an important role in tumor growth. Endothelial cells express the chemokine receptor CXCR4, and interact with its ligand SDF-1/CXCL12. Previous studies have demonstrated that the ERK MAP kinase and the PI3Kinase pathways are activated in response to SDF-1 stimulation. In this study, we investigate the effect of the role of inhibitors of CXCR4, ERK MAP kinase and PI3Kinase on angiogenesis. The AngioKit (TCS Cellworks, U.K) is a 24 well plate in which human endothelial cells are co-cultured with other human myoblasts and fibroblasts in a specially designed medium. Control wells in the kit include media alone, VEGF (+control) and suramin (-control). Test samples were added on the day the kits arrive, then changed on days 4, 7, and 9, and stained on day 11 with CD31 (PECAM). The wells are then photographed and subjected to image analysis. The software measures angiogenesis as total tubule length per well in microns. Test samples can then be compared to the control wells to determine the drugs affect on angiogenesis in vitro. The following drugs were tested in this angiogenesis model system, a human CXCR4 neutralizing antibody (MAB 171, R&D systems, MN), SDF-1, the MAP kinase inhibitor PD098059, and the PI3Kinase inhibitor LY294002. Treatment with the CXCR4 inhibitory antibody, PD098059, and LY294002 caused marked decrease in angiogenesis (below the level of the negative control suramin). Inhibition of angiogenesis below the level of suramin was first detected at 1mg/ml CXCR4 antibody, and10mg/ml CXCR4 antibody resulted in complete inhibition of angiogenesis. The effect of PD098059 on angiogenesis was dependent on its concentration; 20mM PD098059 inhibited angiogenesis while lower concentrations did not. These results are consistent with the drug’s known concentration-dependent inhibition of MEK-1 and indicate that the MEK-1 inhibitor leads to angiostasis secondary to its specific inhibitory effect on MEK-1. The lowest level tested of 1μM LY294002 led to inhibition of angiogenesis, and 50μM of LY294002 led to complete abrogation of angiogenesis. SDF-1 has been reported to be angiogenic. In this model system, the effect of SDF-1 alone on angiogenesis was subtle. However, the endothelial cells used in this model system may be secreting endogenous SDF-1 leading to the saturation of the CXCR4 receptor and minimal effects of exogenous SDF-1 stimulation. This was demonstrated by the significant effect of the CXCR4 inhibitor on angiogenesis without the addition of exogenous SDF-1. These results indicate that CXCR4 inhibition and its downstream pathways PI3K and ERK MAP kinase lead to significant inhibition of angiogenesis, and suggest that selective inhibitors of CXCR4 may be useful agents to inhibit angiogenesis.

Abstract Purpose: Mantle cell lymphoma (MCL) is characterized by a t(11;14) resulting in overexpression of cyclin D1, a member of the phosphatidylinosital 3 kinase (PI3K) pathway. This study tested whether CCI-779, which inhibits the PI3K pathway at the level of the mammalian target of rapamycin (mTOR) could produce tumor responses in patients (pts) with MCL. Patients and Methods: Eligible pts had biopsy-proven, cyclin D1 positive MCL and had relapsed or were refractory to therapy. Pts received CCI-779 250 mg IV every week as a single agent. Pts were re-staged after 1 cycle (4 doses) and every 3 cycles thereafter. Pts with a tumor response after 6 cycles were eligible to continue drug for a total of 12 cycles or 2 cycles after complete remission (CR) and then were observed. Results: Thirty-five pts were enrolled and evaluable for toxicity; 1 patient had MCL by histology but was cyclin D1 negative and ineligible for efficacy evaluation. The median age was 70 years (range, 38–89), 91% were