ALBERT

Description: Key Points Presented are results from the phase 2 dose-expansion study of the combination of carfilzomib, lenalidomide, and dexamethasone (CRd). CRd was well tolerated with robust, rapid, and durable responses.

Description: Carfilzomib is a selective proteasome inhibitor that binds irreversibly to its target. In phase 1 studies, carfilzomib elicited promising responses and an acceptable toxicity profile in patients with relapsed and/or refractory multiple myeloma (R/R MM). In the present phase 2, multicenter, open-label study, 129 bortezomib-naive patients with R/R MM (median of 2 prior therapies) were separated into Cohort 1, scheduled to receive intravenous carfilzomib 20 mg/m2 for all treatment cycles, and Cohort 2, scheduled to receive 20 mg/m2 for cycle 1 and then 27 mg/m2 for all subsequent cycles. The primary end point was an overall response rate (≥ partial response) of 42.4% in Cohort 1 and 52.2% in Cohort 2. The clinical benefit response (overall response rate + minimal response) was 59.3% and 64.2% in Cohorts 1 and 2, respectively. Median duration of response was 13.1 months and not reached, and median time to progression was 8.3 months and not reached, respectively. The most common treatment-emergent adverse events were fatigue (62.0%) and nausea (48.8%). Single-agent carfilzomib elicited a low incidence of peripheral neuropathy—17.1% overall (1 grade 3; no grade 4)—in these pretreated bortezomib-naive patients. The results of the present study support the use of carfilzomib in R/R MM patients. This trial is registered at www.clinicaltrials.gov as NCT00530816.

Description: Background Twenty four-hour urine collection remains one of the most crucial tools for the diagnosis and monitoring of proteinuria and quantification of urinary monoclonal protein in patients with plasma cell dyscrasias (PCD) even though it is cumbersome. Nephrology guidelines recommend replacement of 24-hour urine collection with a spot urine protein/creatinine (Pr/Cr) ratio for the measurement of proteinuria. However, only limited data exist regarding accuracy of spot urine Pr/Cr ratio in patients with PCD and utility of this measure to estimate urinary paraprotein remains uncertain. We conducted a prospective study evaluating the role of spot urine Pr/Cr ratio in patients with PCD. Methods From 04/2012 to 05/2013, a total of 120 PCD patients were prospectively enrolled at Moffitt Cancer Center. Oliguric patients or those requiring dialysis were ineligible. Spot urine was collected on the same day as 24-hour urine collection. Spot urine Pr/Cr ratios were compared to 24-hour urine collections with regard to the amount of (1) total protein and (2) monoclonal protein (M-spike). Results Eighty four out of 120 patients (70%) were evaluable (17 did not provide spot urine samples; no Pr/Cr ratios available in 11; protein below the level of detection in 7; and one without 24-hour urine collection). Evaluable patients had a median age of 68 (range, 36 - 90) years, 63% were male, and 85% were Caucasian. Primary diagnoses were myeloma (n=74; 88%), amyloidosis (n=4), multiple plasmacytomas (n=2), solitary plasmactyoma (n=1) and MGUS (n=3). Prior therapies included chemotherapy in 95% and autologous transplant in 45%. Comorbidities included hypertension (48%), chronic kidney disease (30%), diabetes (15%), coronary artery disease (8%), atrial fibrillation (7%) and congestive heart failure (2%). The median serum creatinine was 0.9 mg/dL (range, 0.5 - 5.1). The median spot urine Pr/Cr ratio was 137 mg/g creatinine (range, 26 - 21,447). The median 24-hour urine protein was 120 mg/24h (range, 27 - 15,092), and the median urine M-spike was 1.2 mg (range, 0 - 8,099). More than half of spot urine samples were collected in the morning (59%). There were strong correlations between (1) spot urine Pr/Cr ratio and 24-hour total urine protein (Spearman correlation coefficient=0.91, p 〈 0.0001), and (2) spot urine Pr/Cr ratio and 24-hour urine M-spike (Spearman correlation coefficient=0.91, p 〈 0.0001). The timing of spot urine sample collection had no significant effects on the correlations (p = 0.46 and 0.95 by Wilcoxon rank-sum test). Conclusions Spot urine Pr/Cr ratio strongly correlates with degrees of proteinuria measured in 24-hour urine collection and may also predict the quantity of urine M-spike in non-oliguric PCD population. Spot urine Pr/Cr ratio is a potentially useful marker as a screening tool or an alternative semi-quantitative measure for rapid estimation of proteinuria which may be used for longitudinal patient follow-up in lieu of cumbersome repeated 24-hour collections. Disclosures: No relevant conflicts of interest to declare.

