Bergsagel, Leif

Abstract Background: The combination of lenalidomide and dexamethasone (Rev/Dex) has been shown to be a highly effective therapy for multiple myeloma (MM). Over half of the patients with newly diagnosed MM achieve a complete response with prolonged therapy. More recently, a phase III trial demonstrated improved survival with use of lenalidomide and low dose dexamethasone with one year survival in excess of 90%. We report the initial results from a phase II trial combining lenalidomide and low dose dexamethasone with cyclophosphamide (CRd) as initial therapy of newly diagnosed MM. Methods: The trial was initiated in July 2006 and completed the target accrual of 33 patients by July 2007. The treatment protocol consisted of lenalidomide given orally at a dose of 25 mg daily on days 1–21 of a 28-day cycle. Dexamethasone (dex) was given orally at a dose of 40 mg on days 1, 8, 15, and 22 of each cycle. Cyclophosphamide at a dose of 300 mg/m2 was given on days 1, 8, and 15 of each cycle. Patients also received an aspirin once daily as thromboprophylaxis. Response was defined as a decrease in serum monoclonal (M) protein by 50% or higher and a decrease in urine M protein by at least 90% or to a level less than 200 mg/24 hours, confirmed at least 4 weeks apart. Results: The median age was 63 years (range, 44–79). Eight patients (24%) had ISS stage III disease. At this time, 19 of the 33 patients are evaluable for confirmed responses (i.e., off-study or completed at least 4 cycles of therapy). Of these, 2 achieved VGPR and 13 had a partial response giving an overall response rate of 79%. The response rate was affected by 5 of 19 patients who went off study within three cycles due to toxicities [interstitial nephritis (1 pt), multiple grade 3 toxicities including infection (1 pt) atrial fibrillation and infection (1 pt)] or alternative treatment [no response and possible renal toxicity (1 pt) and progression at 4 cycles (1 pt)]. Overall, hematological toxicity was the most common with grade 4 toxicity seen in 6 patients (20%). Non-hematological grade 3 or higher toxicities included fatigue (4 pts), thrombosis (3 pts) and renal failure (2 pts). One patient with thrombosis came off study for other toxicities, and the other two continued on study with anticoagulation. Thirteen patients (43%) had dose reductions of both cyclophosphamide and lenalidomide, most commonly due to hematological toxicity. So far, 12 patients have gone off study, 6 went to stem cell transplant, 3 due to adverse events, 1 due to disease progression and 2 patients went to alternate treatment. Conclusions: CRd has excellent activity in newly diagnosed myeloma with an estimated overall response rate of 79%. Response evaluation of the entire 33 patients will be presented at the meeting. We believe that the initial use of a 300 mg/m2 dose of cyclophosphamide resulted in 5 of the first 19 pts experiencing early toxicity and withdrawal. As a result, an expansion of the current trial is evaluating lower doses of cyclophosphamide (300 mg fixed dose). Full analysis of this study will be needed to determine if CRd can improve outcome compared to Rd.

