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Abstract ECOG, CALGB and SWOG (coordinating group) enrolled 899 patients with newly diagnosed MM to receive VAD x 4 (813 were considered eligible), followed by randomization to PBSC-supported MEL-TBI (n=261) versus VBMCP (n=255), using CTX 4.5g/m2 + G-CSF for PBSC mobilization in all patients. Responders to VBMCP or MEL-TBI were randomized to IFN (n=121) or no maintenance (n=121). With a median follow-up of alive patients of 60 mos, median EFS and OS from 1st randomization were 25 and 62 mos for MEL-TBI and 21 and 53 mos for VBMCP (p=.14 for EFS, p=.87 for OS). EFS and OS from IFN randomization were also identical at 23/59 mos for IFN versus 18/74 mos for observation (p=.2/.6). 87 VBMCP failures received salvage autotransplants, with a post-relapse median survival of 24 mos which was similar to the 27 mos observed among the remaining 83 VBMCP failures managed with standard salvage therapies. When examined in the context of 2 other randomized trials (IFM 90, MRC VII), OS from baseline of S932, limited to patients <60 yr (as in IFM 90 + MRC VII), is intermediate between the high dose arms of IFM 90/MRC VII and the standard dose arms of these two trials (randomization at baseline) (Figure 1). OS from the time of randomization to high or standard dose therapy on S9321 (again restricted to age <60 yr) was similar to OS timed from transplant for the high dose arms of IFM 90 and MRC VII. (Figure 2). Figure Figure The comparable outcome after high- versus standard-dose therapies on S9321, interpreted in the context of 2 other published randomized clinical trials, may relate to (1) higher dose intensity with VBMCP than with other standard regimens or (2) a detrimental effect of TBI in S9321. We conclude that with the current follow-up MEL-TBI is no better than VAD/VBMCP with the option of salvage transplant and that IFN maintenance is of no benefit in this maintenance setting. Data from this trial will be provided to MRC to be included in their on-going meta-analyses of high-dose therapies and IFN maintenance.

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 Abstract 429 Background: Patients with MM who have progressed after multiple novel agents have limited treatment options. Pomalidomide (CC4047) is the newest immunomodulatory (IMiD) agent that has demonstrated response rates of 63% in a Phase 2 trial of Pom/dex in patients with relapsed MM (in press, JCO). To better define the response rate in this group, we treated an additional cohort of patients who were truly resistant/refractory to lenalidomide. Methods: To be eligible, patients must:be refractory to lenalidomide therapy defined as relapsing on or within 60 days of stopping lenalidomide. Pomalidomide was given orally 2 mg daily on days 1–28 of a 28-day cycle. Dexamethasone was given orally at a dose of 40 mg daily on days 1, 8, 15 and 22 of each cycle. Response was assessed by the International Myeloma Working Group Uniform Response criteria. All patients received aspirin 325 mg daily as prophylaxis against DVT. Pom/dex regimen would be declared promising if we observed at least 4 confirmed responses out of 34 patients based on a single stage Fleming design. Results: 34 patients (23 male and 11 female) resistant/refractory to lenalidomide were enrolled. The median age was 62 years (range, 39-77). The median time from diagnosis to enrollment was 62 months (range 8-179). 12 patients had high risk molecular markers including loss of 17p (6 pts), t(4;14) (1 pt) and del 13 by cytogenetics (5 pts). 70% had ≥3 prior regimens including 38% with ≥5 prior regimens; 56% had prior thalidomide, 59% had prior bortezomib, and 68% had prior SCT. Toxicity (at least possibly related to treatment) was mild and consisted primarily of myelosuppression: grade 3/4 neutropenia (21%); grade 3 thrombocytopenia (9%); and grade 3 anemia (12%). Grade 3/4 non-hematologic toxicities occurred in 24%: pneumonia (3%), non-infectious pneumonitis (3%), hyperglycemia (3%), edema(3%), fatigue(9%) skin rash(3%). Neuropathy occurred in 18% (12% grade 1; 6% grade 2). No thromboembolic events have occurred so far. Nine (26%) patients were confirmed responders (all PR), surpassing the threshold for a positive trial, and 18 (53%) had SD. With a median follow-up of 4 months (range:0-8), 65% remain progression free; 88% remain alive; and 12% have died, all due to progressive MM. Conclusions: Pom/dex is active and well tolerated in this heavily pre-treated population of lenalidomide-refractory MM patients. The majority of patients have not progressed and objective responses are seen in 26%. This study confirms that pomalidomide has non-cross resistance with lenalidomide and offers benefit for patients who have relapsed after other novel therapies. Disclosures: Lacy: Celgene: Research Funding. Gertz:celgene: Honoraria; millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kumar:celgene: Honoraria; millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bergsagel:Genentech, Inc.: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Merck: Research Funding. Stewart:Takeda-Millenium, Celgene, Novartis, Amgen: Consultancy; Takeda, Millenium: Research Funding; Genzyme, Celgene, Millenium, Proteolix: Honoraria.

Abstract Abstract 616 Background: We have shown the three drug combination of cyclophosphamide, bortezomib and dexamethasone (CyBorD) to be highly active therapy in newly diagnosed myeloma while allowing stem cell harvest and transplantation (Reeder et al. Leukemia 2009; 23:1337-1341). However, twice weekly bortezomib and high dose dexamethasone have been shown to cause significant toxicities. In a prospective Phase II we modified CyBorD by using bortezomib once weekly and reducing the dexamethasone to “low dose dex” after the first two cycles in an attempt to reduce toxicity (cohort 2). We report the efficacy and toxicity of this modified regimen and compare the results to standard CyBorD (cohort 1). Patients and methods: 63 untreated symptomatic patients were enrolled on this Phase II trial (33 on cohort 1 and 30 on cohort 2). All are evaluable for response and toxicity. Treatment on cohort 2 consisted of oral cyclophosphamide 300 mg/m2 and IV bortezomib 1.5 mg/m2 days 1, 8, 15 and 22 and dexamethasone 40 mg by mouth days 1-4, 9-12, 17-20 in cycles 1 and 2, then 40 mg po days 1, 8, 15, 22 in cycles 3 and beyond. Cohort 1 used standard dosing of bortezomib and high dose dexamethasone throughout. Each cycle was 28 days. Acyclovir and a quinolone antibiotic prophylaxis were routinely used. Patients were evaluated for response and toxicity every cycle and were offered stem cell harvest and transplant after the 4th cycle but could continue up to 12 cycles maximum. Results: The median age for all 63 patients was 61 (36-74) years and 48% were female. Durie-Salmon stages were 47% II and 50% III and ISS stages I 43%, II 36%, and III 21%. Twenty-four percent of patients were genetic high risk (t(4;14) or deletion 17). Cohorts I and II were matched for age, gender and ECOG PS. ISS stages were higher in cohort 1 than cohort 2 (II/III 67% vs. 44%). The intent to treat (ITT) ORR (≥PR) for all 63 patients (cohorts 1 and 2) is 90% with 60% ≥VGPR. For cohort 2 (modified CyBorD) the ITT overall response (≥PR) was 93% (CR: 5, nCR: 7, VGPR: 6, PR: 10) with 60% ≥VGPR and 40% ≥ nCR. Those completing all 4 cycles of therapy had 92% ORR (24/26) and 65% (17/26) ≥VGPR. A comparison of response rates in cohorts 1 and 2 is shown in table 1. These high and comparable response rates were associated with fewer grade 3+ adverse events (AE's) than in cohort 1 (37% vs. 48%) and no grade 3 peripheral neuropathy (PN). The median follow-up for all 63 patients is 12.4 (2.8-29.2) months and 95% of patients are alive with 87% free from progression. Conclusions: Modified CyBorD with weekly bortezomib and reduced dexamethasone retains the high activity seen in standard CyBorD, but is less toxic and more convenient. Interestingly, this modified regimen allowed higher overall doses of bortezomib (5.2 vs. 6.0 mg/m2 per cycle) yet overall neuropathy rates were similar. This combination of a weekly alkylating agent with bortezomib and dexamethasone is well tolerated and produces high response rates in newly diagnosed patients with multiple myeloma. Disclosures: Reeder: Millennium (MPI): Research Funding; Celgene: Research Funding. Reece:Ortho Biotech: Honoraria, Research Funding. Chen:Celgene: Honoraria, Research Funding; Ortho Biotech: Honoraria. Hentz:Millennium (MPI): Research Funding. Fonseca:Otsuka: Consultancy; BMS: Consultancy; Amgen: Consultancy; Medtronic: Consultancy; Genzyme: Consultancy. Bergsagel:Merck: Research Funding; Amgen: Consultancy; Genentech: Consultancy; Celgene: Consultancy. Stewart:Proteolix: Honoraria; Millennium (MPI): Research Funding.

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 In approximately 50% of Multiple Myeloma (MM), translocation of non random genes into the IgH locus is believed to be the seminal event in the pathogenesis of the disease. Another 50% of cases are hyperdiploid and trisomic to certain autosomes, but do not harbor any translocation and thus are believed to have genetic alterations in unidentified loci. These observations, together with the finding of somatic mutations in FGFR3, N- and K-RAS, MYC, TP53 and CDKN2C/p18INK4C during the later stages of MM indicate that defective signaling pathways likely play a role in the progression of this malignancy. Of relevance then, in recent years recurrent mutations in kinases have frequently been implicated in malignancies including notably colon cancer and melanoma. We have therefore begun a comprehensive effort to sequence the tyrosine kinome for mutations and genetic polymorphisms in MM. Of particular interest are 90 receptor tyrosine kinases, 43 receptor tyrosine kinase-like, 5 receptor guanylate cyclase, and a lipid kinase. We report here results from our pilot high throughput exon scanning in 32 human MM cell lines which initially focused on 13 kinases known to be somatically mutated in human cancers. To date we have expanded this effort to assess 30 genes with sequence obtained which currently spans 80% coverage of the kinase domains of these genes. A total of 1.9 million bp have been sequenced across 235 exons. No recurrent mutations have been identified in the kinase domains of the cancer-associated genes: SRC, ILK1, KIT, GUCY2F, PDGFRA; in the genetic disorder-associated kinases: BTK, EPHA4, LAMA2, EPHB6, ACVR2; and in the mutation hot spots of frequently mutated cancer gene PIK3CA. A novel missense mutation is however identified upstream of the kinase domain of FGFR3 changing a Ser residue to Arg at codon 433. This residue, which is conserved across species and in FGFR1 and FGFR2, has not been reported in myeloma and in thanatophoric dysplasia, but the biological significance of this mutation is unknown. Several single nucleotide polymorphisms were identified in the coding regions of some of these kinases. Notably, synonymous polymorphisms in the kinase domains of EPHA4, PDGFRA3, KIT, MLK1, ILK1, NTRK3, FLT3, ABL1, FES, MLK4, and EGFR1 were identified that changed a codon but not the amino acid. More importantly, we identified non-synonymous amino acid variations in the kinase domains of EPHA4, GUCY2F, PTK2, and PIK3CA genes that are more likely to effect variability in the activity of these kinases. In summary, no recurrent kinase mutations of significance in Myeloma development or progression have yet been identified. Sequencing of the known cancer associated kinases in MGUS and hyperdiploid MM patients is now underway and our data set is being expanded to include all 139 kinases.

