Bergsagel, P. Leif

Abstract Abstract 445 Background: Carfilzomib is a proteasome inhibitor that irreversibly binds its target and has a favorable toxicity profile that has shown significant activity in relapsed multiple myeloma (MM), leading to recent FDA accelerated approval. To achieve rapid and deep response in patients eligible for stem cell transplant, we combined carfilzomib with the regimen of cyclophosphamide-thalidomide-dexamethasone (CTD). We recently reported the results of the Phase I component of the trial (in which no MTD was reached) followed by the initial Phase II trial; however, with increasing evidence for the safe and effective use of higher doses of carfilzomib, we now report results from dose escalation extension of the Phase II trial. Methods: Newly diagnosed myeloma patients intended for stem cell transplant were eligible. All patients were treated on a 28 day cycle with Carfilzomib IV Days 1,2,8,9,15,16 (see Table 1 below for dosing per cohort) along with Cyclophosphamide 300 mg/m2 PO Days 1,8,15, Thalidomide 100 mg PO Days 1–28 and Dexamethasone 40 mg PO Days 1,8,15,22. We initially conducted a Phase I run in trial of 6 patients with no DLT observed before expanding to the Phase II portion of the study. The initial phase II regimen is shown below – as no DLTs were observed, we have now fully accrued to the Phase II dose level +1. Treatment was for 4 cycles with expected SCT post induction. The primary endpoint of the trial is the proportion of patients who have ≥very good partial response (VGPR) to treatment. All patients received herpes zoster prophylaxis and ASA daily. Results: A total of 38 patients have been accrued to the trial, 6 in the initial Phase 1, 21 in the initial Phase II, and the remaining at dose escalated cohorts. We are reporting the 27 patients who have completed therapy and will update with the dose escalated cohorts. Median age was 65 (range 27–74) and 52% were female. ISS Stage was advanced (II-III) in 56%. Best overall response rate during 4 cycles of CYCLONE at dose level 0 is 96%: CR 29%, VGPR 46%, PR 21% (1 pt achieved MR). Adverse events of grade 3 or higher at least possibly related to CYCLONE occurred in 12 (44%). Most commonly reported non hematological toxicities (all grades) included fatigue (67%), constipation (56%), lethargy (41%) somnolence (37%), malaise (30%) depressed level of consciousness (22%); however, grade 3/4 toxicities occurring in >5% were uncommon: thromboembolic event 11%) and muscle weakness (7%). Two cases of pneumonia required hospitalization. Eight patients (30%) developed grade 1 sensory neuropathy; no higher grade or painful neuropathy was evident. There were no cardiac events seen in greater than 5% of patients. Grade 3/4 hematological toxicities included neutropenia (15%) and lymphopenia (7%). All patients advancing to SCT successfully collected stem cells. One patient died on study from pneumonia. Conclusion: The 4 drug CYCLONE regimen is highly efficaceous with a response rate after only 4 cycles of 96% (75% ≥VGPR, 29% CR) at the current dosing level of carfilzomib IV 20/27 mg/m2 in newly diagnosed myeloma. Toxicities are manageable, with only grade 1 neuropathy and minimal cardiac or pulmonary toxicity. Increasing the dose of carfilzomib is feasible and updated results of dose escalated cohorts will be reported at 20/36 and 20/45 mg/m2. Disclosures: Bergsagel: onyx: Membership on an entity's Board of Directors or advisory committees. Stewart:Millennium Pharmaceuticals: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy.

