Lenalidomide Exposure Research Articles

Targeting Cancer Stem Cell Survival in Plasma Cell Leukemia with a Pan-BCL2 Inhibitor

INTRODUCTIONRepresenting ten percent of all hematologic malignancies, multiple myeloma (MM) is typified by clonal plasma cell proliferation in the bone marrow (BM) and may progress to a therapy-resistant stage characterized by circulating malignant plasma cells, which is termed plasma cell leukemia, (PCL). Notably, copy number amplification involving the myeloid cell leukemia (MCL)-1 locus, and translocations at the BCL2 locus have been described in high-risk MM. Moreover, BCL2 family members, including both MCL1 and BCL2, are highly expressed in relapsed MM. Finally, downregulation of BCL2 family members was reported to increase sensitivity to lenalidomide, a standard treatment for MM, suggesting that BCL2-targeted treatments may obviate therapeutic resistance.Sabutoclax, a potent small molecule inhibitor of 6 anti-apoptotic BCL2 family proteins, was shown to inhibit cancer stem cell (CSC) survival in chronic myeloid leukemia (CML) (Goff et al Cell Stem Cell 2013). Thus, we investigated BCL2 pro-survival splice isoform expression in therapy-resistant MM and PCL and evaluated whether Sabutoclax reduces malignant plasma cell burden in humanized primagraft assays.METHODS AND RESULTSBCL2 pro survival expression in primary myeloma samplesMononuclear cells from MM or PCL samples were isolated by Ficoll-Paque density gradient separation and collected for RNA extraction or FACS Aria purified into CD138+ or CD34-/CD138- cancer stem cells (CSC) subsets. To evaluate BCL2 pro-survival family member expression, splice isoform-specific quantitative PCR was performed to measure expression of pro-survival long isoforms compared with short splice isoforms, which are pro-apoptotic. Interestingly, MM and PCL patients displayed higher levels of BCL2-L, MCL1-L, BCLX-L and BLF1-L compared to normal controls. Moreover, prolonged lenalidomide exposure increased BCL2-L and MCL1-L expression in the myeloma cell line H929, compared to untreated cells.Sabutoclax Treatment of a Novel Humanized Plasma Cell Leukemia Primagraft ModelMononuclear cells from three primary PCL patient samples were stably transduced with a GFP-luciferase lentiviral vector and transplanted intrahepatically in newborn RAG2γ-/- c-/- mice. Engraftment was monitored by peripheral blood free light chain ELISA assays. Flow cytometric analyses revealed robust engraftment of PCL cells in bone marrow, spleen, liver and peripheral blood. Once transplanted mice displayed significant tumor burden above background free light chain levels, animals were randomized by ELISA values in vehicle versus Sabutoclax groups. Sabutoclax was selected because this pan-BCL2 targeted compound, unlike related BCL2 inhibitors such as ABT-199, also inhibits MCL1. Sabutoclax (10mg/kg) was administered intravenously twice weekly for two to four doses. Sabutoclax treated PCL mice showed reduced human plasma cell burden in bone marrow and spleen tissues compared to vehicle controls.CONCLUSIONExpression of pro-survival BCL2 splice isoforms, including BCL2-L and MCL1-L, portends PCL engraftment in immunocompromised mice. Treatment of human PCL engrafted mice with Sabutoclax reduces malignant plasma cell survival in hematopoietic tissues. Thus, selective targeting of pro-survival isoform expressing CSC with a pan-BCL2 inhibitor may abolish BCL2 and MCL1-dependent therapeutic resistance in MM and PCL. DisclosuresJamieson:GlaxoSmithKline: Research Funding; Johnson & Johnson: Research Funding.

Phase 1 Study of the Novel Kinesin Spindle Protein Inhibitor Filanesib + Carfilzomib in Patients with Relapsed and/or Refractory Multiple Myeloma (RRMM)

Phase 1 Study of the Novel Kinesin Spindle Protein Inhibitor Filanesib + Carfilzomib in Patients with Relapsed and/or Refractory Multiple Myeloma (RRMM)

Low-Dose Cyclophosphamide Plus Granulocyte Colony-Stimulating Factor As an Efficacious and Safe Peripheral Blood Stem Cell Mobilizing Regimen in Patients with Hematologic Malignancies

▪Introduction:Collection of adequate numbers of Hematopoietic Stem Cells (HSCs) is a prerequisite for proceeding to autologous bone marrow transplant. However, various studies have showed that approximately 5% to 40% of patients do not meet the minimum threshold of 2×106 CD34+ cells/kg that is associated with timely engraftment and better outcomes. There is little consensus over the optimal mobilization regimen for procurement of peripheral blood CD34 + stem cells (PBSCs). Studies comparing mobilization using high-dose cyclophosphamide(HD-CY) {5-7 g/m2} or intermediate dose cyclophosphamide (ID-CY) {3-4g/m2} plus G-CSF with low dose cyclophosphamide (LD-CY) {1-2g/m2) plus G-CSF have reported higher total PBSC yield with the former but at the cost of higher toxicity [Hiwase et al, Cytotherapy 2007]. Hence the significance of our study to evaluate the efficacy and safety of LD-CY plus G-CSF in the era of novel induction therapies, takes precedence in terms of optimal resource utilization.Methods:We retrospectively analyzed mobilization efficacy and need for supportive care in Myeloma and Lymphoma patients that received LD-CY (2g/m2) plus G-CSF (10µg/kg/day) as the preferred mobilization regimen. Patients treated with novel induction regimens only, with or without radiation were included. LD-CY was given on day 1 with MESNA. G-CSF was started Day +5 from LD-CY and continued until the completion of apheresis. We started to measure peripheral CD34+ (pCD34) when patient's white blood cell count recovered to >1000/µL. When the pCD34 count was ≥10/uL, apheresis was started.Results:Out of 21 patients analyzed, 14 (67%) were Myeloma, predominantly IgG subtype (71%) and 7(33%) Lymphoma predominantly Non-Hodgkins type (71%). Our study population comprised of 53% females, 81% Caucasians with median age of 59 years (30-66). 28.5% patients had radiation treatment in the past. Mean/Median lines of chemotherapy previously received were 2/1 (1-5).71% patients received Lenalidomide (57%) / Thalidomide (14%) based regimens for induction therapies. 52% patients had complete response at the time of chemo mobilization.Successful mobilization (defined as total CD34+ cells collected >2x106/kg) was significantly achieved in 95% patients with mean/ median collection of 12.4/11.5 x106 CD34+ cells/kg (20 out of 21 patients, One sample T- test with significance level, alpha =0.025 [one sided ]showed T statistic 5.36 with p-value <0.0001). First apheresis yielded at least 2 x10 6/kg CD34+ cells in 76 % patients with a mean/median of 5.8/3.8 x106/kg CD34+ cells. 66% of patients were found to be good mobilizers, defined as patients collecting ≥5 x106 CD34+ cells/kg in ≤2 days. 81% patients collected ≥5x106 CD34+ cells/kg and 57 % patients collected ≥10 x106 CD34+ cells/kg. Mean/Median Peak pCD34count was 97.7/60 cells/µL(2-488). Median number of aphereses was 2 (mean 2.6) with the mean and median time to apheresis being 11 days (9-15). None of the patients needed inpatient hospitalization or intravenous antibiotics during mobilization or had any complication related to immunosuppression. 14% and 19% of patients needed packed red blood cell and platelet transfusions respectively during the mobilization period. One Myeloma patient (4.8%) that failed mobilization had previously received five lines of chemotherapy including lenalidomide/thalidomide containing regimens and had stable disease at the time of mobilization. Interestingly, 61% of myeloma patients that successfully mobilized had received lenalidomide containing induction regimen (mean/median no. of cycles- 3.9/4[1-7])Conclusion:Our analysis shows that LD-CY plus G-CSF is efficacious to mobilize sufficient number of stem cells (>2x106 CD34+ cells /kg) in Myeloma and Lymphoma patients treated with novel agents, including the challenging sub-group of lenalidomide treated myeloma patients, with no infectious complications or morbidity noted during the entire period of mobilization. Therefore, we believe LD-CY plus G-CSF may overcome the negative effects of prior lenalidomide exposure on PBSC mobilization [Mark et al, Biol Blood Marrow Transplant 2008] and is superior in terms of optimal resource utilization compared to novel plerixafor based regimens [Chaudhary et al, J Clin Apher 2013]. Prospective studies involving CY based regimens will help elucidate the optimal dose of chemo-mobilization. DisclosuresNo relevant conflicts of interest to declare.