stage 4, and 69% had ≥ 2 extranodal sites. Pts had received a median of 3 prior therapies (range, 1–11) and 54% were refractory to their last treatment. The overall response rate was 38% (13/34) with 1 CR (3%) and 12 PRs (35%), surpassing the pre-defined criteria for a promising agent. Responses tended to occur rapidly with median time to response of 1 month (range, 1–8). To date, 26 patients have progressed, with a median time-to-progression of 6.8 months (95% CI: 3.8 – 9.7). Median duration of response for the 13 responders was 5.7 months (95% CI: 5.2 – 13.2). Overall, 32 out of 35 patients who received treatment had grade 3 or 4 toxicity. The most common toxicities were hematologic with grade 3 (n=24) or grade 4 (n=4). Thrombocytopenia was the most frequent grade 3/4 toxicity (n=25) and the largest cause of dose-reductions, although counts typically recovered within one week. Only 4 patients could tolerate sustained 250 mg per week throughout their treatment (including one who went on to alternate treatment after 1 cycle) and the median dose/month was 175 mg. Conclusions: Single-agent CCI-779 has substantial anti-tumor activity in relapsed MCL. This study demonstrates that agents, which selectively target cellular pathways dysregulated in MCL cells can produce therapeutic benefit. The high response rate warrants further studies of this agent in MCL, but the high incidence of hematologic toxicity suggests that a lower dose should be explored. CCI-779 at 25mg is currently being evaluated in MCL through an NCCTG trial

8043 Background: The mammalian target of rapamycin (mTOR) signal pathway controls cell proliferation and survival. The trial's goal was to determine the anti-tumor activity and safety of single-agent everolimus (TORC1 inhibitor) in patients with relapsed/refractory Waldenstrom macroglobulinemia (WM). Methods: Eligible patients had measurable disease (IgM monoclonal protein >1000 mg/dL with >10% marrow involvement or nodal masses >2 cm), a platelet count ≥75,000 x 106/L, a neutrophil count ≥1,000 x 106/L. Patients received everolimus 10 mg PO daily. Tumor response was assessed after cycles 2 and 6 and then every 3 cycles until progression. Results: 60 pts were treated. The median age was 64 years (range, 43-85). The median number of prior therapies was 3 (range, 1-11). All but two patients (97%) had received prior rituximab based therapy and 64% of patients had received prior alkylator based therapies. The overall response rate (complete response CR+ partial response PR+ minimal response MR) was 73% (95% CI: 60-84%), with a PR of 50% and 23% MR. The median time to progression (TTP), progression-free survival (PFS), and overall survival (OS) for the entire study population is 28 months (mos), (95% CI: 18-not reached (NR)), 22 mos (95% CI: 12 0-NR), and 55 mos (95% CI: 55-NR), respectively. The estimated PFS at 12 and 24 months is 62% (95%CI: 51-75%), and 48% (95%CI: 37-63%), respectively. The 30 patients who achieved a PR responded after a median of 2 months (range, 1-26) of treatment. The median duration of response (DR) for these patients has not yet been reached and 19 of these patients remain in response after a median follow up of 31 months (range, 3-54). All but two patients had a decrease in their serum IgM. Grade 3 or higher related toxicities were observed in 67% of patients. The most common were hematological toxicities with cytopenias. Pulmonary toxicity occurred in 5% of patients. Dose reductions due to toxicity occurred in 63% of patients. Conclusions: Everolimus has high single-agent activity with an overall response rate of 73% and manageable toxicity in patients with relapsed WM, and offers a potential new therapeutic strategy for this patient population.