Abstract We have previously proposed the dysregulation of the D type cyclins by chromosome translocations to the IgH locus or by not fully characterized mechanisms in hyperdyploid patients as a unifying genetic event in the pathogenesis of Monoclonal Gammopathy of Uncertain Significance (MGUS) and multiple myeloma (MM). However, no experimental data are available to evaluate the direct consequences of cyclin Ds overexpression in plasma cells. Similarly, activating mutations of KRAS have been identified in 15% of MM patients, but never in MGUS, and have been proposed to contribute to the progression of MGUS to MM, but this hypothesis has never been experimentally validated. We evaluated the contribution of CyclinD1 and KRAS to the development and progression of monoclonal gammopathy (MG) that develops spontaneously in C57BL/6 mice. We aged a cohort of EuCCND1 mice, and lslKRASV12 mice crossed to three different CRE expressing mice: IgG1CRE, CD19CRE or Vk* CRE (KRAS/CRE). A cohort of Vk* MYC mice, Vk* MYC x EuCCND1 (MYC/CD1) and triple transgenic mice (CD1/KRAS/CRE) was also analyzed. Mice were bled every 10 weeks beginning at 30 weeks, and the presence of MG was evaluated by serum protein electrophoresis. The median Time To M-Spike (mTTS) detection was 97 weeks in C57Bl/6, 63w in EuCCND1, 54w in KRAS/CRE, 54w in Vk* MYC, 38w in CD1/KRAS/CRE and 36w in MYC/CD1 mice. This indicates that acceleration of the development of MG occurs in all these strain of mice. However, we noticed that the spontaneous MG in C57BL/6 wt, EuCCND1 or KRAS/CRE rarely progresses (as determined by detection of a M-spike >7 g/L), while in the other mouse strains it frequently progresses, with a mTTS >7 g/L around 65w. A clear progression of MGUS to MM was observed only in Vk* MYC mice, and was further accelerated in MYC/CD1 mice, which are characterized by extra-medullary disease and shorter overall survival. In conclusion, these data support our hypothesis that Cyclin Ds dysregulation in plasma cells is the initial transformation step leading to MGUS. Further oncogenic events, like activating mutations of KRAS are required to expand the monoclonal plasma cells. Among the oncogenes examined MYC provides the greatest acceleration of the MG in wildtype and EuCCND1 mice, and results in a disease with the malignant features of MM. Disclosures: No relevant conflicts of interest to declare.

Abstract Through the recent elucidation of molecular and cellular processes in multiple myeloma (MM) pathogenesis that effect well-defined proliferative and survival pathways, a number of molecular targets for anti-cancer agents have emerged. This includes proteins involved in cell cycle regulation. A recent model of early MM pathogenesis proposes that at least one of the cyclin D genes is dysregulated in all myeloma tumors facilitating activation of cyclin dependent kinase (CDK)4 (or CDK6), and transition from G1 to S phase. It is hypothesized that this renders myeloma cells more responsive to bone marrow (BM) derived proliferative stimuli including IL-6 that upregulates MYC expression in MM cells and cooperates with cyclin D to promote transit to S-phase. In addition, recent studies have established a causative role of MYC dysreguation in progression from monoclonal gammopathy to myeloma. Thus an approach targeting deregulated cell cycle progression and MYC may prove effective MM therapy. Purvalanol is a potent and selective inhibitor of CDK1, an important regulator of cell cycle progression and has been reported to induce MYC-dependent apoptosis. Activity of purvalanol against a panel of 14 standardized, annotated myeloma cell lines was measured in a 48 hour MTT viability assay. IC50-s of all 14 cell lines ranged between 5 uM to 7.5 uM. Western blot and real-time PCR analysis revealed variability of MYC protein and mRNA levels between the 14 myeloma cell lines. We compared potential therapeutic activity of purvalanol against 3 myeloma cell lines (U266, H929, KMS12PE) with low, intermediate, and high levels of c-MYC expression, respectively. All three cell lines demonstrated G2-M growth arrest however marked apoptosis as determined by PI/annexin V staining was observed only in the cell lines with the highest level of MYC expression. Exposure of MM patient-derived BM mononuclear cells to purvalanol preferentially induced apoptosis of CD138+ MM cells. In contrast, purvalanol was minimally cytotoxic to the non-myeloma cell fraction and to non-transformed fibroblast cell lines (MRC5, IMR90 and W138) and failed to inhibit normal bone marrow-derived CD34 colony formation. Recent studies have demonstrated that the BM microenvironment offers protection of myeloma cells from chemotherapeutic agents by common mechanisms. Myeloma cell lines were cultured in 3 different conditions to mimic the tumor’s protective microenvironment. Soluble factors produced by the BM, IL6 and IGF-1 induced a modest degree of resistance to purvalanol while co-culture on BM stroma cells was completely protective. Studies evaluating the in vivo efficacy of purvalanol in a novel Vk*myc transgenic mouse model that spontaneously develops myeloma with a low proliferative index are ongoing and will be presented. The CDK1 inhibitor, purvalanol demonstrates broad single agent activity against myeloma cells with enhanced activity against MYC overexpressing cells. However, the strong protective effect of the BM microenvironment suggest that combination with agents that can reverse cell-adhesion mediated drug resistance may prove beneficial in optimizing the efficacy of this class of drugs for the treatment of MM.