Description: Relapse of malignancy is frequent in patients transplanted with advanced diseases. There is a relationship between higher Bu exposure and lower risk of relapse, but also higher transplant-related mortality, and data indicated the maximum tolerated daily Bu AUC is 〈 6000 microMol/min when Bu is used in combination with cyclophosphamide (Cy). To reduce transplant-related mortality, Flu has been substituted for Cy in many transplant centers. We hypothesized that pharmacokinetics-targeting of IV Bu, which results in a predictable systemic exposure, would produce values of tolerable Bu AUC that are higher for BuFlu than for BuCy. Our standard HCT regimen since July 2004 consists of once daily IV BuFlu where fludarabine 40 mg/m2 is given intravenously daily for four days and each infusion is followed immediately by an initial IV Bu dose of 130 mg/m2. Pharmacokinetic analysis is performed after the first infusion of busulfan; the goal is to adjust the busulfan doses for days 3 and 4 to achieve an average exposure targeted to an AUC of 4500–5600 microMol/min. Outcomes for the first sixty-nine patients were analyzed. Thirty-nine of 63 (62%) evaluable patients had their Bu doses adjusted, 30 increased [median adjustment 50% (8 – 175%)], and 9 decreased [median 30% (11 – 60%)], while 24 (38%) pts had an AUC within the desired range without adjustment. Most of the patients (98%) had hematological malignancies and 73% had high CIBMTR risk, median age was 48 (22–68) years, donors were HLA-identical siblings (33), matched (23) or mismatched unrelated donors (13). With a median patient follow-up of 392 (248 – 707) days, the non-relapse mortality was 4% at 100 days and 17% at one year. The K-M estimates of survival and event-free survival are 88%±4% and 83%±5% at 100 days, and 61%±6% and 51%±6% at 1 year, respectively. Subsequently, as part of a prospective, targeted AUC-dose finding trial, 20 patients received an initial Bu dose of 170 mg/m2 with subsequent doses targeted to achieve a 4-day average AUC of 5400–6600 microMol/min. Pharmacokinetic analysis was performed after the first and fourth infusion of busulfan. All patients had hematologic malignancies, 65% had high CIBMTR risk, median age was 48 (22 – 61) years, donors were HLA-A, B, C, DRB1, DQB1 matched siblings (11) or matched unrelated donors (9). Thirteen (65%) patients had their doses adjusted, six had it increased [median 37.1% (35.6 – 61.9 %)] and seven decreased [median 41.6% (18.3 – 55.2%)], while seven patients had an AUC within the desired range without adjustment. With a median follow-up of 111 (range 21–312) days, the 100-day K-M estimates of survival are 88%±8% and event-free survival 83%±9%. The complete observation of 100-day safety, relapse and survival will be presented at the meeting and data will determine further AUC escalation.