Contribution of Chemotherapy Mobilization to Disease Control in Multiple Myeloma Treated with Autologous Transplantation

▪Background: Autologous hematopoietic progenitor cells (auto-HC) can be mobilized either by hematopoietic growth factors (GF) alone or cytotoxic chemotherapy + GF (CC+GF). The latter is associated with higher CD34+ yields, but is associated with increased costs and risks of infection and hospitalization. In patients with multiple myeloma (MM) undergoing Auto-HCT, it is uncertain whether mobilization with CC+GF affects post transplant outcomes.Methods: We conducted a retrospective analysis of patients with MM undergoing their first AHPCT following high dose melphalan (≥140 mg/m2) between 2007 and 2012 in US and Canada and registered with the Center for International Blood and Marrow Transplant Research (CIBMTR). Patients who had a planned subsequent allogeneic transplant, received VAD or similar induction therapy, had disease progression prior to transplant, or were mobilized with plerixafor were excluded from the analysis. The primary endpoint was progression-free survival (PFS) and the main secondary end-points were transplant-related mortality (TRM), relapse and overall survival (OS).Results: There were 519 patients in GF and 449 in CC+GF with median follow-up of 42 and 46 months respectively. The most common mobilizing CC regimen was single agent cyclophosphamide (71%). GF and CC+GF groups were similar but there were more patients in GF with only one prior line of therapy, lenalidomide exposure and CR at the time of transplant and more patients in CC+GF with low HCT co-morbidity index, prior thalidomide exposure and planed second autologous transplant (Table 1). Median number of days for auto-HC collection was 2 (IQR 1-3) in GF and 1 (IQR 1-3) in CC+GF (P<0.001). There were fewer days between collection and transplant (median 16 vs. 18, P<0.001) and fewer CD34+ cells (x 106/kg) infused at transplant (median 3.9, IQR=3.1-4.8, vs. 5.1, IQR 3.5-6.9, P<0.001) in GF than in CC+GF. Kinetics of neutrophil engraftment (> 0.5 x 109/L) was similar between groups (13 vs. 13 days, P=0.69) while platelet engraftment (> 20 x 109/L) was faster in CC+GF (median 19 vs. 18 days, P=0.006). There was no difference between groups in number of hospitalization days (14 vs. 14, P=0.7). In univariate analysis, there was no significant difference between the two groups in OS, PFS, or TRM. In multivariate analysis, stage III at diagnosis and Karnofsky status <90 but not modality of mobilization were associated with worse PFS. Similarly HCT-CI >2, Stage III at diagnosis and immunoglobulin isotype (IgG/IgA/Others), but not mobilization were associated with OS. Adjusted 3-years PFS was 43% (95% C.I. 38-48) in GF and 40% (95% C.I. 35-45) in CC+GF, P=0.33 (Figure). Adjusted 3-years OS was 82% (95% C.I. 78-86) vs 80% (95% C.I. 75-84), P=0.43 and adjusted 5-year OS was 62% (95C.I. 54-68) vs. 60% (95% C.I. 52-67), P=0.76, for GF and CC+GF respectively (Figure).Conclusions: MM patients undergoing AHPCT have similar outcomes irrespective of the use of cytotoxic chemotherapy mobilization. We found no evidence that chemotherapy mobilization contributes to disease control in MM.TableCharacteristics of patients and treatmentsGFCC+GFPN=519N=449Lines of therapy<0.0011382 (74)265 (59)2113 (22)136 (30)>224 (5)48 (11)Prior therapy<0.001Thalidomide+Bortezomb+-Steroid74 (14)92 (20)Lenalidomide+Bortezomb+-Steroid121 (23)48 (11)Thaildomide+-Steroid85 (16)106 (24)Bortezomib+-Steroid132 (25)136 (30)Lenalidomide+-Steroid107 (21)67 (15)Disease status at HCT0.05CR84 (16)48 (11)PR407 (78)378 (84)MR/NR/SD28 (5)23 (4)HCT-CI0.0060227 (44)227 (51)1-2147 (29)134 (30)>2145 (28)88(20)Time from diagnosis to HCT<0.001<6 mo221 (43)140 (31)6-12 mo298 (57)309 (69)Year of HCT0.032007-2008289 (56)272 (61)2009-2010105 (20)100 (22)2011-2012125 (24)77 (17)Melphalan dose0.40140-180 mg/m271 (14)70 (16)>180 mg/m2448 (86)379 (84)Transplant type0.02Single transplant417 (80)331 (74)Multiple TransplantsPlanned 2ndAuto (0-6 mo)42 (8)63 (14)Planned 2ndAuto (>6 mo)5 (1)6 (1)Use of maintenance agent198 (38)176 (39)0.29 [Display omitted] DisclosuresCosta:Sanofi: Honoraria.

Lenalidomide Induces Lipid Raft Assembly to Enhance Erythropoietin Receptor Signaling in Myelodysplastic Syndrome Progenitors

Erythropoietin receptor (EpoR) signaling is impaired in patients with Myelodysplastic Syndromes (MDS) despite appropriate growth factor production and cellular receptor display. We previously reported that EpoR signaling is dependent upon receptor localization within membrane lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity (McGraw KL, et al. PLoS One 2012). Here, we show that MDS erythroid progenitors display markedly diminished raft assembly (p=0.005) and smaller raft aggregates (p=0.023) compared to normal controls. Because lenalidomide triggers raft coalescence in T-lymphocytes to promote immune synapse formation, we assessed the effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lipid rafts were isolated from UT7 cells using ultracentrifugation and identified by GM-1 dot blot and Lyn kinase western blot. Lenalidomide rapidly induced lipid raft formation in UT7 cells which was confirmed by confocal microscopy visualization of GM-1 fluorescence. Lenalidomide also significantly induced lipid raft formation in pooled MDS erythroid progenitors (CD71+, cKit+) from 11 patients [mean raft size, control (n=569) vs. lenalidomide treatment (n=659), p<0.001], with no significant change observed in pooled erythroids from 3 normal donors (n= 327 for control and n=365 for lenalidomide treated, p=0.37). Interestingly, lipid rafts were significantly larger in erythroid progenitors from patients who responded (n=5) to lenalidomide treatment compared to non-responders (n=3) (75.52 ±13.68 vs. 35.85 ±8.56, p=0.02). Although lenalidomide increased raft size in erythroid progenitors from both responders (p=0.0007) and non-responders (p=0.013), mean raft size was greater in erythroid precursors from responding patients after treatment (p=0.11). Increased raft aggregation after lenalidomide treatment was accompanied by EpoR recruitment into raft fractions together with STAT5, JAK2, and Lyn kinase, whereas the JAK2 phosphatase, CD45, a negative regulator of EpoR signaling, was displaced from raft fractions. Incubation with lenalidomide prior to Epo stimulation enhanced both JAK2 and STAT5 phosphorylation in UT7 cells and primary MDS erythroid precursors. Bone marrow specimens from 12 non-del(5q) IPSS lower risk, lenalidomide naive MDS patients were analyzed by flow cytometry to compare changes in STAT5 phosphorylation in response to Epo stimulation in the presence or absence of lenalidomide. We found a 79.1% mean increase in p-STAT5 mean fluorescence intensity (MFI 95th percentile) in CD45dim, CD71high, Glylow erythroid precursors in 7 of the 12 patient specimens following lenalidomide exposure. Furthermore, increased STAT5 phosphorylation was accompanied by increased DNA binding of the transcription factor in UT7 cells, and improved erythroid colony forming capacity in both UT7 and primary MDS bone marrow cells. Raft induction was associated with F-actin polymerization that was blocked by Rho kinase inhibition and confirmed by lipid raft isolation followed by dot blot with western blot and confocal microscopy. These data provide new insight into abnormalities in the EpoR signaling platform that underlie impaired Epo responsiveness in MDS erythroid precursors. Our findings that deficient raft integrity impairs EpoR signaling provides a novel strategy to enhance EpoR signal fidelity in non-del(5q) MDS. These data also warrant investigation in a larger data set to determine whether lipid raft size may be a predictive biomarker for lenalidomide response. DisclosuresList:Celgene: Consultancy.

