Fonseca, Rafaël

Abstract Abstract 3854 Poster Board III-790 Background Primary systemic amyloidosis (AL) is an incurable plasma cell disorder. Lenalidomide, especially in conjunction with dexamethasone, has been shown to be active in patients with multiple myeloma. Pomalidomide is a derivative of thalidomide that acts as an immunomodulator. Preliminary data in multiple myeloma demonstrate that this agent is highly active and well tolerated. Methods Eligible patients were accrued into an IRB approved treatment trial with pomalidomide and dexamethasone after signing informed consent. Pomalidomide (2 mg) and dexamethasone (40 mg) were scheduled to be taken orally, the former on a continuous schedule and the latter once weekly. All patients were instructed to take an aspirin daily. Patients were eligible if they had previously treated, biopsy proven, symptomatic AL. Patients were required to have measurable hematologic disease, defined as either a serum M-protein greater than 1 g/dL, a urinary M-protein greater than 200 mg/24-hours, or a serum immunoglobulin free light chain greater than 10 mg/dL. Other eligibility criteria included an ECOG PS>=2, adequate hematologic reserve (ANC>=1000/uL, platelets>=75/uL) and adequate renal function (creatinine <=2.5 mg/dL). They also were required to comply with contraceptive requirements. Patients were excluded if they had uncontrolled infection, another active malignancy, NYHA classification III or IV, a serum troponin T >0.1 ng/mL, >=grade 3 peripheral neuropathy, untreated active thrombosis, or other chemotherapy within 2 weeks of study enrollment. Thirty-four patients are to be enrolled to allow for 2 possible cancels and to provide 90% power to detect a true hematologic response rate of at least 20%. Between 11/24/08-7/28/09, 20 patients have been enrolled. Baseline characteristics and adverse events are available for 17 of the 20 enrolled patients. Results Median age was 63 years (range 52 78), with 60% male. The median time from diagnosis to study entry was 45 months (range 4.5-103). Eighty-two percent had cardiac involvement; 41% renal involvement; 18% peripheral nerve involvement; and 12% autonomic nerve involvement. The cardiac biomarker staging breakdown was: I, 13%; II, 56%; and III, 31%. Patients were heavily treated, with 100% having received prior alkylator (including prior transplant in 9), 44% prior lenalidomide, 31% prior thalidomide, and 37% prior bortezomib. Thirty-three percent of patients had non-hematological adverse events (AEs) >=grade 3 (severe) that were deemed to be at least possibly related to therapy. Thirty-three percent had >=grade 3 hematologic AEs. Neutropenia was most the most common severe hematologic AE; severe thrombocytopenia was observed in only 1 patient, and there was no severe anemia. The most common severe non-hematologic AE was fatigue, which was observed in 3 patients. The only other severe AEs, each of which was observed in one subject each, were pneumonia, weakness, dyspnea, and ascites. As of 8/14/09, 17 patients remain on active therapy. Median time on study is only 3.7 months (range 0.2-8.1). Three patients have discontinued therapy: two progressions; and one death 5 days into treatment, presumably due to cardiac amyloidosis. At the time of this writing response rates could not be calculated given the short follow-up, but hematologic responses have been observed, and details on efficacy will be presented at the meeting. Conclusions Pomalidomide is well tolerated in previously treated AL patients. More information about its activity will be provided at the meeting. Disclosures: Dispenzieri: Celgene: Research Funding. Gertz:Celgene: Honoraria. Kumar:Celgene: Research Funding. Rajkumar:Celgene: Research Funding. Witzig:Novartis: Research Funding. Greipp:Celgene: Research Funding. Fonseca:Celgene: Consultancy. Bergsagel:Celgene: Consultancy. Mikhael:Celgene: Research Funding. Roy:Celgene: Research Funding. Lacy:Celgene: Research Funding.

Abstract Abstract 127 The precise molecular mechanism of action and targets through which thalidomide and related immunomodulatory drugs (IMiDs) exert their anti-tumor effects remains unclear. A landmark paper has recently identified cereblon (CRBN) as a primary target of thalidomide teratogenicity. We hypothesized that this protein would also be required for myeloma (MM) cytotoxicity. To examine this issue we first tested 12 MM cell lines: 4 of which (OCIMY5, OPM1, SKMM2 and KMS12 PE) were very resistant to treatment with both lenalidomide and pomalidomide. Of these, OCIMY5 and OPM1 showed CRBN gene expression levels at the bottom 10% across the human MM cell lines. We next used CRBN shRNA lentiviral expression constructs to knock down CRBN in 5 human-derived myeloma cell lines (HMCLs) (KMS18, MM1.S, H929, OPM2 and JJN3). HMCLs infected with CRBN shRNAs have a significant reduction of CRBN expression and subsequent diminished cell viability (reduced 65–78%). Surviving MM cells with CRBN knock down demonstrate an acquired (essentially complete) resistance to lenalidomide when compared with controls but retained sensitivity to melphalan, dexamethasone and bortezomib. Gene expression changes induced by lenalidomide were dramatically suppressed in those CRBN depleted cells, e.g., OPM2 cells only showed 30 down regulated (3% of control) and 150 up-regulated genes (24% of control) after lenalidomide treatment, further emphasizing that CRBN is required for lenalidomide activity. We next compared by array comparative genomic hybridization the differences between MM1.S (lenalidomide sensitive) and its isogenic lenalidomide resistant line (MM1.S res), generated by culturing MM1.S in gradually increasing concentrations of lenalidomide. Only three structural abnormalities were noted differentiating the two lines one of which was deletion of CRBN. In fact the baseline MM1.S cell line has a minor population of 9% of cells with CRBN depletion which becomes dominant after lenalidomide treatment. However, further scrutiny of CRBN status in additional HMCLs and MM patients (irrespective of therapy and stage) suggested that copy-number abnormalities affecting this gene are uncommon events in MM, with 12% of HMCLs (7 out of 60) and only 1.2% of MM patients (3 out of 238) showing CRBN monoallelic deletion. Next, we examined by quantitative polymerase chain reaction (Q-PCR) ten MM patients who had become resistant to lenalidomide therapy. CRBN expression level by Q-PCR in 8 of 10 patient samples had a significant reduction (20% to 90% reduction compared to baseline) at time of drug resistance, suggesting CRBN expression as a potential biomarker to predict IMiD response. Notably however some clearly resistant patients have normal CRBN, demonstrating that while CRBN is absolutely required for response, multiple resistance mechanisms are likely present. By gene and protein expression downstream targets of CRBN were examined and included interferon regulatory factor 4 (IRF4) previously reported to also be a target of lenalidomide. In summary, we demonstrate here that CRBN is essential for IMiD activity and preliminary data support that low levels of CRBN predict for poor drug response. In addition, our data suggest that CRBN is a critical molecule but not the unique source of IMiD resistance. We suggest that the “IMiDs” be renamed Cereblon binding small molecules to more accurately reflect their mechanism of action. Disclosures: Orlowski: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract Abstract 606 Multiple myeloma is a complex malignancy with multiple underlying genetic events. Our group has spent considerable effort over the last 15 years to elucidate the genetic underpinnings of myeloma. Most recently, we used array-based comparative genomic hybridization (aCGH) as a discovery tool in 62 myeloma patients and 46 myeloma cell lines. In that preliminary screen using the Agilent 44B aCGH platform (∼70kb resolution) we identified a diverse array of abnormalities, which resulted in constitutive activation of the NF-kB pathways. That initial analysis concentrated on the 43 genes we identified as potential targets of the 13 homozygous deletion events detected in the patient samples. A pathway analysis of these genes revealed a single pathway involving TRAF3, TRAF2, BIRC2, BIRC3, and CYLD. This first analysis focused exclusively on abnormalities present in the patient samples as we worried some abnormalities identified exclusively in the cell lines might not be relevant to the pathogenesis of myeloma in patients. However, several abnormalities were equally or more frequent overall but occurred exclusively in cell lines including CDKN2C (14 samples), CDKN1B (4 samples), KDM6A/UTX (4 samples), RB1 (3 samples), TP53 (3 samples). Given the fact that KDM6A/UTX deletions were as frequent as many of the best-described tumor suppressors it seemed like a good candidate but in the absence of patient events or a known function at the time it was not prioritized for further study. Recently, as part of the Multiple Myeloma Research Consortium (MMRC) Genomics Initiative, we have completed the analysis of a cohort of 250 myeloma patient samples by aCGH using the Agilent 244A aCGH platform (∼15kb resolution) and gene expression profiling using the Affymetrix U133Plus2.0 genechip. In this cohort with a significantly improved aCGH platform we identified 17 genes that are recurrently inactivated by homozygous deletions including DIAPH2 (15 samples), CDKN2C (14 samples), TRAF3 (11 samples), CYLD (8 samples), BIRC2/3 (7 samples), KDM6A/UTX (6 samples), and RB1 (5 samples). Based on the significant improvement in resolution and data quality achieved with the Agilent 244A aCGH platform we rescreen all of the cell lines on this improved platform. This significantly changed the frequency of several homozygous deletions in this population with the most frequently targeted genes now being CDKN2C (20 samples), KDM6A/UTX (13 samples), DIAPH2 (7 samples), RB1 (4 samples), TP53 (4 samples), CDKN1B (4 samples), and TRAF3 (4 samples). Moreover, as part of the genomic characterization of a spontaneous myeloma mouse model that we have developed, Vk*-Myc, we have identified recurrent (∼50%) homozygous deletions of Kdm6a/Utx. Therefore, one of the genes most commonly targeted by a homozygous deletion in human and mouse myeloma is KDM6A/UTX. In late 2007 after we had identified the first patients with KDM6A/UTX deletions it was shown that UTX is a functional histone demethylase that removes methyl groups from histone H3 lysine 27 (H3K27me). Given the high incidence of deletions and the fact that MMSET, the overexpressed target gene of t(4;14) in myeloma, is predicted to methylate H3K27, H3K36, and/or H4K20 by evolutionary conservation we developed the hypothesis that myeloma is characterized by abnormalities that result in excessive H3K27me (typically a repressive chromatin mark). Given the extensive whole genome sequencing occurring in the MMRC genomics initiative we elected to focus our resequencing efforts on the cell lines exclusively. These studies identified an additional 4 samples with LOH and an inactivating mutation bringing the total percentage of inactivated cell lines to 33%. Clearly, in the expanded patient and cell line cohorts the inactivation of KDM6A/UTX is not independent of MMSET overexpression suggesting they may act independently or synergistically. We are currently attempting to identify the genes controlled by KDM6A/UTX inactivation to better understand the functional consequences of this highly recurrent event. However, in the mouse model unlike the patient or cell lines, the gene expression profiling has identified a gene expression signature that differentiates UTX inactivated and functional samples suggesting an oncogenic function of inactivation. Disclosures: No relevant conflicts of interest to declare.

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.