Description: This study was designed to determine if macrophage inhibitory protein-1 (MIP-1), a recently described osteoclast (OCL) stimulatory factor,1 was present in marrow from patients with multiple myeloma (MM) and possibly involved in the bone destructive process. MIP-1, but not interleukin-1β (IL-1β), tumor necrosis factor-β (TNF-β), or interleukin-6 (IL-6), messenger RNA was elevated in freshly isolated bone marrow from 3 of 4 patients with MM compared to normal controls. Furthermore, enzyme-linked immunosorbent assays of freshly isolated bone marrow plasma detected increased concentrations of hMIP-1 (range, 75-7784 pg/mL) in 8 of 13 patients (62%) with active myeloma, in 3 of 18 patients (17%) with stable myeloma (range, 75-190.3), as well as in conditioned media from 4 of 5 lymphoblastoid cell lines (LCLs) derived from patients with MM. Mildly elevated levels of MIP-1 were detected in 3 of 14 patients (21%) with other hematologic diagnoses (range, 80.2-118.3, median value of 96 pg/mL) but not in normal controls (0 of 7). MIP-1 was not detected in the peripheral blood of any patients with MM. In addition, recombinant hMIP-1 induced OCL formation in human bone marrow cultures. Importantly, addition of a neutralizing antibody to MIP-1 to human bone marrow cultures treated with freshly isolated marrow plasma from patients with MM blocked the increased OCL formation induced by these marrow samples but had no effect on control levels of OCL formation. Thus, high levels of MIP-1 are expressed in marrow samples from patients with MM, but not in marrow from patients with other hematologic disorders or controls, and support an important role for MIP-1 as one of the major factors responsible for the increased OCL stimulatory activity in patients with active MM.

Description: Abstract 4158 Background: High-dose chemotherapy followed by autologous hematopoietic cell transplantation (HCT) is a preferred primary treatment approach for those patients who have adequate organ function and performance status. We previously reported the use of melphalan + bortezomib conditioning regimen for tandem transplants in a different group of patients with refractory myeloma (Alekshun, et al. ASH 2007). In this study, we examine the effects of adding bortezomib to standard high-dose melphalan in patients with newly diagnosed myeloma who have chemosensitive disease. Methods: Thirty five newly diagnosed multiple myeloma patients with responsive disease (partial response (PR) or better) to induction therapy were enrolled to the study from January 2010 to June 2011. Patients received high-dose melphalan conditioning at 100 mg/m2 IV for 2 days, immediately followed by 1 dose of bortezomib at 1.3 mg/m2 (Mel/Vel). Those patients who achieved PR post induction were considered for tandem autologous transplant with Mel/Vel conditioning. Results: To date, 35 patients received autologous HCT conditioned with Mel/Vel, and 32 are evaluable for response. Median age is 56 years (range, 25 – 72) with the following disease characteristics: IgG (n=21), IgA (n=8), IgD (n=1), light chain (n=5). High-risk cytogenetics/FISH were seen in 13 patients (37%). Median beta-2 microglobulin was 3.5 (range, 1.3 – 34.8). Sixteen patients received bortezomib-based induction, 9 patients received lenalidomide-based therapy and 10 received both bortezomib and lenalidomide. Median time from initiation of induction to transplant was 221 days (range, 134 – 664). Responses to induction therapy were stringent CR (n=10), CR (n=4), very good partial response (VGPR) (n=13), and PR (n=8). Median CD34 cell dose is 4.86 × 106/kg (range, 2 – 20.08). Neutrophil engraftment was achieved after a median of 11 days (range, 10 – 14) and platelet engraftment occurred after a median of 16 days (range, 11 – 19). Two patients received tandem HCT. Best responses post transplant were sCR (n=19), CR (n=3), VGPR (n=7), PR (n=1) and progressive disease (n=2). The one year progression-free survival (PFS) estimate is 77% (95% CI 0.59 – 0.95) and one year overall survival (OS) estimate is 96% (95% CI 0.88 – 1.00) with a median follow-up of 279 days (range, 47 – 515). We did not detect significant differences in OS (p=0.69) stratified by cytogenetic/FISH risk status. Conclusions: The combination of bortezomib and high-dose melphalan (Mel/Vel) as conditioning regimen for autologous HCT is well tolerated and appears to improve responses post transplant. This early result is encouraging and the regimen will be examined in expanded cohort of patients. Disclosures: Baz: Millenium: Research Funding, Speakers Bureau; Celgene: Research Funding. Alsina:Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Allergan: Research Funding.