Phase 1b/2 Study of Carfilzomib, Pomalidomide, and Dexamethasone (KPd) in Patients (Pts) with Lenalidomide-Exposed and/or -Refractory but Proteasome Inhibitor (PI)-Naive or -Sensitive Multiple Myeloma: A Multiple Myeloma Research Consortium Multi-Center Study

Phase 1b/2 Study of Carfilzomib, Pomalidomide, and Dexamethasone (KPd) in Patients (Pts) with Lenalidomide-Exposed and/or -Refractory but Proteasome Inhibitor (PI)-Naive or -Sensitive Multiple Myeloma: A Multiple Myeloma Research Consortium Multi-Center Study

Connect MM®—the Multiple Myeloma (MM) Disease Registry: Incidence of Second Primary Malignancies (SPM)

▪Background: Increased rates of SPM have been observed as newer cancer treatments have improved survival over the past 2 decades (Fraumeni et al. NCI, 2006). Higher incidence of specific types of hematologic SPM following MM, especially acute myeloid leukemia and myelodysplastic syndromes, have been reported relative to the general population (Dores et al. NCI, 2006; Mailankody et al. Blood, 2011; Ravazi et al. Blood, 2011; Landgren and Mailankody. Leukemia, 2014). A complex interplay between myeloma-, host-, environmental-, and treatment-related factors likely contributes to the increased incidence of SPM in MM. Connect MM is the first and largest prospective, observational, US-based, multicenter registry designed to characterize patients (pts), treatment patterns, and outcomes in newly diagnosed MM (NDMM) pts.Methods: Between September 2009 and November 2012,a total of 1493 NDMM pts were enrolled from 234 US sites within 2 mos of the first diagnosis of MM. Patient data were collected at baseline and each subsequent quarter using a standardized form. Invasive SPM included hematologic and solid tumor second cancers and non-invasive SPM were defined as non-melanoma skin cancers (NMSC). SPM incidence and incidence rate (IR; number of pts with SPM per 100 patient-yrs [PY]) were calculated for all pts and by exposure to specific treatments, including lenalidomide (LEN). PYs were calculated as the observation period from the start of treatment until the detection of the first reported SPM (per category), death, or end of follow-up (pt lost or data cutoff).Results: As of Dec 10, 2013, SPM data were available for 1493 NDMM pts. The median age was 67 yrs (range, 24-94 yrs), 82% of patients were white and 57% were male. Median follow-up was 29.0 mos (0-49 mos). The median OS of treated pts was 44.4 mos. Fifty pts did not receive treatment and had no SPM reported. A total of 74 of the 1443 treated pts (5.1%) reported SPM. Invasive SPM were observed in 51 pts (3.5%): 37 pts (2.6%) with solid tumors and 14 pts (1.0%) with hematologic SPM. Lung/bronchus and myelodysplastic syndromes were the most frequently reported solid tumor and hematologic SPM respectively. NMSC were reported for 26 pts (1.8%). 3 pts had both an invasive SPM and NMSC. The IRs for invasive, hematologic, and solid tumor SPM by LEN exposure are listed in Table 1. By multivariate analysis, the only significant risk factor for the occurrence of SPM was prior history of invasive malignancy. Demographics (including age, ethnicity, race, and gender), International Staging System stage, family history of myeloma or other cancers, history of smoldering MM or monoclonal gammopathy of unknown significance, or prior radiation therapy were not associated with the occurrence of SPM.Conclusions: This analysis shows that there was no increased risk of invasive SPM in this disease-specific registry of pts with NDMM. The risk of SPM for LEN exposed pts was not greater than that for pts not exposed to LEN. In addition, multivariate analysis indicated the only significant risk factor for SPM was prior history of invasive malignancy. As additional agents are approved for the treatment of MM and the length of pt survival increases, longer prospective observation with expanded enrollment on the registry will better characterize the occurrence of SPM in this pt population. Correlations with risk factors including age, pre-existing MDS, risk status, as well as type and duration of therapy will continue to be investigated.Table 1Incidence rates (per 100 PYa) by treatment exposureIR per 100 PY (95% CI)SPMLEN-Exposed(n = 977)Non–LEN Exposed(n = 466)Invasive0.85 (0.61-1.19)1.16 (0.72-1.86)Hematologic0.17 (0.08-0.36)0.47 (0.22-0.99)Solid tumor0.67 (0.46-0.98)0.68 (0.36-1.26)NMSC0.50 (0.32-0.77)0.41 (0.18-0.91)a PY of exposure is the sum of exposure of all pts. DisclosuresRifkin:Celgene Corp: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Takeda: Consultancy; Amgen: Consultancy. Abonour:Celgene Corp: Honoraria, Speakers Bureau. Shah:Celgene Corp: Consultancy, Research Funding. Mehta:Celgene Corp: Consultancy, Speakers Bureau. Narang:Celgene Corp: Membership on an entity’s Board of Directors or advisory committees. Terebelo:Celgene Corp: Membership on an entity’s Board of Directors or advisory committees. Gasparetto:Celgene: Consultancy, Honoraria; Millenium: Honoraria. Thomas:Celgene Corp: Membership on an entity’s Board of Directors or advisory committees. Toomey:Celgene Corp: Membership on an entity’s Board of Directors or advisory committees. Hardin:Celgene Corp: Research Funding. Lu:Celgene Corp: Employment. Kenvin:Celgene Corp: Employment. Srinivasan:Celgene Corp: Employment, Equity Ownership. Ricafort:Celgene Corp: Employment. Nagarwala:Celgene Corp: Employment. Durie:Celgene Corp: Expert Board Committee Other; IRC Onyx: Membership on an entity’s Board of Directors or advisory committees; DMC Millennium: Membership on an entity’s Board of Directors or advisory committees; IRC J&J: Membership on an entity’s Board of Directors or advisory committees.

Efficacy and Risk Factors Analysis of Upfront Autologous Stem Cell Mobilization Using Plerixafor and Granulocyte-Colony Stimulating Factor (GCSF) in Patients with Multiple Myeloma

Background: G-CSF with or without cyclophosphamide has been commonly used in multiple myeloma (MM) for stem cell (SC) collection prior to autologous stem cell transplantation (ASCT). Several risk factors including age, prolonged exposure to lenalidomide, and low platelet count are associated with suboptimal stem cell harvest. Plerixafor selectively and reversibly binds to the chemokine receptor CXCR4 and blocks its interaction with stromal cell-derived factor-1α. This drug was approved by the FDA for stem cell mobilization in MM, based on phase III studies demonstrating the superiority of plerixafor and G-CSF over G-CSF alone in achieving a predetermined target yield of 6 × 106CD34+ cells/kg collected in < 2 phereses; however, this outcome is not clinically relevant. The purpose of this retrospective study is to examine the SC mobilization efficiency associated with plerixafor using a more clinically relevant outcome, and to identify risk factors associated with poor SC mobilization, including the potential impact of prior exposure to lenalidomide-containing regimens.Patients and methods: This retrospective study examined MM patients mobilized with plerixafor and G-CSF upfront as part of initial therapy at Memorial Sloan Kettering Cancer Center between 4/1/2009 and 8/1/2013. Baseline characteristics examined included age, race, gender, platelet count, WBC count, and marrow plasmacytosis prior to mobilization. Treatment characteristics examined included type of induction regimen (distinguishing regimens containing lenalidomide only, bortezomib only, or both), number of cycles received, time between last chemotherapy and SC mobilization, time between start of treatment and SC collection, and prior radiation. The primary endpoint was the rate of SC collection success, defined as the ability to collect at least 5 x 106CD34+ cells/kg during the first set of phereses, which would be sufficient for two ASCT. The secondary endpoint was SC collection efficiency, measured by the number of CD34+ cells yielded per pheresis performed during the first set of stem cell collection. Linear regression was used to examine the univariate effect of baseline and treatment variables on SC collection efficiency. Variables significant at the 0.05 level were entered into a multivariable model.Results: A total of 138 MM patients were mobilized with plerixafor and G-CSF before proceeding to stem cell collection. The rate of SC collection success was 92.8%. Due to this high success rate, we could not identify independent risk factors associated with poor SC mobilization. When considering SC efficiency as outcome, the average efficiency for the entire cohort was 7.25 x 10^6 CD34+ cells/kg/pheresis. Increased age (p=0.005), shorter time between treatment start and SC collection (p=0.05), higher number of cycles received during induction treatment (p=0.042), lower platelet count (p=0.009) and lower WBC prior to G-CSF (p=0.01), and exposure to lenalidomide only (p=0.011) were all identified as risk factors by univariate analysis. Only lower WBC prior to GCSF (p=0.009) maintained statistical significance after multivariate analysis.Conclusions: This retrospective study shows that plerixafor is a highly effective agent for SC mobilization and collection in MM patients, with only few patients failing to achieve the target collection regardless of baseline or treatment characteristics including prior lenalidomide exposure. However, we identified WBC prior to G-CSF administration as a predictor of SC collection efficiency. These results provide strong support to the upfront use of plerixafor and G-CSF for SC mobilization in MM patients. A cost analysis is currently in progress comparing this SC collection modality with other conventional means currently being employed in patients with MM. DisclosuresNo relevant conflicts of interest to declare.