Abstract Introduction. Growing evidence suggests that immune cells that reside within the tumor microenvironment are dysregulated and functionally impaired, leading to defective anti-tumor immunity of the host. One of the major immunosuppressive mechanisms during tumor progression is expansion of regulatory immune cells. Here, we analyzed the immune cells within the bone marrow (BM) and the peripheral blood (PB) of 2 immunocompetent multiple myeloma (MM) mouse models. We next studied the role of regulatory T cells (Tregs) in MM pathogenesis. Materials and methods. To study the immune cell populations of the BM and PB, we used two immuncompetent mouse models and transplanted VK*MYC cells or 5TGM1 cells into C57BL/6 and C57BL/Kalwrij mice respectively. The immune cell populations and checkpoint receptor expressions were analyzed by CyTOF mass cytometer or flow-cytometry. Treg induction assay was performed in vitro to study the mechanism of Treg increase in the BM of myeloma injected mice. CD4+ CD25- cells were obtained from C57BL/Kalwrij mice and were co-cultured with 5TGM1 cells or B cells from C57BL/Kalwrij mice in vitro. Treg induction was compared by flow-cytometry. Transplantable VK*MYC cells were injected into "depletion of regulatory T cell" (DEREG) mice, which expresses a simian diphtheria toxin (DT) receptor-enhanced GFP fusion protein under the control of the FOXP3 gene locus, or their wild type littermates. DT injection into these mice leads to depletion of Tregs as previously described (J Exp Med. 2007; 204: 57-63). DT was given once every week for a total 3 times i.p to the DEREG mice or the littermate controls to specifically deplete Tregs and to study the role of Tregs during MM progression. Tregs (CD4+ FOXP3-GFP+ cells) were sorted from VK*MYC injected mice or non-injected DEREG mice BM using FACSAria cell sorter. Cells isolated were subjected to RNA sequencing. Gene Set Enrichment Analysis (GSEA) was performed to define differences in molecular signatures between MM-associated and normal Tregs. Results. The Treg proportion was significantly increased within the CD4+ T cells in the BM of myeloma cell injected mice from the early stage of disease compared to control mice, while in the PB, the increase was observed only at the late stages of disease progression. The effector T cell (Teff)/Treg ratio was significantly decreased in the BM at the end-stage myeloma bearing mice (P<0.01). Checkpoint related molecules (PD-1, LAG-3 and Tim-3) on Tregs and Teffs were up-regulated at the protein level in the BM of myeloma injected mice compared to control (P<0.01). These data indicate the activation of Tregs and decrease of Teff activity, leading to suppression of anti-myeloma T cell activity in the MM BM. Under in vitro co-culture conditions, 5TGM1 cells induced a significant increase in the number of Tregs from non-Treg CD4+ T cells compared to controls (P<0.01). 5TGM1 cell-induced Tregs presented with enhanced ki-67 expression, thus suggesting the ability of MM cells to induce Treg proliferation. Additionally, the trans-well co-culture experiment showed that the major mechanism for Treg induction by 5TGM1 was by direct contact with T cells rather than secreting factors. Significant increase in survival was observed in the VK*MYC injected DEREG mice under Treg depletion compared to the DEREG mice without Treg depletion and wild type littermates under DT treatment (P<0.001). The Treg depleted DEREG mice were accompanied with an increase of Teffs (P<0.01), indicating recovery of anti-myeloma T cell activity. RNA sequencing of BM Tregs from VK*MYC injected mice showed increased expression of immune checkpoint related molecules and increased Treg effector molecules (IL-10, Granzyme B) compared to BM Tregs from control mice, indicating a more functionally active phenotype of VK*MYC associated Tregs. GSEA showed an enrichment of genes involved in type-1 interferon signaling in VK*MYC associated Tregs. Conclusions. We used 2 immunocompetent MM mouse models to study the characterization and significance of Tregs in MM progression. CyTOF analysis and RNA sequencing data indicated Treg activation in the MM BM microenvironment. The Treg in vivo depletion experiment showed that Tregs have a significant role in MM progression. These data indicate that immunotherapy targeting Tregs may represent a novel therapeutic strategy for MM. Studies are ongoing to understand the roles of Tregs in human MM patients. Disclosures No relevant conflicts of interest to declare.