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 Background: MYC rearrangement is a recurrent somatic event in MM and results in super-enhancers repositioning within 3Kb (centromeric or telomeric) of the MYC locus. BRD4, a member of the bromodomain family, binds H3K27Ac and regulates enhancers activity, RNA polymerase II transcription initiation and elongation and in particular it regulates MYC transcription in MM cells. Immunomodulatory drugs (IMiDs) promote the proteasomal degradation of IKZF1/3 and the transcriptional repression of MYC and IRF4. However the exact mechanisms through which IMiDs downregulate MYC are not well understood. Methods and Results: Using a extended panel of 12 MM cell lines we have first confirmed our previous observartion that IMIDs universally degraded IKZF1/3, however unlike in IMiDs-sensitive cell lines it failed to downregulate MYC in resistant cells. Therefore, this finding suggests that MYC transcriptional regulation rather than IKF1/3 degradation is the "bottleneck" in IMiDs anti-myeloma activity or resistance. In addition, RNA-seq of paired and serially collected primary myeloma cells with acquired resistance to IMIDsconfirmed the enrichment of genes signatures associated with MYC at the time of acquired resistance. Of note, in a library of t(4;14) MM cell lines lenalidomide significantly downregulates FGFR3 while the expression of WHSC1 was not affected. As in t(4;14) cells FGFR3 is driven by the 3' IGH enhancer while WHSC1 is under the control of the intronic Em enhancer, and in view of the known role of IKZF1 in CSR, we posit that IKZF1 regulates plasma cells enhancer activity and hence MYC expression. To validate this hypothesis we first established the genome-wide distribution of IKZF1 in MM1S, RPMI8226 and OPM2 cells by ChIP-Seq. This analysis revealed the distribution of IKZF1 across the genome, predominantly to intronic an intragenic loci with only ~ 8% or reads mapping within +/- 5 kb of genes TSS. Importantly IKZF1 was also enriched at known myeloma cells enhancers or super-enhancers such as IGLL5, TXNDC5, BCL2L1, and DUSP22. We also mapped BRD4 binding to the genome in 8226 and MM1S cell lines and confirmed previously described mapping to known B-cell enhancers. Of note IKZF1 nearly universally overlapped with all BRD4 enhancers and superenhancers loci. These findings lead us to propose that IKZF1 depletion in response to IMiDs treatment will result in enhancer destabilization and displacement of BRD4 from enhancer loci. Indeed, Chip-Seq analysis of IKZF1 and BRD4 genomic distribution pre- and post- treatment with lenalidomide (10 uM for 4 or 8h) in IMiDs sensitive cell lines significantly depleted IKZF1 and BRD4 at superenhancer loci. In contrast, in IMiDs resistant cell lines (RPMI8226) and despite IKZF1 depletion, BRD4 was largely retained at these loci. These findings suggest that in IMiDs sensitive cells, IKZF1 is the major transcription factor mediating BRD4 recruitment to enhancer and superenhancer loci. In contrast, in IMiDs resistant cells and similar to BET-bromodomain inhibitor (JQ1) resistance, transcriptional plasticity or hyper-phosphorylation of BRD4 attributable to the phosphatase PP2A inactivation may also be mediating IMiDs resistance. Indeed the levels of p-BRD4 determined by western blotting were significantly higher in IMiDs resistant INA6 and RPMI8226 cells compared to MM1S cells. Furthermore, treatment of INA6 cells with the PP2A activator phenothiazine significantly sensitized INA6 cells to lenalidomide. Lastly, in order to investigate whether IMiDs resistance was associated with acquired new MYC rearrangements with an "IKZF1_resisant enhancer," we performed mate-pair sequencing of IMiDs resistance patients (18 post acquisition of IMIDs resistance and 7 pairs of pre- and post- acquisition of IMIDs resistance). MYC structural rearrangements (indels, translocations, inversions) were detected using DELLY and MANTA. Of note several MYC rearrangement were identified including MYC duplication in 1 patient, MYC structural rearrangement (±3Kb of TSS) in 9 patients (enhancer partner: TXNDC5, IGHa2, NSMCE2, IGLL5) and a newly acquired IRF4-IGH a2 rearrangement in 1 patient with pre-existing MYC-IGLL5 rearrangement. Conclusion:We have demonstrated that IMIDs alter enhancer activity in myeloma cells by depleting IKZF1 / IKZF3 and disrupting BRD4 binding to enhancers and super-enhancers, stalling of RNA polymerase II and MYC transcription. Disclosures Neri: Celgene and Jannsen: Consultancy, Honoraria. Bergsagel:Amgen, BMS, Novartis, Incyte: Consultancy; Novartis: Research Funding. Bahlis:Onyx: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau; BMS: Honoraria; Celgene: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau.