Genome-wide miRNA profiling in myelodysplastic syndrome with del(5q) treated with lenalidomide.

Lenalidomide is a potent drug with pleiotropic effects in patients with myelodysplastic syndrome (MDS) with deletion of the long arm of chromosome 5 [del(5q)]. We investigated its effect on regulation of microRNA (miRNA) expression profiles in del(5q) patients with MDS in vivo. We used miRNA expression microarrays to study changes in miRNA levels in peripheral blood CD14+ monocytes collected from patients before and during lenalidomide treatment and compared them with those from healthy donors. Before treatment, we observed strong upregulation of pro-apoptotic miR-34a and miR-34a* that diminished during lenalidomide exposure. Upregulation of HOX-related miR-196b and erythroid-specific miR-451 seen in untreated patients remained unchanged after the treatment. At the time of hematologic response, expression of several miRNAs clustering to the 14q32 locus was reduced. Additionally, we focused more deeply on miRNAs from the 5q commonly deleted region and found that levels of miR-378 and miR-378* followed haploinsufficiency trend. This report describes changes in miRNA expression in del(5q) patients with MDS treated with lenalidomide, likely arising from deregulation of pathways implicated in lenalidomide action.

Predicting Autologous Stem Cell Mobilization Failure In Hematologic Malignancies

BackgroundAutologous stem cell transplantation (ASCT) after high-dose chemotherapy improves remission rates and prolongs survival in a number of hematologic malignances including multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), and Hodgkin's lymphoma (HL).The prerequisite to ASCT is the collection of an adequate number of CD34+ cells; the minimum dose needed to ensure hematopoietic recovery is 2.0x106cells/kg, but recent studies have suggested that 5.0x106cells/kg is the optimal dose as it results in more rapid and sustained engraftment. An estimated 15-30% of patients fail to mobilize the required number of cells for ASCT, although the number of mobilization failures is declining due to advances in mobilization regimens. Previously identified variables for predicting mobilization failure include: advanced age, the type of chemotherapy and number of cycles administered, and prior radiotherapy. Patients/MethodsWe performed retrospective chart review of 803 consecutive patients who underwent apheresis for autologous stem cell collection at the Siteman Cancer Center from January 2008 through December 2011. Patients were considered eligible for analysis if they had a diagnosis of MM, NHL or HL and had not undergone previous stem cell collection. Univariate analysis was performed to identify predictors associated with successful collection of > 2.0x106 cells/kg and > 5.0x106 cells/kg. Variables analyzed were: mobilization regimen; age; gender; BMI; weight/ideal body weight; diagnosis; prior radiation; number of chemo cycles administered; alkylating agent exposure; lenalidomide exposure; blood glucose level; blood triglycerides level; glucocorticoid use at apheresis; statin use at apheresis; and history of diabetes mellitus, coronary artery disease, hypertension, or hyperlipidemia. Results706 patients met the eligibility criteria for analysis. The median age was 57.9 years (range 18.5-74.6) and 58% (411) were male. 53% (373) had MM, 38% (266) had NHL, and 9% (67) had HL. 63% (444) of patients were mobilized with plerixafor and G-CSF, 17% (117) with chemotherapy and G-CSF, and 21% (145) with G-CSF alone.After one apheresis procedure, 75% (531) of patients collected > 2.0x106 cells/kg and 50% (356) collected > 5.0x106 cells/kg. 42% (295) of patients underwent more than one apheresis procedure. During their mobilization, 94% (664) of patients were able to collect > 2.0x106 cells/kg and 68% (480) collected > 5.0x106 cells/kg.Data was available on >99% of the population for all variables with the exceptions of number of chemo cycles administered (n= 667), blood triglyceride level (n= 358), and glucocorticoid use at apheresis (n= 660). Significant results from univariate analysis are summarized in table 1. ConclusionThe most significant predictors of mobilization failure identified were mobilization regimen, diagnosis, alkylating agent exposure, and number of chemo cycles. Lenalidomide exposure was associated with a reduced risk of mobilization failure, but this is likely attributed to the reduced risk of mobilization failure for MM, as 99% (255) of patients with lenalidomide exposure had MM. Interestingly, diagnosis of hypertension was associated with a reduced risk of mobilization failure. The mobilization failure rate in the study population is 6%, which is below the expected rate found in historical studies. This is likely a result of advances in mobilization regimens as only 21% of the study population was mobilized with G-CSF alone.Table 1Day 1Total> 2.0x106cells/kg> 5.0x106cells/kg> 2.0x106cells/kg> 5.0x106cells/kgMobilization RegimenPlerixafor+G-CSF (n= 444)83.6%58.8%96.6%74.8%Chemo+G-CSF (n= 117)69.2%59.0%93.2%70.9%G-CSF (n= 145)54.5%17.9%86.9%44.8%p value<0.001<0.001<0.001<0.001DiagnosisMM (n=373)84.2%61.7%97.3%83.9%NHL (n=266)66.9%39.1%91.0%52.3%HL (n= 67)58.2%32.8%88.1%41.8%p value<0.001<0.001<0.001<0.001Alkylating Agent ExposureYes (n= 356)66.3%39.3%91.0%52.8%No (n= 350)84.3%61.7%97.1%83.4%p value<0.001<0.0010.001<0.001Number of Chemo CyclesMedian Cycles Successful5454Median Cycles Failure878.59p value<0.001<0.0010.003<0.001Lenalidomide ExposureYes (n= 258)81.8%56.2%96.5%80.6%No (n= 448)71.4%47.1%92.6%60.7%p value0.0020.0200.036<0.001HypertensionYes (n= 305)79.7%96.1%No (n= 399)71.9%NS92.5%NSp value0.0180.047 Disclosures:Abboud:Novartis: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees; Alexion: Membership on an entity's Board of Directors or advisory committees; Teva: Speakers Bureau. Schroeder:Celgene: Research Funding; Sanofi: Research Funding. Uy:Celgene: Research Funding; Sanofi: Research Funding. Vij:Celgene: Research Funding, Speakers Bureau; Onyx: Research Funding, Speakers Bureau; Millennium: Speakers Bureau; BMS: Honoraria; Lilly: Honoraria. Westervelt:Celgene: Honoraria, Research Funding. Stockerl-Goldstein:Celgene: Speakers Bureau; Millennium: Speakers Bureau.