Background The precise molecular mechanism and targets of lenalidomide specific anti-Multiple Myeloma (MM) activity remain largely unknown, although, cereblon (CRBN) has been identified as a central mediator of activity with IRF-4 acting as a downstream molecule. Lenalidomide resistance in MM cells which, despite CRBN depletion, are able to restore their IRF-4 levels suggests the existence of alternative pathways. Methods To measure apoptosis and gene expression analysis 1x 106cells were incubated with 10μm to 50μm of lenalidomide for 24 to 96 hours before they were examined by annexin-PI and FACS analysis. Gene and protein expression were measured by RT-PCR, western blot, and immunohistochemistry. For lenalidomide followed bortezomib serial studies, cells were incubated with 10μm of lenalidomide for 48 hours and later treated with bortezomib for 24 to 48 hrs. CRBN and immunoglobulin light chain knock-down were performed by lentiviral mediated shRNA. Results We found lenalidomide induces Endoplasmic reticulum stress (ERS) via accumulation of intracellular immunoglobulin proteins, but only in the presence of CRBN in a panel of MM cell lines. This accumulation of intracellular proteins resulted in ERS as documented by western blot and RT-PCR in OPM2 and MMIS cell lines. We next utilized OPM2 cells knocked down for CRBN and control, non-target (NT), and treated with different doses of lenalidomide. After three days of treatment an ERS response was occurring in OPM2-NT cells but not in CRBN knockdown cells. Using Western blot we found decreased XBP-1u and increased XBP-1s, ERS marker proteins, in CRBN positive (NT) cells after 72 hours of lenalidomide treatment, but not in CRBN knock-down cells. Another ERS marker GRP78/BiP was also clearly accumulated after lenalidomide treatment in CRBN positive cells. Inactivation of the tumor suppressor p53 by degradation is a mechanism utilized by cells to adapt to ERS has been reported. We also found that p53 protein down regulated after lenalidomide treatment in CRBN positive OPM2 and MMIS cells. We have analyzed other isogenic cell lines MM1.S (lenalidomide-sensitive) and isogenic MM1.Sres (lenalidomide-resistant) HMCLs and found that lenalidomide induces ERS in MM1.S but not MM1.Sres cells. CRBN is important for lenalidomide to induce immunoglobulin protein accumulation and ERS was further proved by over-expressing CRBN in OCI-My5 MM cell line and subsequent lenalidomide treatment. To test the effect of light chain accumulation as ER stressors, we knocked down immunoglobulin light chain lambda in human MM cell line OPM2. Knock-down of lambda light chain in OPM2 cells resulted in lenalidomide resistance and it induced endoplasmic reticulum stress mediated anti-myeloma activity. Our preliminary data reveals that lenalidomide mediated progressive ERS can positively enhance bortezomib induced apoptosis in an in-vitro MM model. We pre-treated myeloma cell lines with lenalidomide and subsequently treated with bortezomib. MM cells pre-treated with lenalidomide for two days and there after treated with bortezomib clearly shown increased sensitivity to bortezomib induced apoptosis than non-pre-lenalidomide treated cells. Conclusion Here we show that lenalidomide causes accumulation of immunoglobulin light chains, inducing progressive ER stress, resulting in anti-myeloma activity and that effect depends on the presence of CRBN. Proteasome inhibitors such as bortezomib also sensitize MM cells to endoplasmic reticulum stress-mediated apoptosis. Our data demonstrated the synergistic effect of lenalidomide and proteasome inhibitors, likely due to the augmented protein stress. This data support the use of serial therapy utilizing pre-lenalidomide treatment followed by bortezomib or carfilzomib treatment. Disclosures: Fonseca: Medtronic: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Genzyme: Consultancy; BMS: Consultancy; Lilly: Consultancy; Onyx: Consultancy, Research Funding; Binding Site: Consultancy; Millennium: Consultancy; AMGEN: Consultancy; Cylene: Research Funding; Prognostication of MM based on genetic categorization of the disease: Prognostication of MM based on genetic categorization of the disease, Prognostication of MM based on genetic categorization of the disease Patents & Royalties.

Abstract Abstract 5093 A relative increase in proliferation is now accepted as the most important measure in risk stratification of myeloma. Attempts to include measures of proliferation in different stratification strategies have been met with obstacles largely influenced by their practicality and economic viability in the clinical setting. The plasma cell labelling index (PCLI) is the gold standard used in myeloma; however it is still only used in a minority of centers across the globe. New surrogate markers of proliferation, such as serum biomarkers and transcription-based methods, are under investigation but few have evolved to become part of a uniform strategy in the stratification of myeloma. The gene-expression based proliferation indices (gPI) described by Bergsagel et al, Shaughnessy et al and Hose et al all show similar correlation with PCLI. Studies comparing all currently available methods are lacking. In a cohort of 70 patients, PCLI and gPI were compared with two immunohistochemistry-based methods of measuring proliferation, Ki67 and a more specific measure of S-phase entry – phosphorylation of minichromosome maintenance protein 2 (pMcm2) at serine 40/41, combined with dual staining of CD138. We observed a similar correlation of gPI wth PCLI to that previously reported (r value; CI – 0.43;0.21–0.61). Both Ki67 and pMcm2 staining showed a stronger correlation with PCLI than with gPI and PCLI (r value; CI – 0.81;0.71–0.88 and 0.86; 0.78 – 0.91 respectively). PCLI and gPI have already been shown to be an independent predictor of time to progression and overall survival. Although this sample size was too small to test for time to progression or overall survival and thus independence, a logical extrapolation would indicate similar independence and predictions on survival and time to progression. As Ki67 is routinely used for more proliferative cancers in the clinic, the extension of its use as part of a new stratification system in myeloma provides interesting possibilities for further exploration. Disclosures: O'Dwyer: Novartis: Honoraria, Research Funding.

Abstract Background Carfilzomib is a proteasome inhibitor that irreversibly binds its target and has been validated as both relapse and upfront therapies for multiple myeloma. To optimize pre transplant response, we combined carfilzomib with the widely accessible backbone of cyclophosphamide-thalidomide-dexamethasone (CTD). We previously reported results of the Phase I component of the trial (in which no MTD was reached); we now report results for the MTD and fully accrued expansion cohorts at the MTD of the CYCLONE trial NCT01057225. Methods Newly diagnosed myeloma patients intended for stem cell transplant were eligible. All patients were treated on a 28 day cycle with carfilzomib IV Days 1,2,8,9,15,16 (see Table below for dosing per cohort) along with cyclophosphamide 300 mg/m2 PO Days 1,8,15, thalidomide 100 mg PO Days 1-28 and dexamethasone 40 mg PO Days 1,8,15,22. The initial phase I/ II regimen is shown below – as no DLTs were observed, we increased dosing of carfilzomib to 36 and then 45mg/m2 and have now nearly fully accrued to the Phase II dose level +1 (27mg/m2) and expansion MTD (36mg/m2). Treatment was for 4 cycles with planned SCT post induction. The primary endpoint of the trial is the proportion of patients who have ≥very good partial response (VGPR) to treatment. All patients received herpes zoster prophylaxis and ASA daily. Results A total of 54 patients have been accrued to the trial, 6 in the initial Phase 1, 21 in the initial Phase II (CFZ 27mg/m2), and 27 at CFZ 36-45mg/m2. Median age was 63 (range 27-77) and 52% were male. ISS Stage was advanced (II-III) in 57% The overall partial response rate or better following 4 cycles of CYCLONE at dose level 0 or greater is 91%: CR 18%, VGPR 58%, PR 16%, MR 2%, SD 4%. One patient withdrew from the trial after cycle 1. At dose level 2, 3/7 patients experienced dose-limiting toxicity in cycle 1 including: grade 3 alanine aminotransferase increase (possibly related); grade 3 infusion reaction (probably related); and grade 4 heart failure with grade 3 dyspnea, atrial fibrillation, and fatigue (all possibly related). No dose-limiting toxicity was observed in 6 patients at dose level 1 and thus DL1 was deemed the MTD. Across dose levels, adverse events are fully evaluable in 48 patients. AEs of grade 3 or higher at least possibly related to CYCLONE occurred in 26 (54%) – 35% non hematological and 33% hematological. Most commonly reported non hematological toxicities (all grades) included fatigue (63%), constipation (46%), elevated creatinine (27%), hyperglycemia (27%), lethargy (25%) peripheral sensory neuropathy (25% - all grade 1), and somnolence (21%); however, grade 3/4 toxicities occurring in >5% were uncommon: hypertension (8%), thromboembolic event (6%) and hyperglycemia (6%). Three cases of pneumonia required hospitalization. There were no cardiac events seen in greater than 5% of patients. Grade 3/4 hematological toxicities included lymphopenia (23%) and neutropenia (17%). All patients advancing to SCT successfully collected stem cells. One patient died on study from pneumonia. 