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 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 3853 Poster Board III-789 Background Light chain (AL) amyloidosis is a clonal plasma cell disorder characterized by progressive multiorgan deposition of amyloid fibrils derived from immunoglobulin light chains. Treatment strategies for AL have typically targeted the clonal plasma cell and have paralleled that for myeloma, with recent clinical trials evaluating the role of novel agents such as immune modulatory drugs (IMiDs). Lenalidomide has activity in patients with AL, and given the synergy seen with alkylating agents, we designed a phase 2 clinical trial of cyclophosphamide added to lenalidomide and dexamethasone. Methods Patients (Pts) with newly diagnosed or previously treated AL, with adequate hematological and organ function, were enrolled between December 2007 and November 2008. Treatment consisted of 28-day cycles with Lenalidomide at 15 mg daily (days 1-21), oral cyclophosphamide 300 mg (days 1, 8, 15) and dexamethasone 40 mg weekly. All patients received aspirin for thromboprophylaxis. Hematological and organ responses were assessed using conventional response criteria. Results Thirty-five pts, 11 (31%) previously treated, with a median age of 64 yrs (range 44-82) were enrolled. Median time from diagnosis to trial enrollment was 1.6 mos (range; 0.1- 129) and 23 (66%) were alive at the time of analysis with a median follow up of 8.7 mos (range; 1.5-17.6). Three or more organs were involved in 10 (28%) pts with cardiac and renal involvement present in 22 (63%) and 27 (77%) respectively. Fifteen pts (44%) had advanced stage III disease by the troponin/ NT-ProBNP staging system. Seven (20%) pts had previous stem cell transplant, one pt had thalidomide and others had melphalan, dexamethasone or a combination of the two. The median duration on study was 6.5 months and the median number of cycles administered was 6, with 27 (77%) patients completing at least 3 cycles of therapy. Thirteen patients still remain on study, with the most common reason for study discontinuation being toxicity (10 pts, 45%). There were 15 (43%) and 24 (69%) episodes of Grade 3 or 4 hematologic and non-hematologic adverse events respectively. The most common non-hematologic toxicities seen were fatigue, edema, constipation and rash. Six patients died while on study due to sepsis (1), multiorgan failure (1), arrhythmia (1), or heart failure (3). The overall hematologic response rate was 60% (21 pts; CR (2), VGPR (10) and PR (9)); 79% for stage I and II pts and 40% for stage III patients. Among those patients receiving at least four cycles, the response rate was 20/23 (87%); 2CR, 10 VGPR and 8 PR. At least one organ response was documented in 8 (24%) patients. The median overall survival (OS) for all enrolled patients is 16.1 mos (95% CI; 10.8-NR). The median OS was not reached for stage I and stage II patients, and was 7 mos (95% CI; 4.6 - 16.1) for stage III pts. The median time to hematologic progression was 16.1 mos and to hematologic or organ progression was 6.7 mos (95% CI; 6.3, 16.1). The median OS among those with a hematologic response was 16.1 mos, and not reached among the group with a VGPR or better. Among the 31 patients with measurable FLC, those (n=24) with a ≥50% decrease in the difference between light chains had superior OS compared to the rest (16.1 mos vs. 4.3 mos). The six patients who died on study all had cardiac involvement and significantly higher NT-ProBNP/ BNP compared to the rest. Conclusion The combination of cyclophosphamide, lenalidomide and dexamethasone has significant activity in patients with light chain amyloidosis. Toxicity is manageable with dose reductions, but patients with advanced cardiac involvement are at a higher risk for toxicity and early discontinuation of therapy. Future trials with this combination should compare this regimen with other treatment approaches in selected patients, as well as pursue a dose-adapted strategy in those with advanced cardiac involvement. Disclosures: Kumar: CELGENE: Research Funding; MILLENNIUM: Research Funding; BAYER: Research Funding; GENZYME: Research Funding; NOVARTIS: Research Funding. Gertz:celgene: Honoraria; millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees. Dispenzieri:Celgene: Research Funding.