Lenalidomide Promotes The Expansion Of CD8 T Cells With An Effector Memory Phenotype In a Murine Xenograft Model Of Chronic Lymphocytic Leukemia

Lenalidomide (Revlimid®), a thalidomide analogue, is an orally administered second generation immunomodulator with anti-angiogenic and anti-neoplastic properties. Initial studies treating patients with chronic lymphocytic leukemia (CLL) suggest that lenalidomide can have considerable efficacy and that its mode of action is mainly indirect, affecting non-malignant cells in the microenvironment, in particular T lymphocytes. Because a recently described xenograft model for CLL has highlighted the importance of CLL-derived, autologous T cells in promoting leukemic B-cell engraftment and growth in vivo, we have studied the influence of lenalidomide on the expansion of CLL B- and T-lymphocytes in this model.After an initial 12 day culture of FACS-isolated CLL-derived T cells with or without anti-CD3/CD28 beads plus IL-2 (30 IU/ml), T lymphocytes were transferred into alymphoid NSG mice via the retro-orbital plexus (day 0). On day 7, CLL cells were delivered retro-orbitally. These recipient animals are referred to as “T + PBMC mice”. Mice that did not receive T cells on day 0 but were given CLL PBMCs at day 7, with or without lenalidomide, served as controls (“PBMC only mice”). Recipient mice received lenalidomide (10mg/kg/day) or vehicle control daily by gavage starting at day 0. All mice were sacrificed at day 28 (28 days after T-cell and 21 days after B-cell transfer), and blood, spleen, and bone marrow were collected. On this material, four analyses were performed: [1] level of human CD45+ cell engraftment; [2] numbers and types of CLL-derived T cells; [3] numbers of CLL B cells; and [4] levels of cytokines reflective of Th1 and Th2 immune responses.There was a clear enhancement in human hematopoietic (CD45+) cell engraftment in those mice exposed to lenalidomide. This was most marked for the PBMC only mice (vehicle: 10.64%; lenalidomide: 38.53%), although it was also evident for T + PBMC mice (vehicle: 55.96%; lenalidomide: 69.65%).T-cell phenotyping was carried out, before and after cell culture and also at sacrifice. Prior to culture, CLL samples contained on average ∼96% CD5+CD19+ cells and ∼3% CD5+CD19- cells; for the latter, ∼67% were CD4+ and ∼33% CD8+. After 12-day culture, these percentages remained largely unchanged. However, the numbers and types of T cells recovered from the spleens at sacrifice were quite different after in vivo exposure to lenalidomide. For the PBMC only, the percentages of CD4+ and CD8+ cells in the spleens differed somewhat based on lenalidomide exposure (CD4: Vehicle 86% vs. Lenalidomide 61%; CD8: Vehicle 10% vs. Lenalidomide 28%). However, this change was dramatic for the T + PBMC mice (CD4: Vehicle 64.1% vs. Lenalidomide 28.9%; CD8: Vehicle 34% vs. Lenalidomide 62%). Furthermore, when the CD8+ cells from these animals were subsetted based on antigen-experience and function, it appeared that lenalidomide exposure had led to the outgrowth of a greater number of effector memory (CD45RO+ CD62L-) than central memory (CD45RO+ CD62L+) T-cells.For CLL-derived B cells, the numbers differed, based not only on lenalidomide exposure but also on prior in vitro activation. Specifically, in PBMC only mice, the addition of lenalidomide led to increased numbers of CLL B cells in the spleen (Vehicle: 7.81% vs. Lenalidomide: 14%). Conversely, in the T + PBMC mice, the numbers of B cells decreased (Vehicle: 2.36% vs. Lenalidomide: 0.34%).An analysis of Th1 and Th2-related cytokines in the plasmas of the mice at sacrifice revealed a fall in IL-4, IL-5, and IL-10 and a marked increase in IFNg, consistent with a Th2 to Th1 transition.The above data suggest that administration of lenalidomide permits greater engraftment of human hematopoietic cells in alymphoid mice. Although this enhancement involves all members of the hematopoietic lineage, T cells, in particular CD8+ effector memory T cells, emerge in excess over time. This CD8 expansion is associated with diminished levels of CLL B cells suggesting that the decrease is due to T-cell mediated cytolysis. In contrast, in the absence of prior T-cell activation, CLL T cells appear to support better CLL B-cell growth. These findings suggest that lenalidomide alters B-cell expansion in vivo depending on the activation and differentiation state of the autologous T-cell compartment. They also implicate the generation of cytolytic T cells as one mechanism whereby lenalidomide leads to clinical improvement in CLL. Disclosures:Allen:Celgene Corporation: Honoraria.

Lenalidomide Inhibits Proliferation Of Chronic Lymphocytic Leukemia Cells Via a Cereblon/p21WAF1/Cip1-Dependent Mechanism

Lenalidomide has demonstrated clinical activity in patients with chronic lymphocytic leukemia (CLL). However, its mechanism of action is not fully elucidated. Lenalidomide is not directly cytotoxic to CLL cells in vitro, but can alter the capacity of CLL cells to interact with cells in its microenvironment. This has led to the speculation that its primary activity is indirect, although direct effects on CLL cells have been observed.In this study, we examined the direct effects of lenalidomide on CLL cells. We performed transcriptome analysis on primary CLL cell samples exposed to lenalidomide in vitro for 6 and 24hrs and found a significant upregulation of the cyclin-dependent kinase inhibitor p21WAF1/Cip1 (p21), which was also confirmed at the protein level. Since p21 can inhibit the progression of cells from G1 to S phase of the cell cycle, this suggests that lenalidomide may render CLL cells less responsive to proliferation stimuli from the microenvironment. To test this hypothesis, we induced CLL cells to proliferate in vitro using accessory cells made to express CD154 and media containing human interleukin (IL)-4 and IL-10. We monitored for proliferation of CLL cells cultured with and without lenalidomide using carboxyfluorescein succinimidyl ester (CFSE). In repeated experiments, we observed that lenalidomide consistently and significantly inhibited the proliferation of CLL cells in a dose-dependent manner, at concentrations that are achieved in treated patients. Evaluation of the DNA content of CLL cells using propidium iodide and flow cytometry also revealed that lenalidomide significantly decreased the fraction of CLL cells in S and in G2/M phases of the cell cycle and concomitantly increased the percentage of CLL cells in G0/G1 phases. This block in cell proliferation also was accompanied by the upregulation of p21, and the extent of which correlated with the degree to which leukemia-cell proliferation was inhibited. In additional studies, we used small interfering RNA (siRNA) to silence p21 in CLL cells and measured the effect of silencing on cell proliferation in the presence of lenalidomide. We observed that the ability of lenalidomide to inhibit CLL cell proliferation was significantly reduced in CLL cells silenced for p21 compared to CLL cells transfected with control siRNA, supporting a role for p21 expression in mediating the proliferation block induced by lenalidomide. We did not however observe the induction of p53 expression following lenalidomide exposure, suggesting that lenalidomide may upregulate p21 via a p53-independent mechanism.Cereblon (CRBN) is the only known molecular target of lenalidomide. To functionally interrogate the potential role of CRBN in lenalidomide activity on CLL cells, we used siRNA to silence CRBN in primary CLL cells and monitored the impact of silencing on the ability of lenalidomide to upregulate p21 and to inhibit proliferation. We observed lower levels of p21 expression in CRBN-silenced cells exposed to lenalidomide compared to cells transfected with non-specific siRNA, suggesting that CRBN may play a role in p21 upregulation. Furthermore, we observed that CRBN silencing significantly abrogated the anti-proliferative effect of lenalidomide. These results indicate the involvement of CRBN in the anti-proliferative activity of lenalidomide, which is mediated, at least in part, by p21.This study demonstrates that lenalidomide inhibits the proliferation of CLL cells in a CRBN/p21-dependent manner. This direct effect of lenalidomide on CLL cells may account in part for the highly infrequent observation of disease progression in patients receiving long-term maintenance therapy with this agent (Blood, 2013, PMID:23801633). Disclosures:Fecteau:Celgene: Research Funding. Corral:Celgene: Employment. Gaidarova:Celgene: Employment. Cathers:Celgene: Employment. Lopez-Girona:Celgene: Employment. Messmer:Celgene: Research Funding. Kipps:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Phase 1 Study Of The Novel Kinesin Spindle Protein Inhibitor ARRY-520 + Carfilzomib (Car) In Patients With Relapsed and/Or Refractory Multiple Myeloma (RRMM)