Abstract INTRODUCTION: mTOR inhibitors have been associated with responses in Mantle cell lymphoma and other lymphomas (J Clin Oncol23; 5347, 2005). Phase I and II trials with the oral everolimus (RAD001, Novartis Pharmaceuticals) have demonstrated a favorable side effect profile. This phase II study tested everolimus in patients with relapsed/refractory Waldenstrom Macroglobulinemia (WM). METHODS: Patients who had at least one previous therapy for WM and who had relapsed or refractory disease were eligible. Other eligibility criteria included symptomatic disease, > 2 weeks from last therapeutic agent used, >10% involvement with lymphoplasmacytic cells in the bone marrow, IgM paraprotein > 2x upper limit of normal, absolute neutrophil count >1000/mm3 and platelet count >75,000/mm3, serum creatinine of < 2 x upper limit of normal. NCI CTCAE v3.0 was used for toxicity assessment. Response was assessed by criteria established at the second consensus panel for WM. All patients received 10mg RAD001 daily for 28 days per cycle for 6 cycles; response was assessed after 2 cycles. RESULTS: 10pts (8 men and 2 women, median age is 62 years, range 52–71) have been treated to date. The median number of lines of prior treatment was 3 (range 1 – 5). The median IgM at baseline was 3970 mg/dL (range 3120– 7410); median M-spike at baseline was 2.07 gm/dL (range 1.41 – 4.02); and median hemoglobin was 11.3 g/dL (9.4–13.3). The median follow up was 4 months (range 1 – 7 months). Prior therapy included rituximab, nucleoside analogues (fludarabine and 2-CDA), combination chemotherapy (e.g CHOP, CVP), chlorambucil. and bortezomib. Seven patients are currently evaluable for response. The best response to single agent RAD001 after 2 cycles is presented in Table 1. Median duration of response has not been reached. Patients tolerated RAD001 well without significant toxicities. The main toxicities observed were apthous ulcers in 4 patients. One patient required dose reduction due to mucositis. One patient stopped therapy within the first month of treatment and went on to hospice care before succumbing to his disease after one month. Attributable toxicities otherwise proved manageable with appropriate supportive care and RAD001 was generally well tolerated, with no peripheral neuropathy. CONCLUSIONS: Oral RAD001 as monotherapy has been well tolerated and demonstrates exciting activity achieving PR + MR in 57%, and/or stabilization of disease in 43% of evaluable patients with relapsed WM. Accrual of a total of 30 patients is rapidly ongoing. Updated data will be presented at the meeting. Table 1 Response N=7; ORR (CR+PR+MR)= 57% Median time to best response (months) Partial Response 1 (14%) 5 Minimal Response 3 (43%) 4 (3–6) Stable Disease (SD) 3 (43%) NA Progressive Disease 0

Abstract From 2013-2017, numerous new approvals and expanded indications have dramatically changed the treatment (tx) landscape for MM. Since that time, experienced MM physicians from leading academic institutions and cancer centers (experts) have annually updated and contributed tx recommendations for an online decision support tool designed to provide healthcare providers (HCPs) with patient-specific tx guidance. This MM tool is freely available online at www.clinicaloptions.com/myelomatool and HCPs can use the tool to enter details of a patient case, indicate their intended tx plan, and then expert tx recommendations for that case are provided. Methods This study includes analysis of tx recommendations from 5 MM experts for 270 unique MM case scenarios made during development of the 2018 update to this tool. We also aim to compare the evolving tx recommendations among the experts over time, from 2013-2017, and to determine practice patterns of HCPs using the tool over the same time span. Results Based on analysis of these annually updated MM tools, consensus (≥ 3 of 5 experts choosing the same tx for the same case) among experts has been increasing for induction tx since 2013. In ASCT-eligible patients with no specific comorbidities, experts recommended bortezomib/lenalidomide/dexamethasone (VRd) in 100% and 90% of tool cases in 2018 and 2017, respectively, which increased from 65% to 75% consensus in 2013-2015. In previous analyses, the percentage of HCPs using VRd has also increased, albeit more slowly than with experts. In 2017, 42% of HCPs indicated that they are using VRd as induction in this setting compared with 22% in 2015-2016 and 9% in 2014-2015. For ASCT-ineligible patients with ECOG > 2 but no specific comorbidities, all 5 experts recommend triplet therapy for all cases regardless of risk in 2018, but in 2017, 3 of 5 recommended triplet therapy and 2 of 5 recommended doublets for normal-risk disease. Expert recommendations for induction change, however, based on specific patient comorbidities. For patients with peripheral neuropathy, 4 of 5 experts recommended carfilzomib/lenalidomide/dexamethasone instead of VRd. For patients with renal insufficiency, 4 of 5 experts recommended bortezomib/cyclophosphamide/dexamethasone. For patients with cardiac dysfunction, all 5 experts continue to recommend VRd for patients with ECOG ≤ 2 while 2 