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 Background: Thalidomide and its analogue lenalidomide have high response rates among patients with newly diagnosed as well as previously treated myeloma. Pomalidomide (CC4047) is the newest immunomodulatory (IMiD) agent that has shown single-agent activity in phase I studies. We report on the first Phase 2 trial of pomalidomide combined with low dose dexamethasone (Pom/dex) in patients with relapsed or refractory multiple myeloma. Methods: 37 patients (21 male and 16 female) were enrolled. Pomalidomide was given orally 2 mg daily on days 1–28 of a 28-day cycle. Dexamethasone was given orally at a dose of 40 mg daily on days 1, 8, 15 and 22 of each cycle. Response was assessed by the International Myeloma Working Group Uniform Response criteria. All patients received aspirin 325 mg daily as prophylaxis against DVT. Results: The median age was 66 years (range, 40 – 88). All patients were evaluable for response and toxicity, and all analysis were done on intent to treat basis. All patients had received prior therapy; 38% had 3 prior regimens; 35% had 2 prior regimens and 27% had one prior regimen. 76% had previous autologous stem cell transplant (ASCT) and 24% had 2 prior ASCT. 62% had previous IMiD therapy. Toxicity was mild and consisted primarily of myelosuppression. Grade 3 neutropenia occurred in 31%; grade 3 thrombocytopenia 3%; grade 3 anemia 3 %. Other grade 3/4 toxicities seen in less than 5% pts included: diarrhea, atrial fibrillation, pneumonia, dehydration and renal insufficiency. 16 % had grade 1/2 neuropathy. No grade 3 neuropathy was seen and there have been no thromboembolic events. Thirty (81%) patients are continuing study treatment. Seven patients have discontinued treatment due to: disease progression (5), died on study (1) and the medical doctor’s discretion (1). Twenty three of 37 patients (62%) achieved an objective response to therapy; including 9 (24%) with VGPR; 14 patients (38%) with PR; 6 (16%) with stable disease. Objective responses were seen in 4 of 13 patients (29%) who were refractory to lenalidomide. Conclusions: Pomalidomide plus dexamethasone (Pom/dex) is highly active and well tolerated for treatment of relapsed/refractory multiple myeloma with an objective responserate of 62%, including a 29% response rate among patients who are lenalidomiderefractory.

Abstract Purpose: Flavopiridol is a cyclin-dependent kinase inhibitor with preclinical activity against multiple myeloma cells in vitro and ex vivo. It can induce apoptosis in non-cycling human cell lines, including RPMI-8226, a myeloma cell line. Suggested mechanisms of cytotoxicity include down regulation of anti-apoptotic regulators, direct binding to duplex DNA, disruption of transcription factor/DNA binding, upregulation of p53, inhibition of transcription, and inhibition of angiogenesis. A Phase II multicenter trial was conducted to determine the activity of flavopiridol in patients with relapsed or refractory multiple myeloma. Experimental Design: Eighteen patients were treated with 1 hour flavopiridol infusions (50 mg/m(2)/day) for 3 consecutive days every 21 days. Responses were assessed according to IBMTR criteria each cycle. Serial bone marrow aspirates were collected before and immediately after flavopiridol treatment to measure in vivo and ex vivo effects of flavopiridol on patient plasma cells. Immunoblotting for Mcl-1, Bcl-2, p53, cyclin D, phosphoRNA polymerase II and phosphoSTAT 3 was conducted on total cellular proteins isolated from sorted plasma/myeloma cells. Ex vivo assessment of flavopiridol sensitivity of freshly collected myeloma cells was performed for 5 patients pre-therapy. Results: Median age of patients ranged from 49 to 81 years, with a median of 65 years. Patients had received a median of 3 (range, 1–5) treatment regimens prior to enrollment. Sixty-one percent of patients were refractory to prior therapy and fifty-five percent had received prior high dose therapy with hematopoietic stem cell support. The immunoglobulin isotypes of the patients were as follows: IgG (10), IgA (4), light chain (3), and non-secretory (1). At enrollment, 13 patients had a beta-2 microglobulin greater than 3.5. Seven had a plasma cell labeling index greater than or equal to 1%. Four had an elevated LDH. The trial was stopped at time of interim analysis due to a lack of clinical efficacy. Of eighteen treated patients, 15 progressed (including 1 death on study) and 3 discontinued due to toxicity. All patients have ended the active treatment phase, and the mean number of cycles administered for all enrolled patients was 1.6 cycles (range 1–3). No objective responses were observed. The most frequent adverse events were leukopenia and diarrhea (83% each), followed by thrombocytopenia, nausea, and fatigue (61% each). Ex vivo flavopiridol treatment of pre-therapy patient plasma/myeloma cells led to cytotoxicity, but only after longer exposure times at higher flavopiridol concentrations than were anticipated to be achieved in vivo. Further, immunoblotting demonstrated no indication that known in vitro cellular effects of flavopiridol were recapitulated in vivo. Conclusions: Flavopiridol has little single-agent activity in relapsed or refractory multiple myeloma when administered at this dose and schedule, which is likely due to the inability to achieve biologically relevant concentrations in patients. .

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.