Phase 1 Study Of The Novel Kinesin Spindle Protein Inhibitor ARRY-520 + Carfilzomib (Car) In Patients With Relapsed and/Or Refractory Multiple Myeloma (RRMM)

Effect Of Cumulative Adverse Clinical Risk Factors On Patients With Multiple Myeloma Undergoing First GCSF Apheresis

With the new treatments for multiple myeloma (MM), increasing numbers of patients fail to mobilize sufficient peripheral blood stem cells (PBSC) for autologous stem cell transplant (ASCT). Multiple studies have identified clinical and laboratory factors, such as age, number of lines of chemotherapy, radiation exposure, bone marrow involvement, and low PLT count as risks for poor mobilization. However, there are fewer studies that analyze only the effect of multiple clinical risk factors on mobilization outcomes. We retrospectively analyzed 259 MM patients who underwent first apheresis after GCSF mobilization between December 2000 and December 2012. Clinical risk factors analyzed include age, number of lines of chemotherapy, number of cycles of chemotherapy, number of doses of cyclophosphamide, number of doses of lenalidomide, and prior external beam radiation. The standard dose of GCSF was 10 mcg/kg/day, however the exact dose for a significant number of patients was not known. Patients were assessed as to whether optimal (≥8x106 CD34+ cells/kg) or minimal (≥4x106 CD34+ cells/kg) number of stem cells for two ASCTs were collected. The median age of the entire cohort was 59.7 years (27.9-76.0). Overall 10.8% and 32.6% failed to collect the minimal and optimal number of stem cells after one round of apheresis. Of the twenty patients who underwent a second round of apheresis, 9 (45%) collected minimal and 7 (35%) collected optimal total number of stem cells, with an overall failure rate of 4.6% and 29.8%, respectively. The effect of the number of clinical risk factors on the mobilization failure during first apheresis is summarized in Table 1. For each additional clinical risk factor, the likelihood of collecting the minimal and optimal number of CD34+ cells is reduced by 34% (CI=0.484-0.893, p=0.0072) and 32% (CI=0.538–0.860, p=0.0013) respectively. On univariate analysis, all risk factors were analyzed as continuous variables, except for prior radiation which was analyzed as a categorical variable. Prior lenalidomide exposure (odds ratio=0.502, CI=0.297–0.845, p=0.0096), and prior radiation therapy (odds ratio=0.502, CI=0.293–0.861, p=0.0123) had the greatest negative predictive value. Of the 38 patients who were exposed to lenalomide (median 4 cycles; range 1-24 cycles), 13% and 42% failed to collect minimal and optimal number of stem cells in the first apheresis cycle, respectively. An association was seen between number of days required to collect target number of stem cells and number of risk factors (p≤0.001). Median number of days required to collect target number of stem cells for 0, 1 or 2+ clinical risk factors was 2, 2, and 4 days, respectively. When the effect of clinical risk factors were analyzed according to number of CD34+ cells/kg collected on each day of apheresis, statistically significant differences in collection efficiency were seen on the first 3 days of apheresis (Figure 1). In summary, clinical characteristics of patients with MM can potentially be used to predict mobilization failure. The presence of 2 or greater clinical risk factors adversely affect the ability to successfully collect the target stem cell dose. These risk factors may help in identifying high-risk MM patients who may benefit from alternative mobilization regimens that can be tested in prospective clinical trials.Number of Clinical Risk Factors012+N1128561% Patients not collecting optimal # of cells on 1st apheresis cycle23.231.850.895% CI15.8-32.122.1-42.833.7-63.9p-value0.19710.0003% Patients not collecting minimal # of cells on 1st apheresis cycle5.411.818.095% CI2.0-11.35.8-20.69.4-30.0p-value0.11950.0096Median number of days of collection224Range1-81-91-10p-value0.4233&lt;0.0001 Disclosures: No relevant conflicts of interest to declare.

A Phase I Trial Of Anti-KIR Monoclonal Antibody IPH2101 and Lenalidomide For Multiple Myeloma

IntroductionMultiple myeloma (MM) remains an essentially incurable plasma cell malignancy. MM utilizes specific immunoevasive strategies to avoid natural killer (NK) cell immune surveillance and cytotoxicity. Immunomodulatory agents such as lenalidomide (LEN) may exert indirect anti-MM efficacy via expansion and activation of NK cells. However, these favorable effects may be diminished when LEN is co-administered with high doses of dexamethasone (DEX). IPH2101 is a monoclonal anti-inhibitory KIR antibody which prevents negative signaling in NK cells and enhances NK cell recognition and killing of MM cells. A single-agent, phase I study of IPH2101 demonstrated full KIR blockade with encouraging safety and tolerability, and 34% of heavily pre-treated patients achieved disease stabilization (Blood 2012;120:4324-33). Preclinical data demonstrate that LEN and IPH2101 exert anti-MM effects via complementary NK-cell immunomodulatory mechanisms (Blood 2011;118:6397-91). Herein, data are presented from the first clinical experience with IPH2101 and LEN in combination in patients with MM. MethodsA 3+3 phase I dose-escalation trial was conducted. Patients (age 18-80) with measurable, progressive MM were enrolled having received one or two prior lines of therapy. Prior LEN exposure was permitted unless resistance or intolerance was observed. Patients must have had ECOG performance status ≤ 2, creatinine clearance ≥ 60 ml/min, platelets ≥ 75,000/uL (or ≥ 30,000/uL if > 50% bone marrow plasma cells), absolute neutrophil count ≥ 1,000/uL, bilirubin < 1.5 ULN, and ALT / AST < 3 ULN. Patients must have adhered to standard prescribing guidelines for LEN. Three dose levels included: IPH2101 0.2mg/kg IV q 28 days + LEN 10 mg PO days 1-21; IPH2101 0.2 mg/kg + LEN 25 mg, and IPH2101 1mg/kg + LEN 25 mg for 4 cycles. Responding patients were allowed to receive 4 additional cycles. Patients completing all 8 cycles were maintained on LEN thereafter. No administration of DEX or other systemic corticosteroids was permitted. Dose reductions of LEN were permitted per prescribing information. The primary objective was to determine the safety and tolerability of IPH2101 + LEN, the secondary objectives included pharmacokinetics (PK) and pharmacodynamics (PD) of IPH2101 and biologic correlates with LEN as well as to determine clinical activity by standard IMWG uniform response criteria. Results15 patients (10 M, 5 F, median age 60) were enrolled, 8 in first relapse and 9 in second relapse. 9 had prior LEN exposure. Cohorts 1 and 3 were expanded to n=6 patients respectively due to occurrence of possible dose-limiting toxicity. In both cases, a patient experienced a similar, apparent infusion reaction on cycle 1, day 1, characterized by fever, chills, cytokine release, and leucopenia. Events resolved with supportive care and both patients continued on trial without recurrence. The protocol was amended to include premedication with anti-histamine and acetaminophen,and no further infusion reactions were observed. Most other observed adverse events were of low grade and generally investigator-attributed as possibly or probably related to LEN. IPH2101 PD were not affected by co-administration of LEN. Full KIR occupancy was achieved in cohort 3 across the dosing interval. Five patients achieved a response (2 VGPR, 3 PR) with a median duration of 15+ months (3-26+). ConclusionThe combination of IPH2101 + LEN appears to be a safe and well tolerated, and steroid-free combination in MM patients. Infusion reactions have not been observed since the addition of premedication prior to IPH2101 dosing. IPH2101 PD do not appear to be altered by co-administration of LEN, and full KIR blockade over the dosing interval has been achieved. Although the study is small, response rate and response duration are encouraging. These findings support further investigation of antiKIR therapy with LEN as the first, steroid-sparing, dual immunotherapy for MM. Disclosures:Benson:Innate Pharma: Research Funding. Off Label Use: Lenalidomide without concomitant dexamethasone. Zerbib:Innate Pharma: Employment. Andre:Innate Pharma: Employment. Caligiuri:Innate Pharma: Membership on an entity's Board of Directors or advisory committees.