Description: Abstract 813FN2 Introduction: Carfilzomib is a next-generation proteasome inhibitor that selectively and irreversibly binds to its target, resulting in sustained inhibition absent of off-target effects relative to bortezomib. Carfilzomib has demonstrated durable anti-tumor activity and an acceptable tolerability profile in patients with multiple myeloma (MM). Final results will be presented for the bortezomib-naïve group of PX-171-004, a phase 2 study of single-agent carfilzomib in patients with relapsed and/or refractory MM. Herein we report the most recent data analysis available at time of abstract submission. Updated final results for the bortezomib-naïve group of PX-171-004, including OS data, will be presented at the meeting. Methods: Patients received either 20 mg/m2 for all treatment cycles (Cohort 1) or a stepped-up dose-escalating regimen of 20 mg/m2 for Cycle 1 and 27 mg/m2 for all treatment cycles thereafter (Cohort 2). Carfilzomib was administered over 2–10 minutes on Days 1, 2, 8, 9, 15, and 16 of every 28-day cycle, for a maximum of 12 cycles. The primary endpoint was the best overall response rate (ORR; [CR + VGPR + PR]) determined according to the IMWG Uniform Response Criteria. Secondary endpoints included the clinical benefit response rate (CBR; [ORR + MR per EBMT criteria]), progression-free survival (PFS), time to progression (TTP), duration of response (DOR), OS, and safety. The result of efficacy analysis from disease assessment by the Independent Review Committee is presented in this abstract. Results: 127 of 129 enrolled bortezomib-naïve patients were evaluable for response. Prior therapies included thalidomide (59%), lenalidomide (59%), alkylating agents (81%), and stem cell transplant (73%). Patients had received a median of 2 prior regimens (1 in 54 patients, 2 in 40 patients, 3 in 28 patients, and ≥4 in 7 patients). 84 patients (65%) were disease refractory to their most recent therapy, defined as ≤25% response or progression during or within 60 days after completion of therapy. The median duration of carfilzomib treatment is 7 cycles (range 1−12) in Cohort 1; 8 patients were receiving drug as of February 2011 in Cohort 2 with a median treatment of 6.5 cycles (range 1−13) at that time. Best ORR was 42% in Cohort 1 and 52% in Cohort 2. Median TTP was 8.3 months and median DOR was 13.1 months in Cohort 1. The median TTP and DOR for Cohort 2 have not been reached at the time of this interim analysis; the lower bound of the 95% CI for the median TTP was 10.2 months, and 84% were estimated to have DOR ≥1 year at the time of data cutoff. Higher response rates for Cohort 2 compared with Cohort 1 do not appear to be associated with higher toxicities. Patients with unfavorable cytogenetic characteristics (≥1 abnormality) per mSMART criteria had an ORR of 37% and CBR of 42% compared with 50% and 65%, respectively, for patients with no abnormality. The most common treatment-emergent adverse events (AEs), regardless of relationship to carfilzomib in Cohorts 1 and 2, respectively, were fatigue (71%, 54%), nausea (54%, 43%), anemia (46%, 37%), and dyspnea (49%, 33%). These were primarily ≤Grade 2 in severity. The most common Grade 3/4 AEs were anemia (15%), lymphopenia (15%), thrombocytopenia (13%), pneumonia (12%) and neutropenia (12%). Treatment-emergent peripheral neuropathy (PN) was mild and infrequent (16%). Only 1 case of Grade 3 PN (0.8%) was observed. Overall, 38 patients (30%) completed 12 cycles and 22 of these patients continued to receive carfilzomib therapy under a safety extension protocol (PX-171-010), an ongoing, multicenter, open-label phase 2 study to monitor long-term use of single-agent carfilzomib. Conclusions: To date we have seen robust and durable single-agent activity for carfilzomib in bortezomib-naïve patients with relapsed and often refractory MM with an ORR of 42−52% and a CBR of 59−63% from 2 separate dose cohorts in a population wherein 92% received an immunomodulatry drug and 73% had undergone an autologous stem cell transplant previously. These data are suggestive of a dose–response relationship and are being further evaluated in the exploratory phase 1b/2 study PX-171-007. Carfilzomib was associated with minimal PN and excellent long-term tolerability, with nearly one-third of patients completing 12 cycles and 22 of these continuing treatment beyond 1 year in the extension protocol PX-171-010. Disclosures: Vij: Onyx Pharmaceuticals: Consultancy, Research Funding; Celgene: Research Funding, Speakers Bureau; Millennium: Speakers Bureau. Kaufman:Millenium: Consultancy; Onyx Pharmaceuticals: Consultancy; Novartis: Consultancy; Keryx: Consultancy; Merck: Research Funding; Celgene: Research Funding. Jakubowiak:Ortho Biotech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Exelixis: Consultancy, Honoraria. Wang:Onyx Pharmaceuticals: Research Funding. Jagannath:Millennium: Honoraria; Celgene: Honoraria; Onyx Pharmaceuticals: Honoraria; Merck: Honoraria; OrthoBiotec.Imedex: Membership on an entity's Board of Directors or advisory committees; Medicom World Wide: Membership on an entity's Board of Directors or advisory committees; Optum Health Education: Membership on an entity's Board of Directors or advisory committees; PER Group: Membership on an entity's Board of Directors or advisory committees. Kukreti:Celgene: Honoraria. Alsina:Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Allergan: Research Funding. Belch:Celgene: Research Funding; Onyx: Research Funding. Gabrail:Millennium: Research Funding. Matous:Celgene: Speakers Bureau; Millenium: Speakers Bureau; Cephalon: Speakers Bureau. Vesole:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Speakers Bureau. Orlowski:Onyx Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kunkel:VLST biothech: Consultancy; Threshold: Consultancy; Onyx Pharmaceuticals: Consultancy. Wong:Onyx Pharmaceuticals: Employment, Equity Ownership. Stewart:Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding.

Description: INTRODUCTION: Perifosine (Peri) a novel, oral signal transduction modulator with multiple effects including inhibition of Akt and activation of JNK, has demonstrated clinical activity when combined with dexamethasone (Dex) in patients (pts) with relapsed/refractory MM (ASH 2007 #1164). Lenalidomide (Revlimid®, Rev) a novel, oral immunomodulatory drug has single-agent activity against MM and additive effects when combined with Dex. We previously reported encouraging safety data and observed clinical activity of the oral triplet combination in the first 12 pts (ASH 2007 # 1169). We now report the phase I results of this study which aimed to determine the MTD and activity of Peri + Rev + Dex, as an oral combination in pts with relapsed or refractory MM. METHODS: Four cohorts (≥6 pts each) were planned, dosing Peri at 50 or 100mg (daily), Rev 15 or 25mg (d 1–21) and Dex 20mg (d 1–4, 9–12 and 17–20 for 4 cycles, then 20 mg d 1–4) in 28-d cycles. To limit dex-related toxicities, the protocol was amended to use weekly dex (40 mg), applying to cohorts 3, 4, and the MTD cohort. Toxicity was assessed using NCI CTCAE v3.0; DLT was defined as grade (G) 3 non-hematologic toxicity, G4 neutropenia for 5 d and/or neutropenic fever, or platelets 1 occasion despite transfusion. Response was assessed by modified EBMT criteria. Pts had to have received at least 1 prior therapy and no more than 4. Pts refractory to Rev/Dex were excluded. RESULTS: 32 pts (17 men and 15 women, median age 61 y, range 37 – 80) were enrolled; 6 pts in cohort 1 (Peri 50mg, Rev 15mg, Dex 20mg); 6 pts in cohort 2 (Peri 50mg, Rev 25mg, Dex 20mg); 8 pts in cohort 3 (Peri 100mg, Rev 15mg, Dex 40mg/wk); 6 pts in cohort 4 (Peri 100mg, Rev 25mg, Dex 40mg/wk) and 6 pts at MTD (Cohort 4). Median prior lines of treatment was 2 (range 1–4). Prior therapy included dex (94%), thalidomide (83%), bortezomib (47%), and stem cell transplant (47%). 37% of pts had progressed on prior Thal/Dex. Two pts did not complete one full cycle (noncompliance and adverse event not related to study drugs – both in cohort 3) and were not included in the safety and efficacy analysis. Of the 30 pts evaluable for safety, the most common (≥ 10%) grade 1/2 events included nausea (13%); diarrhea (17%); weight loss (17%); upper respiratory infection (23%); fatigue (30%); thrombocytopenia (20%); neutropenia (20%); hypophosphatemia (23%); increased creatinine (23%); anemia (36%); hypercalcemia (47%). Grade 3/4 adverse events ≥ 5% included neutropenia (20%); hypophosphatemia (17%); thrombocytopenia (13%); anemia (10%), fatigue (7%). There was one reported DLT in cohort 3 (Nausea). Rev was reduced in 8 pts, Peri reduced in 8 pts and Dex reduced in 6 pts. All 30 pts in the analysis are evaluable for response, with best response as follows: Response: N = 30 N (%) Duration (wks) ORR (≥PR) stable disease: 〈 25% reduction in M-protein Near Complete Response (nCR) 2 (7%) 79+, 15+ Very Good Partial Response (VGPR) 3 (10%) 62+, 34, 17 15 (50%) Partial Response (PR) 10 (33%) 26+ (range 11 – 54+) Minimal Response (MR) 6 (20%) 17+ (range 9 – 30+) Stable Disease (SD) 7 (23%) 14+ (range 8 – 19) Progression (PD) 2 (7%) 8, 4 As of August 2008, the median time to progression (TTP) for pts achieving ≥ PR is 31 wks (range 11–79), and the median TTP for all 30 pts is 23 wks (range 4 – 79). The median TTP has not been met with 11/30 pts continuing on active treatment. Survival for all study pts remains at 90%. CONCLUSIONS: Patients have tolerated Peri + Rev + Dex well with manageable toxicity, and with encouraging clinical activity demonstrated by an ORR (≥ PR) of 50%. Accrual is complete and the final analysis for all pts will be presented at the meeting.

Description: Rituximab is a chimeric murine/human IgG1 kappa anti-CD20 monoclonal antibody that has revolutionized treatment of patients with CD20+ malignancies because of its non-cross resistance with chemotherapy and favorable toxicity profile. There is limited data evaluating the feasibility of combining rituximab with conditioning chemotherapy (or chemo-radiotherapy) for patients undergoing allogeneic hematopoietic cell transplantation (allo-HCT) for advanced CD20+ expressing malignancies. Twelve patients (M= 7; F=5), with a median age of 54.5 (41–66) years, underwent allo-HCT for the following diagnoses (disease status at time of transplantation) [chronic lymphocytic leukemia (CLL)= 7 (CR2=2; PR2= 3; ≥PR3=2); mantle cell lymphoma (MCL)= 3 (CR1=1; ≥PR2= 2); follicular NHL=1 (CR3=1), diffuse large B-cell (DLBC)=1 (〉PR3=1)]. Donors were as follows: matched-related donors (MRD) = 7; matched-unrelated donors (MUD) =3; mismatched unrelated donor (MMUD)= 2. Fludarabine-based conditioning regimens comprised fludarabine plus targeted doses of intravenous busulfan (FLU-BU=8), or total-body irradiation (FLU-TBI=3), or cyclophosphamide (FLU-CY=1). Anti-thymocyte globulin (ATG) was administered in two cases of MMUDs. GVHD prophylaxis consisted of tacrolimus plus mycophenolate mofetil= 8, or methotrexate = 4. Nine (75%) of 12 patients received rituximab 375 mg/m2 on day +1 (±3 days) and all (100%) patients received rituximab 375 mg/m2 on day +8 (± 3 days) without serious infusion reactions. All patients engrafted. Median time to neutrophil engraftment for the entire cohort was 15.5 (12–27) days. Median time to platelet engraftment in nine patients was 14.5 (10–17) days. Three patients never dropped their platelet counts below 20,000/uL. Median donor chimerism at day +90 (±10 days) for unsorted BM, CD3 and CD33 by PCR/STR method were 94% (70–100%), 86% (59–100%) and 100%, respectively. At a median follow up of 5 (0.6–30.3) months, the 100-day non-relapse mortality (NRM) in 11 evaluable patients was 10%. Acute GVHD, grades II, III, and IV, developed in 50%, 8.3% and 8.3%, respectively, at a median time of 27 (16–77) days. These findings demonstrate that administration of rituximab as part of conditioning chemotherapy is feasible and does not affect timely hematopoietic engraftment of allograft recipients with advanced CD20+ malignancies.

Description: Multiple Myeloma (MM) is an incurable disease that warrants novel treatments. MM survival is dependent on resistance conferred by interactions with the bone marrow (BM) microenvironment (stroma and/or extracellular matrix proteins): Environment Mediated-Drug Resistance (EM-DR). We have shown that adhesion to fibronectin inhibits CD95-induced caspase-8 activation resulting in apoptosis by regulating the cellular localization and availability of c- FLIPL (Shain K. et al. J Immunol. 2002;168(5):2544–53). TNF-related apoptosis inducing ligand/Apo-2L (TRAIL/Apo-2L) is a member of the TNF superfamily of death ligands that induce apoptosis of resistant MM cell lines and patient’s cells while cultured in suspension. TRAIL/Apo-2L also inhibits the growth of human plasmocytomas in xenografted NOD/SCID mice (Mitsiades C. et al. Blood2001; 98(3):795–804). To explore if the tumor microenvironment confers resistance to TRAIL/Apo-2L mediated apoptosis, we treated RPMI 8226 cells with recombinant human killer TRAIL (Alexis Biochemicals). In a series of 3 experiments, we observed a 50.8 ± 10 % specific apoptosis, measured by annexin/PI staining, when cells were treated with TRAIL (10ng/mL) in suspension (Su) media. In contrast, a dramatic resistance to TRAIL mediated apoptosis (6.9 ± 1.2 %) was observed when RPMI 8226 cells were adhered (Ad) to stroma cells (HS-5 and HS-5 transfected with green fluorescent protein-GFP) suggesting the EM-DR phenotype. We hypothesized that EM-DR can potentially be reversed by Bortezomib, a proteosome inhibitor, via NF-κB inhibition resulting in reduced transcription of cellular adhesion proteins genes (Mitsiades et al. Blood2002; 99(11): 4079–4086). Resistance to drug-induced apoptosis was also observed when RPMI 8226 cells were adhered to HS-5 and HS5-GFP and treated with Bortezomib alone at 5, 10 and 15nM (55.6 ± 11.6 % Su and 12.1 ± 1.6 % Ad). Microarray analysis of RPMI 8226 cells treated with Bortezomib showed that death receptor 4 (DR4) and DR5 expression was up-regulated suggesting a potential interaction between Bortezomib and TRAIL pathway. When RPMI 8226 cells were treated with Bortezomib (10nM) for 20 Hrs and subsequently treated with TRAIL for additional 4 Hrs, we observed an enhancement to 77.7 ± 3.64 % (Su) and 47.22 ± 13.6 % (Ad) specific apoptosis. In addition, in a series of 3 experiments using transwell inserts, RPMI 8226 cells (upper well) separated from HS-5 (lower well) were treated with Bortezomib (10nM), TRAIL (10ng/ML) or Bortezomib and TRAIL combined as described above. Results were comparable with direct contact experiments described, suggesting that cytokines and/or chemokines released by stroma may be responsible for EM-DR phenotype (Figure 1). Further mechanistic studies elucidating TRAIL and Bortezomib combination are currently under investigation. These preclinical studies provide the basis for clinical trial testing the combination of these agents. Figure Figure