Abstract Abstract 3884 Poster Board III-820 Background and Objective Thalidomide/dexamethasone (thal/dex) combination has shown high activity in newly diagnosed multiple myeloma (MM) (Rajkumar SV. at al, J Clin Oncol 2006;24:431-436). In newly diagnosed patients, lenalidomide/dexamethasone (len/dex) has demonstrated superiority compared with high-dose dexamethasone alone (Zonder JA et al, Blood 2007;110:77). Although both thal/dex and len/dex are active in newly diagnosed MM, no randomized trial has been reported comparing these two regimens, and unfortunately none are ongoing or planned. We compared the efficacy and the toxicity of thal/dex and len/dex as primary therapy in 411 newly diagnosed MM patients treated at the Mayo Clinic. Patients and methods 411 consecutive patients seen at Mayo Clinic between 2001 and 2008, who received induction with thal/dex (n=183) or len/dex (n=288) were retrospectively studied. Thalidomide was given at a dose ranging from 100 mg/day to 400 mg/day continuously; the lenalidomide dose was 25 mg/day, days 1-21 on a 28-day cycle. All patients received dexamethasone, either at high-dose (40 mg orally on days 1-4, 9-12, and 17-20) or at low-dose (40 mg orally day 1, 8, 15, 22); each cycle was repeated every 4 weeks. In addition, a case-matched subgroup analysis that adjusted for age, gender and transplantation status was performed among patients who received high-dose dexamethasone comparing the thal/dex (n=72) and len/dex (n=72) groups. Outcome was analyzed on an intention-to-treat basis. The Chi-square or the rank sum tests were used to compare variables. Time-to-event analysis was performed using the Kaplan-Meier method and all comparisons were determined by the log-rank test and by the Cox proportional hazards model. Results On intention-to-treat analysis, of 411 patients, 80.3% versus 61.2% patients, respectively in the len/dex group and in the thal/dex group (p < 0.001), achieved at least a partial response. A significant difference between the 2 groups was found in terms of both very good partial response or better (34.2% vs 12.0%, p < 0.001) and complete response rate (13.6% vs 3.3%, p < 0.001). Duration of therapy was significantly longer in len/dex patients as compared to thal/dex patients: 36.7% vs 12.6% of patients who did not stop treatment to receive SCT were still receiving therapy at 1 year (p < 0.001).Time-to-progression was significantly better in the len/dex group than in patients receiving thal/dex (median 27.4 vs 17.2 months, HR 0.64; 95% CI 0.44-0.93; p = 0.019). Similarly, progression-free-survival was significantly higher in len/dex patients (median 26.7 vs 17.1 months, HR 0.69; 95% CI 0.48-0.98; p = 0.036). This translated into an increase in overall survival (OS) (median not reached for len/dex group compared to 57.2 months in thal/dex patients, HR 0.60; 95% CI 0.40-0.92; p = 0.018). Survival advantages were evident in patients presenting with International Staging System Stage (ISS) I/II (HR 0.57; 95% CI 0.32-1.00; p = 0.052) at diagnosis but not in patients with ISS stage III in subgroup analysis. There was a trend toward better OS in len/dex group compared to thal/dex group both for patients who underwent transplant and for patients who did not. A similar rate of patients experienced at least one grade 3 or higher adverse event (57.5% vs 54.6% in len/dex and thal/dex groups, respectively, p = 0.568). However, the toxicity profile was different in the two groups: major grade 3-4 toxicities of len/dex were hematological, in particular neutropenia (14% with len/dex vs 0.6% with thal/dex, p<0.001) while the most common toxicities in thal/dex were venous thromboembolism (15.3% vs 9.2%, p = 0.058) and peripheral neuropathy (10.4% vs 0.9%, p < 0.001). The data on efficacy and safety shown above were also confirmed in the subgroup case-matched analysis which included only high-dose dexamethasone patients. Conclusions This cohort study shows the superiority of len/dex in terms of response rates and survival, compared to thal/dex. The toxicity profile of the 2 regimens is different and len/dex treatment, although more active, was not associated with increased toxicity (grade 3-4 AEs). These data need to be carefully evaluated and randomized prospective phase III studies are necessary to confirm these results and determine the optimal initial therapy for MM. Disclosures: Off Label Use: research drugs in combination to standard care. Lacy:celgene: Research Funding. Gertz:celgene: Honoraria; genzyme: Honoraria; millenium: Honoraria; amgen: Honoraria. Kumar:celgene: Research Funding; millenium: Research Funding; bayer: Research Funding; Novartis: Research Funding; Genzyme: Research Funding. Dispenzieri:celgene: Research Funding. Bergsagel:amgen: Membership on an entity's Board of Directors or advisory committees; genetech: Membership on an entity's Board of Directors or advisory committees; merck: Research Funding; celgene: Membership on an entity's Board of Directors or advisory committees. Witzig:celgene: Research Funding. Fonseca:medtronic: Consultancy; genzyme: Consultancy; celgene: Consultancy; amgen: Consultancy; BMS: Consultancy; otsuka: Consultancy. Greipp:celgene: Research Funding.