Lenalidomide at Therapeutic and Supratherapeutic Doses Does Not Prolong QTc Intervals in the Thorough QTc Study Conducted in Healthy Men

The effect of lenalidomide on the corrected QT (QTc) interval was evaluated in healthy men and extended to patients based on the lenalidomide concentration–QTc (C–QTc) relationship. A rigorous assessment of the effect of lenalidomide on QTc intervals was conducted in healthy volunteers who each received, in randomized order, a single oral dose of 10 mg lenalidomide, 50 mg lenalidomide, 400 mg moxifloxacin (positive control) and placebo. Plasma lenalidomide exposure was compared between healthy volunteers and patients with multiple myeloma or myelodysplastic syndromes. In healthy volunteers, moxifloxacin produced the expected significant prolongation in QTcI (individual correction). For lenalidomide 10 mg and 50 mg, the time-matched changes from placebo in the baseline-adjusted least-squares mean QTcI were <3 ms with the upper limit of the two-sided 90% confidence interval for the change <10 ms at all time-points. No subjects experienced QTcI >450 ms or change from baseline >60 ms after lenalidomide administration. Similar results were seen with QT interval data corrected by Fridericia and Bazett methods. The C–QTc analysis yielded no significant association between lenalidomide concentrations and QTcI changes up to 1522 ng/mL; this range was close to that observed in patients receiving lenalidomide doses up to 50 mg, including those with reduced drug clearance due to renal impairment. In conclusion, single doses of lenalidomide up to 50 mg were not associated with prolonged QTc intervals in healthy males. The C–QTc analysis further assured that lenalidomide doses up to 50 mg are not expected to prolong QTc intervals in patients.

Evaluating the effects of lenalidomide induction therapy on peripheral stem cells collection in patients undergoing autologous stem cell transplant for multiple myeloma

Lenalidomide (LEN) is a relatively new and very effective therapy for multiple myeloma (MM). Prior LEN therapy is associated with an increased risk of peripheral blood stem cell collection (PBSC) failure, particularly with filgrastim (G-CSF) alone. We performed a retrospective chart review of 319 consecutive MM patients who underwent apheresis to collect PBSCs for the first autologous stem cell transplant (ASCT). The median number of PBSCs collected in the LEN (+) group was significantly less than the LEN (-) group (6.34 vs. 7.52 × 10(6) CD34(+) cells/kg; p = 0.0004). In addition, the median number of apheresis sessions required for adequate PBSCs collection were significantly more in the LEN (+) group as compared to LEN (-) group (2 vs. 1 sessions; p = 0.002). In the LEN (+) group, there was a negative correlation between PBSCs collected and prior number of cycles of LEN (p = 0.0001). Rate of PBSC collection failure was 9% in the LEN (+) group and 5% in the LEN (-) group (p = 0.16). Only six patients who failed PBSC collection with G-CSF were able to collect adequate PBSCs with G-CSF + plerixafor. LEN exposure had no effect on neutrophil or platelet recovery post-ASCT. Up to four cycles of LEN exposure have minimal negative impact on PBSC collection. Despite prolong exposure of LEN, PBSC collection was adequate for two ASCTs in the majority of patients and post-ASCT engraftment was not longer than expected; however, clinical relevance (complication rate, quality of life, cost) of prolonged LEN exposure on both PBSC and ASCT, should be evaluated in prospective clinical trials.

Secondary Myelodysplasia-Associated Metaphase Cytogenetic Abnormalities in Newly Diagnosed Multiple Myeloma Treated On Total Therapies 2 & 3– Influence of Cumulative Dosing of Maintenance Drugs

AbstractAbstract 324▪▪This icon denotes a clinically relevant abstract Background:There has been recent evidence that lenalidomide, while used in the treatment of myelodysplastic syndromes (MDS), also has been associated with an enhanced risk of secondary malignancies including MDS. Here we are examining the impact of maintenance on the development of metaphase-based MDS-associated cytogenetic abnormalities (MDS-CA), and subsequent development of therapy related MDS (t-MDS) and acute leukemia (t-AL) in the two arms of Total Therapy 2 (TT2) and two successive Total Therapy 3 trials (TT3A, TT3B). Methods:The details of TT2/TT3A/TT3B trials and patient outcomes have been reported previously. TT2 (median follow-up 118 months) patients were then randomized to receive thalidomide maintenance (TT2+Thal) for 3 years versus no thalidomide (TT2-Thal). Maintenance in TT3A (median follow-up 78 moths) consisted of VTD in the first year and TD for years 2 and 3 in TT3A, while TT3B (median follow-up 48 months) employed VRD for all 3 years. The patient populations were as follows: TT2-Thal arm (n=283), TT2+Thal arm (n=270), TT3A (n=279) and TT3B (n=162). Bone marrow examination with conventional cytogenetics was performed at baseline, post-induction, post-transplant and then at least semi-annually from maintenance in all patients prospectively. Chi-square and Fisher's exact tests were used to compare baseline characteristics between protocols. Univariate and multivariate Cox proportional hazard regression were used to model associations between baseline covariates and onset of MDS-CA and t-MDS/t-AL. Kaplan and Meier method was used to model cumulative incidence of MDS-CA and t-MDS/t-AL. Results:Baseline characteristics were fairly comparable between the two arms of TT2 and the two TT3 studies, except a higher proportion of GEP70 high risk patients in TT3B. Cumulative incidence of MDS-CA was significantly higher in TT3B protocol from time of first transplant (Figure 1), with the most common cytogenetic abnormality being deletion of chromosome 20 (del20, 35%), followed by monosomy/deletion 7 (27%) and monosomy/deletion 5 (17%) across all protocols. Of the 71 patients with an MDS-CA, 29 were also diagnosed with clinical MDS or AML; for these 29 patients, the median time to this diagnosis from first transplant was 58 months. The cumulative incidence of MDS-CA was higher and achieved more statistical significance (Figure 2) in TT3B compared to TT3A when landmarked from the start of maintenance. In univariate analysis for all patients, TT3B emerged as a significant variable in the univariate Cox regression model for MDS-CA, and in both the univariate and multivariate models for del20. Univariate and multivariate modeling within TT3B patient population did not reveal significance with cumulative dosing of different maintenance drugs. Discussion:Three randomized phase III trials (IFM-2005-02, CALGB 101104 and MM-015) have demonstrated an increase incidence of second malignancies in patients receiving lenalidomide maintenance after having been exposed to alkylating agent melphalan. In our present study, we are reporting an increased incidence of MDS-CA in the TT3B trial that employed VRD maintenance when compared with predecessor TT3A and TT2 trials. The data suggests that the risk is associated with lenalidomide exposure rather than its cumulative dosing in patients with prior exposure to therapy with alkylating agents. [Display omitted] Pairwise log-rank comparisons:TT3B vs. TT3A:p-value = 0.0206TT3B vs. TT2-Thal:p-value = 0.0134TT3B vs. TT2+Thal:p-value = 0.0178TT3A vs. TT2-Thal:p-value = 0.1502TT3A vs. TT2+Thal:p-value = 0.3134TT2-Thal vs. TT2+Thal:p-value = 0.8863 [Display omitted] Disclosures:Sawyer:University of Arkansas for Medical Sciences: Employment, under panding process, under panding process Patents & Royalties.

Priming of Mir-181a in Acute Myeloid Leukemia (AML) to Increase Chemosensitivity: A Phase I Trial of Lenalidomide (LEN) Followed by Idarubicin and Cytarabine.