of 5 change from VRd to Rd for patients with ECOG > 2. For patients who achieve VGPR or better to induction, all 5 experts recommend lenalidomide maintenance for standard risk disease. But for patients with high risk disease who achieve VGPR or better to induction, 3 of 5 experts recommend bortezomib/lenalidomide combination maintenance therapy regardless of induction regimen, 1 expert recommends single-agent bortezomib regardless of induction regimen, and 1 expert recommends bortezomib after a PI-based induction regimen or lenalidomide for an IMiD or IMiD/PI-based induction regimen. In the 2018 tool update, the number of R/R case scenarios with 100% expert consensus increased over previous years: 20% of cases had all 5 experts agreeing on tx vs 12% in 2017. In addition, 29% of cases had 3 of 5 experts agreeing and 8% had 2 pairs of experts agreeing in 2018 vs none with 3 of 5 experts agreeing and 21% with 2 pairs of experts agreeing in 2017. In 2018, experts recommended daratumumab/pomalidomide/dexamethasone all cases with 100% consensus, which comprises patients who are refractory to multiple agents including lenalidomide, bortezomib, and/or carfilzomib. Overall, 23 different regimens were recommended for R/R MM; and additional majority expert consensus included daratumumab/lenalidomide/dexamethasone, daratumumab/carfilzomib/dexamethasone, and carfilzomib/pomalidomide/dexamethasone (Table 1). Conclusions Practice patterns are changing rapidly and are more complex, particularly in the R/R setting, in response to the evolving tx landscape for MM. This analysis highlights several areas of expert consensus, but disparities remain for select cases. The newest approved therapies continue to have a strong impact on experts' recommendations as they have been fully integrated into their tx approaches. These analyses provide a real-time assessment of changing tx patterns among experts as well as HCPs. We will present analyses of cases entered in the 2018 online tool by HCPs and detailed comparisons of expert and the intended tx of clinicians using the tool. Disclosures Ghobrial: Takeda: Consultancy; Janssen: Consultancy; Celgene: Consultancy; BMS: Consultancy. Lonial:Amgen: Research Funding. Raje:Research to Practice: Honoraria; Takeda: Consultancy; AstraZeneca: Research Funding; Celgene: Consultancy; BMS: Consultancy; Merck: Consultancy; Janssen: Consultancy; Amgen Inc.: Consultancy; Medscape: Honoraria. Kumar:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; KITE: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding.

Abstract Abstract 1409 MYC is among the most prolific oncogenes in cancer, yet pharmacologic strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have undertaken to target c-Myc transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative co-activator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain-containing proteins as transcriptional regulatory factors for c-Myc. BET inhibition with JQ1 rapidly downregulates c-Myc transcription, followed by depletion of chromatin-bound c-Myc and genome-wide downregulation of Myc-dependent target genes. In translational model systems of multiple myeloma and Burkitt's lymphoma, both canonical MYC -dependent hematologic malignancies, JQ1 treatment produces a potent antiproliferative effect associated with cell cycle arrest. In multiple myeloma, adhesion to bone marrow stroma is associated with upregulation of BRD4, a BET bromomdomain coactivator protein. Inhibition of Myc function with JQ1 leads to impaired adhesion to stroma and cellular senescence, a classical Myc-specific phenotype. Mechanistically, JQ1 treatment depletes BRD4 from IgH enhancers, leading to prompt and robust downregulation of MYC transcription. In vivo efficacy of JQ1 in two disseminated models of multiple myeloma and in a Burkitt's lymphoma human xenograft establishes the therapeutic rationale for BET bromodomain inhibition in these diseases. Together, these studies identify a mechanistic rationale for targeting c-Myc in human cancer, and potentially other undruggable oncogenes driven by immunoglobulin rearrangement. Note: G.C.I. and J.E.D. have made equal contributions to this research; C.S.M. and J.E.B. are jointly senior authors Disclosures: Richardson: Millennium: Advisory Board; Celgene: Advisory Board; Johnson & Johnson: Advisory Board; Novartis: Advisory Board; Bristol Myers Squibb: Advisory Board. Ghobrial:Bristol-Myers Squibb: Research Funding; Millennium: Research Funding; Noxxon:; Millennium:; Celgene:; Novartis:. Anderson:Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Kung:Novartis Pharmaceuticals: Consultancy, Research Funding. Mitsiades:Millennium Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Kosan: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centocor: Consultancy, Honoraria; Amnis Therapeutics: Consultancy, Honoraria; PharmaMar:; OSI Pharmaceuticals: Research Funding; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding; Genzyme: Research Funding; Johnson & Johnson: Research Funding. Bradner:Acetylon Pharmaceuticals: Scientific Founder; SHAPE Pharmaceuticals: Scientific Founder; Tensha Therapeutics: Scientific Founder.

Abstract Waldenström macroglobulinemia (WM) is a lymphoid neoplasm associated with accumulation of lymphoplasmacytic cells in the bone marrow and an elevated monoclonal IgM in the serum. Although the genetic and molecular mechanisms behind the pathogenesis of WM are not well understood, losses of the long arm of chromosome 6 have been found as the only recurrent genomic aberration. These deletions, mostly starting at 6q21, are observed in upwards of 50% of patients and involve the majority of the clonal cells. To better characterize pathologic gene expression signatures in different types of WM at the molecular level (6q deleted versus not), we examined gene expression level changes of more than 20000 human gene probes in bone marrow tissues of clinically proven WM patients. We used Affymetrix HG-U133A v2 chips (n = 24). Clonal cells were purified using a combination of anti-CD138 and anti-CD19 Miltenyi™ magnetic microbeads. Based on cIgM-FISH results, we classified samples into two groups - 6q normal (ND) group and 6q deletion (D) group. We then proceeded to identify genes that showed differential expression between the two groups. We selected 165 differentially expressed genes (DEG) using SAM, and underwent further investigation of biological annotations of them using Gene Ontology™ (GO) terms. Among165 DEG, only 32 showed high level of expression in ND group compared to D group while 133 showed high level of expression in D group. As expected, all genes that reside in 6q region showed under-expression in D group, constituting 53% (17 genes) of genes under-expressed in D group (32 genes). According to GO analysis, the 6q-deletion-induced under-expression affects many biological processes, and is associated with a transcriptional upregulation of downstream genetic cascades. This likely indicates release of regulatory control by a tumor suppressor gene. Genes involved in cell growth and maintenance, cell communication and morphogenesis are also over-expressed in D group. Interestingly, a gene that is involved in innate immune response and inflammatory response shows under-expression in D group but is not accounted for by other mechanisms. In similar vein, a gene that is involved in negative regulation of cytokine biosynthesis showed over-expression in D group. We think these transcriptional characteristics can provide basic framework for differentiating subtypes of WM and search for genes associated with WM pathogenesis

Abstract Abstract 587 Background: The phosphatidylinositol 3-kinase/mammalian target of rapamycin (mTOR) signal transduction pathway controls cell proliferation and survival. Everolimus is an oral agent targeting raptor mTOR (mTORC1). The trial's goal was to determine the anti-tumor activity and safety of single-agent everolimus in patients with relapsed/refractory Waldenstrom's macroglobulinemia (WM). Patients and Methods: Eligible patients had measurable disease (IgM monoclonal protein >1000 mg/dL with >10% marrow involvement or nodal masses >2 cm), a platelet count ≥75,000 × 106/L, a neutrophil count ≥1,000 × 106/L, and a creatinine and bilirubin ≤2x laboratory upper limit of normal. Patients received everolimus 10 mg PO daily and were evaluated monthly. Tumor response was assessed after cycles 2 and 6 and then every 3 cycles until progression. Results: 50 pts were treated. The median age was 63 years (range, 43-85). The overall response rate (CR+PR+MR) was 70% (95% CI: 55-82%), with a PR of 42% and 28% MR. The median duration of response and median progression-free survival (PFS) has not been reached. The estimated PFS at 6 and 12 months is 75% (95%CI: 64-89%) and 62% (95%CI: 48-80%), respectively. Grade 3 or higher related toxicities were observed in 56% of patients. The most common were hematological toxicities with cytopenias. Pulmonary toxicity occurred in 10% of patients. Dose reductions due to toxicity occurred in 52% of patients. Conclusions: Everolimus has high single-agent activity with an overall response rate of 70% and manageable toxicity in patients with relapsed WM, and offers a potential new therapeutic strategy for this patient group. Disclosures: Ghobrial: Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Gertz:celgene: Honoraria; millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees. Richardson:Millennium Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Johnson and Johnson: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Keryx: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Treon:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau; Genentech: Honoraria, Research Funding, Speakers Bureau. Witzig:Novartis: Research Funding.