90% completed at least 4 cycles. With median follow-up of 16 months (range 0-64), 1-year PFS was 90% and 1-year OS was 98%. Conclusion The 4 drug CYCLONE regimen is highly efficacious with an overall response rate after only 4 cycles of 91% (76% ≥VGPR) at the dosing level of carfilzomib IV 20/27-36 mg/m2 in newly diagnosed myeloma. Toxicities are manageable, with only grade 1 neuropathy and minimal cardiac or pulmonary toxicity. CYCLONE is a combination that results in rapid and deep responses with minimal neuropathy prior to stem cell transplant. Comparative studies of this regimen with longer duration of therapy (as induction or consolidation) at the defined MTD should be considered. Disclosures: Mikhael: Celgene: Research Funding; Onyx: Research Funding; Sanofi: Research Funding. Reeder:Millenium: Research Funding; Celgene: Research Funding; Novartis: Research Funding. Costa:Otsuka: Research Funding. Stewart:Celgene: Honoraria; Millenium: Honoraria, Research Funding; Onyx: Research Funding.

Abstract Abstract 2881 Multiple myeloma (MM) is characterized by a remarkable heterogeneity in outcome following standard and high-dose therapies. Significant efforts have been made to identify genetic changes and signatures that can predict clinical outcome and include them in the routine clinical care. Gene expression profiling (GEP) studies have achieved a central role in the study of multiple myeloma (MM), as they become a critical component in the risk-based stratification of the disease. To molecularly stratify disease-risk groups, we performed GEP on purified plasma cells (obtained from the immunobead selection of CD138+ cells) from 489 MM samples in different stages of the disease using the Affymetrix U133Plus2.0 array. A total of 162 probes were analyzed using an in house automated script to generate a GEP report with the most used risk stratification indices and signatures, including the UAMS 70-gene, UAMS class, TC classification, proliferation and centrosome signature, and NFKB activation indices. In a subset of 57 samples, IgH translocations were analyzed using FISH and results were correlated with GEP data. A macrophage index was calculated and used as a surrogate measurement of non-plasma cell contamination. A total of 49 samples (10%) were excluded from subsequent analysis as the macrophage index indicated a significant contamination with no plasma cells, hence potentially compromising the results. The percent of high-risk disease patients identified from different signatures ranged from 26.4% by using high proliferation index to 28.8% with high centrosome signature and 31.3% with high 70-gene index. This percent of high-risk cases based on the 70-gene index is similar to what was found in Total therapy 2 (TT2) and TT3 cohorts. A third of patients (33.2%) were classified as D1 in the TC class, followed by 11q13 (19.3%), D2 (16.4%), 4p16 (13.8%), MAF (6.1%), None (4.7%), D1+D2 (4.5%) and 6p21 (1.8%). The NF-kB pathway was likely activated in 45.5% to 59.5% of cases, depending on the index used for its calculation. High proliferation index and high centrosome signature significantly correlates with 70-gene high-risk group (p<0.0001). Conversely, the activation of NF-kB pathway was not significantly different between high- and low- risk subgroups. TC subgroups D1 (p<0.0001) and 11q13 (p=0.01) were significantly more common in the 70-gene low-risk group. Similarly, TC subgroups 4p16 (p=0.0004), Maf (p=0.02) and D2 (p=0.05) were enriched in the high-risk group. Translocations t(4;14)(p16;q32), t(11;14)(q13;q32) and t(14;16)(q32;q23) were precisely predicted by the TC classification (100% correspondence). Cases with IgH translocations with unknown partner were classified in subgroups D1 (33%), D2 (25%), 6p21 (25%) and Maf (16%). Here we summarized the associations between the most significant gene expression indices and signatures relevant to MM risk-stratification. The multiple variables simultaneously analyzed in an automated way, provide a powerful and fast tool for risk-stratification, helping in the therapeutic decision-making. Disclosures: Stewart: Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding. Fonseca:Consulting :Genzyme, Medtronic, BMS, Amgen, Otsuka, Celgene, Intellikine, Lilly Research Support: Cylene, Onyz, Celgene: Consultancy, Research Funding.

Abstract Human myeloma cell lines (HMCL) provide both a discovery and validation platform to improve our understanding of the molecular pathogenesis of multiple myeloma. We have completed a project to characterize the underlying genetics of all commonly used HMCL with a primary goal of identifying appropriate model systems for findings from large scale patient studies like the multiple myeloma genomics initiative (MMGI). We first purchased all 37 commercially available HMCL from DSMZ, JCRB, ECACC, and ATCC. An additional, 32 non-commercial cell lines were obtained from Diane Jelinek and Leif Bergsagel. Subsequently each HMCL was thawed and cultured under strict parameters, which yielded cells for analysis, by comparative genomic hybridization, whole exome sequencing (Agilent SureSelect V4+UTR) and mRNA sequencing. The combination of these two assays provides a detailed map of the genetic complexity underlying this deadly disease. For variant discovery, alignment was done using BWA followed by realignment, quality recalibration and duplicate removal. High quality calls were identified using Mutect by requiring a minor allele frequency of 0.25 and a minimum depth of coverage of 10x. This identified a median of 81999 mutations per sample with 25th and 75thpercentile values of 76621 and 87440 respectively. To identify likely somatic mutations, we filtered out variants found in dbSNP, 1000 genomes project and the NHLBI Exome Sequencing Project when any of the SNPs within each database had a global minor allele frequency of greater than 0.1%. In addition, we included only those variants in gene space defined by RefSeq or GENCODE and targeted by Agilent exome capture V4+UTR. After these filtering steps a median of 1389 putative somatic mutations remained. To identify key events responsible for mutagenesis, we extracted non-synonymous variants in genes, which show substantially expression in RNASeq(FPKM>5). This identified a median of 371 and 135 variants per sample respectively. Overall, across all the 66 samples, there were at total of 9074 non-synonymous mutations in 4453 genes, which are significantly expressed. To identify potential oncogenes we focused on mutations that occurred at the same position in the genome or altered the same amino acid in expressed genes. This identified 63 genes including KRAS(n=16) and NRAS(n=11) as expected but also BRD9, CCNE1 and MAP2K3. To identify putative tumor suppressors, we looked for genes with homozygous mutations or genes with more than one mutation in the same sample. This identified 112 genes including TP53, TRAF3, FAM46C and DIS3 as expected along with LTB and KDM6B. Independently, to attempt to identify genes involved in MM pathogenesis, genes with mutations having a SIFT score of less than 0.05 which occurred in at least 5%(n>3) of the cell-lines were identified. This identified 185 genes of which 25 are in our putative oncogene list and 37 are in the tumor suppressor list. These study results provide the myeloma community with the unique resource of a fully characterized series of models that can be used for laboratory-based tests. To increase the utility of the dataset to the community all these data are freely available at www.keatslab.org or by request. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2992 Background: Patients with myeloma receiving initial therapy with lenalidomide-based regimens can have difficulty collecting adequate stem cells for an autologous transplant. Stem cell collection in these patients can be significantly enhanced by addition of the CXCR-4 antagonist plerixafor to the mobilization regimen. Plerixafor is typically given subcutaneously (SQ), with collection approximately 11 hours after injection to obtain maximum yield. Intravenous administration can potentially allow more rapid and predictable mobilization compared to the SQ route. We designed this trial to prospectively assess the efficacy of intravenous plerixafor administration in patients undergoing lenalidomide therapy. Patients and methods: Patients who were receiving initial therapy with a lenalidomide-based regimen and were undergoing stem cell collection within 12 months of their myeloma diagnosis were enrolled. Patients received GCSF at 10 μg/kg/day for 4 days followed by addition of plerixafor at 0.24 mg/kg/dose starting on day 5. Plerixafor was administered intravenously early morning (6–7 am) followed by apheresis beginning 4–5 hours later. Plerixafor was administered for a maximum of 4 days; but patients could continue apheresis beyond the 4th day at treating physician discretion. The aims of the study were to determine the proportion of patients reaching a stem cell yield of at least 3 million CD34 cells/kg by second day of apheresis, the safety and tolerability of intravenously administered plerixafor, and the overall rate of failure to mobilize (defined as less than 2.5 million CD34 cells/kg in 4 collections). Results: Thirty-seven patients were accrued between December 2009 – April 2011, and 36 were eligible for analysis. The median age was 61 years (range; 28–73); 61% were male. The median time from start of initial therapy to enrollment was 4.6 months (range; 2.6 to 11.1) and the median cycles of lenalidomide were 4 (range; 3–11). Thirty-four (94%) of the patients achieved at least 3 million CD34 cells/kg within 2 days of apheresis. The median CD34 cells/kg after 1 day of collection was 3.9 million (range; 0.7 to 9.2) and after two days of collection was 7.02 million (range: 1.1–16.5). Two patients failed the mobilization (<2.5 million CD34 cells/kg). There were no grade 3 or 4 non-hematological adverse events and one patient experienced grade 4 thrombocytopenia. The most common grade 1 or 2 adverse events seen were gastrointestinal, namely nausea, diarrhea and abdominal pain or bloating. Grade 1 dizziness was reported in 8 patients. Overall, the infusion was well tolerated. Conclusion: Intravenously administered plerixafor is an effective strategy for mobilization in this patient group with low rate of failure to mobilize. It is well tolerated with toxicity comparable to the SQ administration. It also offers flexibility in patient scheduling with a convenient schedule for early morning infusion followed by apheresis later in the day. Disclosures: Kumar: Merck: Consultancy, Honoraria; Celgene: Consultancy, Research Funding; Millennium Pharmaceuticals, Inc.: Research Funding; Genzyme: Research Funding; Novartis: Research Funding. Off Label Use: Use of the investigational agent MLN9708 for the treatment of previously untreated multiple myeloma. Lacy:Celgene: Research Funding.

Abstract To further understand the molecular biology of mucosa-associated lymphoid tissue (MALT) lymphoma, we conducted a comprehensive gene expression study on the Affymetrix platform using total RNA extracted from biopsy specimen of 35 well-characterized pulmonary MALT lymphoma samples and compared them to gene expression profiles of a range of B and T cell lymphoma, such as Burkitt’s lymphoma, diffuse large B-cell lymphoma, Waldenstrom macroglobulinemia, chronic lymphocytic leukemia, multiple myeloma, and peripheral T-cell lymphoma, and normal cellular counterparts, such as different B-cell and T-cell populations, normal plasma cells and lung tussue. Analytical strategies were adopted to negate tissue specific effect such that our results can be generalized to MALT lymphoma from other anatomical sites. We further analyzed the data using functional tools such as gene-set enrichment analysis and gene ontology. Network/pathway analysis was performed using Metacore. Unsupervised clustering of whole dataset (including other lymphoid malignancies and normal cellular counterpart) using genes with variable expression, showed that compared to other B-cell lymphoid malignancies, MALT lymphoma have a prominent T-cell signature. Using established gene expression of different B-cell compartment, we confirmed that MALT has a marginal zone B-cell and memory B-cell cell-of-origin signature. Using ANOVA with post-hoc pair-wise comparison, we identified 4 novel transcripts over-expressed in MALT but not other B-cell tumors, and validated one of these using immunohistochemistry. MALT lymphoma with t(11;18) or t(14;18) over-expressed genes enriched for pathways other than the NFKB pathway such as JAK-STAT and SRC signaling pathways, which may represent novel therapeutic targets. We identified a number of genes with ‘outlier’ expression including RARA and RGS13. However, translocation involving RARA and the immunoglobulin heavy chain locus was not detected by FISH, suggesting other mechanisms of deregulating gene expression were involved. Using unsupervised clustering of only the MALT samples, we found that the molecular heterogeneity within MALT is composed of a group characterized by MALT1 translocations and another group with plasmacytic differentiation. Interestingly, further refinement of this grouping can be achieved by clustering the samples based on ‘outlier’ gene expression. Samples with MALT1 translocations have high expression of RARA, samples with plasmacytic differentiation have high FKBP11 expression and samples with high RGS13 expression tend to have trisomy 3 and reactive follicles. Our study therefore identified novel molecular markers, deregulated pathways and genetic defects in MALT. Furthermore, subgroups with distinct pathological features and anchored by unique pattern of ‘outlier’ gene expression were identified.