Abstract Background: The combination of lenalidomide and dexamethasone (RD) has been shown to be highly effective as initial therapy for multiple myeloma. More recently, a phase III trial demonstrated improved survival with use of lenalidomide and low dose dexamethasone (Rd). Further improvements by incorporating other active agents may increase the depth of response and may improve long-term outcome. We report the results from a phase II trial combining lenalidomide and low dose dexamethasone with cyclophosphamide (RCd) as initial therapy for newly diagnosed MM. Methods: The trial was initiated in July 2006 and completed the initial target accrual of 33 patients by July 2007 (Cohort 1). Due to high rate of dose reductions in cohort 1, between July 2007 and May 2008 an additional 20 patients were enrolled with lower doses of cyclophosphamide (Cohort 2). The treatment protocol consisted of lenalidomide given orally at a dose of 25 mg daily on days 1–21 of a 28-day cycle. Dex was given orally at a dose of 40 mg on days 1, 8, 15, and 22 of each cycle. Cyclophosphamide at a dose of 300 mg/m2 was given on days 1, 8, and 15 of each cycle in cohort 1 and 300 mg on days 1, 8, and 15 of each cycle in cohort 2. Patients also received an aspirin once daily as thromboprophylaxis. Response was defined as a decrease in serum monoclonal (M) protein by 50% or higher and a decrease in urine M protein by at least 90% or to a level less than 200 mg/24 hours. Responses were assessed on intent to treat basis. Results: 53 patients were enrolled. The median age was 64 years (range, 37–82). 14 patients (29%) had ISS stage III disease. The median number of cycles was 4 (range: 1 – 20). The best response based on all enrolled patients on an intent to treat basis was 83%, including CR:1 (2%), VGPR: 20 (38%), PR: 23 (43%), and less than PR: 9 (17%). The CR plus VGPR rate was 40%. Among the 20 patients in cohort 2 who were enrolled after optimization of the cyclophosphamide dosing, 16 (80%) have already achieved a partial response or better with 17 patients in this cohort still receiving study treatment. Overall, 25 of the 53 enrolled patients have discontinued study treatment. Reasons for ending treatment are: completed study per protocol (14), disease progression (5), adverse event (3) and alternate treatment (3). This includes 3 patients from cohort 2, one completed treatment per protocol, one went to alternate therapy and one patient went off for progression. Hematological toxicity was the most common with grade 4 toxicity seen in 8 patients (only 1 from cohort 2). Non-hematological toxicities included neuropathy, diarrhea, cystitis, and thrombosis. There were 6 patients with grade 4 non-hematological toxicities (none from cohort 2) attributed to the drug (thrombosis, confusion, depression and sepsis). Thirteen patients had dose adjustments, most commonly due to hematological toxicity attributed to lenalidomide or cyclophosphamide. One patient has died off study, of intracranial hemorrhage. The median follow-up for the remaining patients is 7.8 months. Conclusions: The combination of lenalidomide, cyclophosphamide, and dexamethasone (RCd) has excellent activity in the setting of newly diagnosed myeloma. Seventeen patients continue on study and the response rates are likely to be higher with additional followup. Myelosuppression was a significant toxicity leading to dose reductions and cycle delays, and is lower with decreased dose of CTX in cohort 2 without any apparent loss of responses.

Abstract Abstract 604 We have conducted a pilot study to interrogate both CpG hyper- and hypomethylation in Multiple Myeloma (MM) using the Illumina GoldenGate (GG) BeadArrayTM high-throughput assay. Specifically, we utilized the Illumina Methylation Cancer Panel I, which spans 1,505 CpG loci selected from 807 genes including known tumor suppressor genes and oncogenes. To date we have profiled 50 CD138+ MM specimens, 4 MM cell lines and 6 normal CD138+ plasma cell samples (each representing a pool of 3-5 individuals) obtained from the bone marrow of patients who have undergone hip or knee replacement surgeries. We have obtained highly reproducible and reliable DNA methylation profiles with an average r2 of greater than 0.98 when the β values were compared between technical replicates. Further, we generated an internal control panel for the array by performing methylation specific PCR (MSP) to determine the methylation status of several genes found on the array (CDKN2A, E-Cadherin, MGMT, hMLH1) in 4 MM cell lines. A differential methylation analysis was conducted between normal plasma cells and MM samples using the BeadStudio Methylation Module v3.2. Differential scores for each CpG locus on the array were generated that correspond to significant differences between groups. Scores greater than (hypermethylation) or less than (hypomethylation) 13 represent ‘Diff scores' that are statistically significant (p values < 0.05) and this cut-off threshold is used to determine differentially methylated loci. Globally, when comparing MM to normal plasma cells, hypomethylation occurred at 42% of probes on the array, and was more common than hypermethylation, which occurred at only 21% of probes. Interestingly, 77% of probes situated in non-CpG islands were hypomethylated compared to 26% or probes in CpG islands. On the contrary hypermethylation occurred mainly in CpG islands (26% of probes) whereas only 7.4% of probes in non-CpG islands were considered hypermethylated. A total of 946 CpG loci were differentially methylated between normal plasma cells and MM. Among the most significantly differentially methylated CpG loci between normal plasma cells and MM were FZD9, ALOX12, MMP-3, MMP-8, and p16. Array comparative genomic hybridization (aCGH) data availabel for this same sample set was used to generate a hyperdiploid index for each sample by identifying those samples with whole chromosome gains at minimally three odd numbered chromosomes. There were 751 CpG loci that were differentially methylated between hypderdiploid and non-hyperdiploid tumors with WEE1, FRZB, ELK1, CLDN4, and SHH being among the most significant. We also applied the pathway enrichment analysis tool GeneGo v5 (Agilent) to these data, which identified cell adhesion/ECM remodeling, developmental pathways and several immune response pathways as the most significantly enriched pathways between normal and plasma cells with ephrin signaling pathways in the context of cell adhesion as the most significantly enriched pathway between hyperdiploid and non-hyperdiploid tumors. We hope to integrate methylation data with orthogonal datasets including gene expression, aCGH, and DNA sequence data being developed as part of the MMRF Genomics Initiative. Further validation of hits will also be followed up by functional studies to assess the true biological relevance of differentially methylated genes. This study represents one of the first methylome interrogation studies in MM and points towards global hypomethylation as possibly an important mechanism driving myelomagenesis. Our data also suggest that differential methylation of genes may be a significant determinant of hyperdiploid biology. Determining the set of critical genes and pathways based on the myeloma methylome is likely to lead to an improved understanding of biological mechanisms involved in myelomagenesis, ultimately leading to new and improved methods of diagnosing and treating this disease. Disclosures: No relevant conflicts of interest to declare.

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.