AbstractAbstract 2619Expression of miR-181a confers better prognosis in cytogenetically normal (CN) AML (Schwind et al, JCO 2010). Laboratory studies by our group have demonstrated that miR-181a is upregulated by C/EBPα-p30. CN AML patients (pts) harboring CEBPA mutations, the majority of which block C/EBPAα-p42 expression but allow C/EBPα-p30 expression, have elevated miR-181a and more favorable clinical outcome (Marcucci et al, JCO 2008). We hypothesized that pharmacologic potentiation of C/EBPa-p30 expression, and in turn miR-181a upregulation, would increase chemosensitivity and improve clinical outcomes in AML. Based on prior in vitro and in vivo studies, LEN was selected as a candidate agent to achieve these pharmacodynamic (PD) endpoints. Pharmacokinetic (PK) and PD studies from our prior single-agent, high-dose LEN trial demonstrated upregulation of miR-181a in leukemic blasts at tolerable doses; preclinical studies showed that this effect is dose dependent. Given these results and previously demonstrated LEN clinical activity in AML (Blum et al, JCO 2010; Fehniger et al, Blood 2011; Ades et al, ASH 2010; Lancet et al, ASH 2011), a phase I trial was conducted of LEN as miR “priming” with intensive chemotherapy in two parallel cohorts of AML: Prior Therapy (PT) Cohort for relapsed/refractory pts <65 years of age, and Untreated (U) Cohort for newly diagnosed pts >60 years. In both groups, LEN was given for 14 days(d) starting at 25mg/d; cytarabine and idarubicin (starting dose 12/mg/m2 × 3d) began on d5. PT Cohort received cytarabine at a starting dose of 1.5 g/m2/d over 24 hours for 4d; U Cohort received 100mg/m2 for 7d in standard fashion. 31 pts enrolled, 17 with relapsed/refractory AML and 14 with previously untreated AML. PT Cohort pts had median age 53 years (range, 22–64) and median presenting WBC count of 4.2 × 109 (range, 0.9–22.7 × 109). U Cohort pts had median age 69 years (range, 48–77), median presenting WBC count of 3.1 × 109 (range, 0.5–31.9 × 109), and median marrow blasts of 36%. None of the U Cohort pts had favorable risk disease by ELN classification, 6 had adverse risk. Note that one patient <60 years of age was enrolled in U Cohort due to a recent amendment expanding eligibility. Dose limiting toxicities included rash (4), delayed hematologic recovery (1), mucositis (1), and cardiomyopathy (1). One pt died within 30d of starting treatment (surgical complication unrelated to treatment); however, two additional pts died of therapy-related complications within 60d of induction [intracranial hemorrhage with refractory thrombocytopenia (1) and cardiomyopathy (1)]. The maximum administered dose of LEN was 30mg/d, and we are currently expanding the 25mg dose level in both groups. PT Cohort is being expanded at LEN 25mg with idarubicin 8mg/m2/d and cytarabine at 1mg/m2/d. U Cohort is being expanded at the MTD of LEN 25mg with idarubicin 12mg/m2/d and cytarabine at 100mg/m2/d. Complete remission (CR) was achieved in 41% (7/17) in the PT Cohort and 69% (9/13, all >60 years) in the U Cohort. One pt in U Cohort was not evaluable for response as the pt discontinued treatment before chemotherapy was given. Preliminary PK parameters and LEN exposure (Figure 1) are consistent with what we reported previously. Observed PK of idarubicin was similar to expected and did not suggest an interaction with LEN. There was no apparent difference in PK of LEN in those who experienced skin rash (or other toxicities/clinical outcomes) compared with those that did not. Preliminary PD testing of LEN-induced modulation of miR-181a expression was consistent with the hypothesis of the trial. Results in a small subset of pts (N=6) tested to date showed a trend for greater post-LEN increase in miR-181a(pretreatment vs. d5 before chemotherapy began) in pts who achieved CR compared with those who did not (Figure 2). In conclusion, LEN priming with chemotherapy is reasonably well tolerated and active in AML. We are planning a randomized phase II study in previously untreated patients to test the hypotheses and compare efficacy of LEN priming vs. post-chemotherapy LEN. For the current trial, an amendment to shorten the course of LEN in order to facilitate dose escalation above 25mg is planned. Updated correlative results for all enrollees will be presented. Supported by NCI U01 CA 76576-05, NIH/NCI K23CA120708, Leukemia and Lymphoma Society SCOR 7004-11, and SPORE P50-CA140158. [Display omitted] [Display omitted] Disclosures:Off Label Use: Lenalidomide in AML.

Phase 1 Study of the Novel Kinesin Spindle Protein Inhibitor ARRY-520 + Carfilzomib in Patients with Relapsed and/or Refractory Multiple Myeloma

AbstractAbstract 4082 Background:ARRY-520, a novel kinesin spindle protein (KSP) inhibitor, has been studied as a single agent and in combination with dexamethasone, and demonstrated promising clinical activity in patients with bortezomib- and lenalidomide-refractory multiple myeloma (MM). Carfilzomib, a novel irreversible proteasome inhibitor (PI), has also demonstrated single agent activity in relapsed and refractory MM, and recently received regulatory approval for this indication. Preclinical data support the presence of synergy with the combination of a PI and a KSP inhibitor via the latter's ability to down-regulate Mcl-1, supporting our hypothesis that the combination of carfilzomib and ARRY-520 (Car-ARRY) would be highly active in relapsed and/or refractory myeloma. We therefore aimed to combine these two agents for the first time, and here report the initial findings from the phase I dose-escalation in patients with relapsed and/or refractory MM. Methods:The primary objective was to determine the maximum tolerated dose (MTD) and the safety/tolerability of the Car-ARRY combination. Secondary objectives were to determine efficacy as measured by the overall response rate, time to progression, progression free survival and time to next therapy. Patients had to have myeloma that was relapsed and/or refractory, be ineligible for autologous stem cell transplant, bortezomib refractory/intolerant, and prior lenalidomide exposure. ARRY-520 was administered intravenously over 1 hour on days 1, 2, 15 and 16, while carfilzomib was administered intravenously over 30 minutes on days 1, 2, 8, 9, 15 and 16 on a 28 day cycle. All patients received growth factor support with filgrastim.Dose-escalation used a standard 3+3 schema proceeded based on dose-limiting toxicities (DLTs) during cycle 1, with planned escalation of the dose of ARRY-520. Dose level 1 was ARRY-520 0.75 mg/m2, and carfilzomib was dosed at 20 mg/m2 for cycle 1 on days 1 and 2 and all subsequent dose were at 27 mg/m2. Adverse events (AEs) were graded by NCI-CTCAE v4, while responses were assessed by the modified International Uniform Response Criteria. Results:To date, 8 patients have been enrolled in the ongoing dose escalation phase. The median age was 66 (range 47–80), 6/8 were males, and the median number of prior therapies was 4 (range 2–10). 7/8 patients had undergone prior autologous stem cell transplant, and all patients were bortezomib refractory or intolerant. In the first cohort, 3 patients were enrolled and no dole limiting toxicity (DLT) was observed. During the second cohort, ARRY-520 was escalated to 1 mg/m2 with carfilzomib at 20/27 mg/m2, and among the first 3 patients, one patient suffered a DLT in the form of an admission for influenza pneumonia with non-neutropenic fever. Expansion of cohort 2 is currently underway.Among the 6 patients who completed the first cycle of therapy, 5 remain on study. In the first cohort, one patient remains on study with 6 cycles and achieved a near complete remission, 1 patient achieved stable disease, and 1 patient suffered disease progression after first cycle. In the second cohort, all three patients who completed the first cycle have stable disease and remain on trial.In the first 6 toxicity-evaluable patients who have completed one cycle, grade (G) 3 events included one each of pneumonia, diarrhea, and hyperglycemia. There was limited hematologic toxicity with 4/6 patients with G1/2 thrombocytopenia, 3/6 patients with G1/2 anemia, and 1/6 patient with G1/2 neutropenia. Additional G1/2 non-hematologic toxicity included 3/6 patients with diarrhea, 3/6 patients with dyspnea, 3/6 patients with transient elevations in creatinine and 3/6 patients with aspartate aminotransferase elevations.An MTD has not been established and enrollment is ongoing in cohort 2 with carfilzomib at 20/27mg/m2 and ARRY-520 at 1.0 mg/m2. Conclusions:The combination of ARRY-520 and carfilzomib is well tolerated with limited hematologic toxicity and a manageable side effect profile. Notably, in this patient population, with patients who have bortezomib refractory/intolerant myeloma, the combination has demonstrated early signals of activity. Updated safety and efficacy data for all patients will be presented at the meeting. Disclosures:Shah:Onyx: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Array BioPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau. Off Label Use: This presentation will include information about Arry-520 which is not yet approved for use in patients with multiple myeloma. Wang:Pharmacyclic: Research Funding; onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hilder:Array BioPharma: Employment. Orlowski:onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; array biopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees.