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Dysregulation of cyclin-dependent kinases is a hallmark of myeloma, and specifically, cdk5 inhibition can enhance the activity of proteasome inhibitors in vitro. Dinaciclib is a novel potent small molecule inhibitor of cyclin-dependent kinases (CDK)1, CDK2, CDK5, and CDK9. Patients with relapsed multiple myeloma and ≤5 prior lines of therapy, with measurable disease, were enrolled. Dinaciclib was administered on day 1 of a 21-day cycle at doses of 30 to 50 mg/m(2). Overall, 27 evaluable patients were accrued; the median number of prior therapies was 4. The dose level of 50 mg/m(2) was determined to be the maximally tolerated dose. The overall confirmed partial response rate (PR) was 3 of 27 (11%), including 1 patient at the 30 mg/m(2) dose (1 very good PR [VGPR]) and 2 patients at the 40 mg/m(2) dose (1 VGPR and 1 PR). In addition, 2 patients at the 50 mg/mg(2) dose achieved a minimal response (clinical benefit rate, 19%). Leukopenia, thrombocytopenia, gastrointestinal symptoms, alopecia, and fatigue were the most common adverse events. The current study demonstrates single agent activity of dinaciclib in relapsed myeloma, with 2 patients achieving a deep response (VGPR) and 10 patients obtaining some degree of M protein stabilization or decrease. This trial was registered at www.clinicaltrials.gov as #NCT01096342.

Previous studies demonstrated that ataxia telangiectasia mutated– and Rad3-related (ATR) kinase and its downstream target checkpoint kinase 1 (Chk1) facilitate survival of cells treated with nucleoside analogs and other replication inhibitors. Recent results also demonstrated that Chk1 is depleted when cells are treated with heat shock protein 90 (Hsp90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). The present study examined the effects of 17-AAG and its major metabolite, 17-aminogeldanamycin (17-AG), on Chk1 levels and cellular responses to cytarabine in human acute myelogenous leukemia (AML) cell lines and clinical isolates. Cytarabine, at concentrations as low as 30 nM, caused activating phosphorylation of Chk1, loss of the phosphatase Cdc25A, and S-phase slowing. Conversely, treatment with 100 to 300 nM 17-AAG for 24 hours caused Chk1 depletion that was accompanied by diminished cytarabine-induced S-phase accumulation, decreased Cdc25A degradation, and enhanced cytotoxicity as measured by inhibition of colony formation and induction of apoptosis. Additional studies demonstrated that small inhibitory RNA (siRNA) depletion of Chk1 also sensitized cells to cytarabine, whereas disruption of the phosphatidylinositol 3-kinase (PI3k) signaling pathway, which is also blocked by Hsp90 inhibition, did not. Collectively, these results suggest that treatment with 17-AAG might represent a means of reversing checkpoint-mediated cytarabine resistance in AML.

Rituximab is a chimeric murine/human monoclonal antibody that binds to CD20 on B lymphocytes. Although binding of the Fab domain may induce apoptosis, the Fc domain recruits immune effector functions to mediate cell lysis. Interleukin-12 (IL-12) facilitates cytolytic T-cell responses, enhances the lytic activity of natural killer (NK) cells, and induces the secretion of interferon γ (IFN-γ) by both T and NK cells. Therefore, the hypothesis was considered that combining IL-12 with rituximab would augment the immune-mediated cell lysis induced by rituximab. A phase 1 study of IL-12 in combination with rituximab was conducted in 43 adults with B-cell lymphoma to determine the optimal immunologic dose of this combination. Rituximab was administered at a dose of 375 mg/m2 by intravenous infusion weekly for 4 weeks, and IL-12 was given subcutaneously twice weekly. The starting dose of IL-12 was 30 ng/kg and this was escalated to 500 ng/kg. Constitutional symptoms and liver enzyme elevations at 500 ng/kg of IL-12 were dose limiting. A greater than 20-fold increase in the serum levels of IFN-γ and a 2.5- to 5-fold increase in inducible protein 10 (IP-10) levels was seen at IL-12 doses of 100 ng/kg or greater. Objective responses occurred in 29 of the 43 patients (69%), with 8 of 11 complete responses seen at IL-12 doses of 300 ng/kg or greater. The optimal immunologic dose of IL-12 in combination with rituximab was determined to be 300 ng/kg subcutaneously twice weekly starting on day 2. These data suggest that IL-12 and rituximab is an active combination and further studies of this combination in B-cell non-Hodgkin lymphoma are warranted.

Background: Bruton’s tyrosine kinase (BTK) is an essential component of the B-cell receptor signaling pathway, a pathway critical to the survival and proliferation of malignant B cells. Ibrutinib, a small molecule inhibitor of BTK, has shown significant activity in several B-cell malignancies and is currently FDA approved for the treatment of patients (pts) with CLL or mantle cell lymphoma who have received at least one prior treatment.In a Phase 1 dose-escalation study, ibrutinib induced a response in 6 (38%) of 16 patients (pts) with relapsed/refractory FL and 6/11 (54%) of pts treated at doses ≥ 2.5 mg/kg (Advani JCO 2013, Fowler ASH abstr 156:2012). 560 mg daily was identified as a well-tolerated dose in lymphoma resulting in full-target occupancy. On the basis of these results, the Mayo Clinic and Princess Margaret P2Cs conducted a CTEP-sponsored Phase 2 trial of single-agent ibrutinib in relapsed/refractory FL (NCI 9271). Methods: Eligible pts had relapsed or refractory FL (Gr 1, 2, or 3a) that had progressed during or after 1 or more prior chemotherapy regimens. Therapy consisted of ibrutinib, 560 mg daily, until progression or unacceptable toxicity. Response was assessed by CT at 8 weeks and then every 12 weeks. All pts were required to have an on-study biopsy and a second biopsy at progression after response with fresh tissue collected for correlative studies. The primary endpoint was overall response rate (ORR) and secondary endpoints were safety and tolerability, overall survival, time to response, time to treatment failure, duration of response, and progression-free survival (PFS). Planned exploratory correlative studies included C1D8 and C3D1 FDG-PET response evaluation in a subset of 20 patients, and whole-transcriptome sequencing for correlation of mutations with response and resistance. Results: 40 patients (38 evaluable for response) were accrued from April 2013 to April 2014. Median age was 64 years (range 46-82), 70% were men, 55% had FLIPI ≥ 3, median number of prior regimens was 3 (range 1-11), 20% had a prior stem cell transplant, 45% were rituximab refractory, and 36% were refractory to their most recent treatment. At a median follow-up of 6.5 months (mo) (range 1.8-14.6+), the ORR for the 40 intention-to-treat pts is 30% (95% CI: 17-47%) with 1 CR and 11 PRs by CT criteria (4/12 responders had a negative restaging PET/CT). 26 (65%) patients have exhibited tumor size reduction. Only 2/18 (11%) pts with rituximab-refractory disease responded, compared to 8/19 (42%) pts with rituximab-sensitive disease (P=0.06) and 2/3 who were rituximab-naïve. There was no correlation between response and number of prior regimens. Median time to response was 2.4 mo (range 1.8-12.9 mo). Median PFS is 9.9 mo (95% CI: 6 mo, not reached). One of 12 responders has progressed after 9.9 mo on treatment. Median treatment duration was 5 mo (range 1-15+ mo). Dose delays (2 pts) and reductions were infrequent (4 pts). 17 pts have discontinued treatment due to PD (12), refusal (2), AEs (2), and death (1). Gr 3-4 AEs occurred in 30% of pts, including the following AEs in more than 1 pt: anemia (2), neutropenia (3), and infection (2). 3 pts have died (1 PD, 1 pneumonia, 1 gastric hemorrhage). Of the 20 pts with C1D8 PET scans, 19 also had C3D1 PET scans and were evaluable for response. Six of these 19 pts had objective responses by CT criteria at week 8 or beyond. Of the 6 responders, C1D8 PET/CT showed qualitative (visual) improvement in 3 pts, no visual change in 1 pt, and qualitative worsening in 2 pts. On the C3D1 PET/CT, 4 of 6 had qualitative improvement and 2 had no change. Evaluation of quantitative PET data including SUVs and total lesion glycolysis is ongoing and will be presented. Conclusions: Single agent ibrutinib is well tolerated with a modest ORR in relapsed/refractory FL at this early assessment. Continued follow-up is warranted to capture late responders and to establish response duration. Ibrutinib appears less active in FL than in MCL and CLL. Early PET scans (C1D8) do not reliably predict for response. Support by N01-CM-2011-00099 Disclosures Bartlett: Gilead: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Astra Zeneca: Research Funding; Janssen: Research Funding; ImaginAb: Research Funding; Genentech: Research Funding; Millennium: Research Funding; Pfizer: Research Funding; Pharmacyclics: Research Funding; Novartis: Research Funding; Medimmune: Research Funding; Celgene: Research Funding. Off Label Use: PF-05082566 for B-NHL. Kuruvilla:Janssen: Honoraria. Reeder:Millennium, Celgene, Novartis: Research Funding.

Backgrounds B cell receptor (BCR) signals and microenvironment protection have been recognized to have key influences on CLL survival and activation. Akt activation is down-stream of the BCR signaling cascade and is critical in the mediation of the bi-directional interactions between CLL B-cells and bone marrow stroma. MK2206 is an allosteric Akt inhibitor previously tested for safety in solid tumor patients. Our preclinical data has demonstrated the synergy of MK2206 with bendamustine (B) to induce CLL apoptosis in vitro. MK2206 also abolished the signal activation and cytokine production induced by BCR ligation in CLL leukemic cells (Ding, ASH 2012). Therefore, we conducted a phase 1 trial testing the safety and efficacy of MK2206 in combination with bendamustine and rituximab (BR) for patients with relapsed/refractory CLL. Methods A phase 1 dose-escalation study was designed to define the maximum tolerated dose (MTD) as well as the safety and efficacy of 3 dose levels (90 mg or 135 mg or 200 mg weekly) of the oral Akt inhibitor MK2206 in combination with bendamustine (70 mg/m2 daily for 2 days in each cycle) and rituximab (cycle 1: 375 mg/m2, cycle 2-6: 500 mg/m2) therapy in relapse/refractory CLL patients. To test the targeting efficacy of MK2206 on Akt and downstream signals, we designed a one-week “run-in” of single agent MK2206 before the initiation of BR during cycle 1. MK2206 was then given along with BR on day 1 of cycles 2-6. Tumor response was evaluated 2 months after the 6th/last cycle of therapy based on IWCLL 2008 criteria. Correlative laboratory studies included assessment of Akt signaling and the plasma cytokine levels (multiplex beads assay (Invitrogen)) at baseline and after one week of first dose of single agent MK2206. Results Nine patients (median age 68) were enrolled in the phase I portion of this trial. All 9 patients had unmutated immunoglobulin heavy chain variable region genes (IGHV) and 4 had del(11q). Six had received prior chemoimmunotherapy (CIT: FCR or PCR or PCO) and 5 had high risk disease defined as early progression within 26 months of the last CIT. Three had received alemtuzumab containing regimen. We have observed potent lymphocyte mobilization in all evaluated patients (n=7) after one-dose of MK2206 during week 1 (mean increase ALC: 35%) as well as reductions of plasma CCL3, CCL4 and CCL2 in the majority of the patients. A single dose-limiting toxicity (DLT) (febrile neutropenia and hemolytic anemia) was observed at dose level 1 (90 mg weekly) among 6 patients treated at this dose. Two patients with dose-limiting toxicities at dose level 2 (135 mg weekly) were observed where one patient had a rash and one patient had dehydration and febrile neutropenia among three patients treated at this dose. Accordingly, the 90 mg once weekly dose was determined to be the MTD for phase 2 trial testing. The most frequent grade 3 or 4 adverse events observed to date were neutropenia (44%) including febrile neutropenia (22%), rash (22%), diarrhea (22%), nausea and vomiting (11%), dehydration (11%), hemolytic anemia (11%) and thrombocytopenia (11%). The frequency of cytopenias was similar to the published German experience with BR (50% grade 3 or 4 hematological toxicities compared to 44% in N1087) in relapsed CLL patients (Fischer, JCO 2011). The median number of cycles received was 6 (range 1-6). Using an intent to treat analysis, two patients achieved complete response (CR, n= 2, 22%), one patient achieved nodular partial response (nPR, n=1, 11%) and five patients achieved a partial response (PR, n=5, 56%). The overall response rate (ORR) is 89%. Conclusion The Akt inhibitor MK2206 in combination with BR is well tolerated in patients with relapsed/refractory CLL. Single dose of MK2206 inhibits Akt phosphorylation, mobilizes leukemic cells and decreases plasma cytokines involved in BCR signals. The preliminary efficacy of this combination demonstrated promising results with a 89% ORR and 22% CR albeit in the small phase 1 cohort. These results compare favorably to BR alone (9% CR and 59% ORR) in relapsed/refractory CLL patients. Phase 2 testing of this combination is now underway. Acknowledgment This work was supported by the funding from NCI grant K23CA160345 (WD), NCI grant CA95241 (NEK) and Alliance for Clinical Trials in Oncology (CA025224 and CA33601). Disclosures: Off Label Use: MK2206, used in a phase 1 trial of relapsed CLL disease. Shanafelt:Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding. Zent:Novartis: Research Funding; GlaxSmithKline: Research Funding; Biothera: Research Funding; Genzyme: Research Funding; Genentech : Research Funding. Reeder:Celgene: Research Funding; Novartis: Research Funding; Millenium: Research Funding.

Abstract 3928 Background: Accumulating data support the critical role of PI3K/Akt in CLL B cell receptor (BCR) mediated signal transduction, cell proliferation and survival. In addition recent preclinical and clinical studies indicate that specific PI3K blockade results in robust preclinical and clinical efficacy in CLL. In our model system of CLL B cell-stromal cultures which feature their interaction, platelet derived growth factor (PDGF) present in CLL culture medium drives VEGF production through PI3K/Akt activation in stromal cells (Blood. 2010. 116:2984). Indeed Akt was found to be activated in leukemic cells during the CLL-stroma interaction (Leuk Res. 2008. 32:1565). Therefore, we hypothesized that Akt inhibition should promote CLL B cell apoptosis and abrogate BCR mediated cytokine production. MK2206 is an orally bioavailable highly specific allosteric Akt inhibitor. It has been tested in patients with refractory solid tumors and was demonstrated to be safely administered in a phase I trial. Therefore the goal of this study was to test the preclinical efficacy of MK2206 on both the survival and the BCR mediated cytokine production of CLL leukemic B cells. Methods: Peripheral blood mononuclear cells isolated from CLL patients (n=37) were treated with escalating concentrations of MK2206 (1–16 μM) for 24 hours, 48 hours or 72 hours. The levels of leukemic B cell viability were tested using an (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. The potential impact (antagonistic/additive/synergistic) of Bendamustine in combination with MK2206 was also tested by using the MTT assay. We used the Calcusyn system to calculate the effect of drug interactions. The combination index (CI) as calculated by the program usually indicates synergy when ≤ 0.8 and indicates additive outcomes when between 0.8–1.2. A CI >1.2 indicates antagonism. Downstream signals of Akt activation in CLL B cells were evaluated by testing their expression of Mcl-1, 4EBP1 and p70S6K using immunoblot. The impact of Akt inhibition by MK2206 on cytokine production in response to B-cell receptor ligation with anti-IgM was also tested using a multiplex cytokine analysis (Invitrogen) in a time-course experiment. Results: MK2206 treatment induced concentration- and time-dependent apoptosis in CLL leukemic cells. At 72 hours, the IC50 of MK2206 in the experiments using CLL leukemic cells in vitro is ∼8 mM. MK2206 incubation at 1 or 5 mM cultured with CLL B cells over a 48-hour period abolished of Akt and p70S6K phosphorylation while native PARP was cleaved into the 85 kD polypeptide fragment. However, the expression level of the upstream signal molecule, PI3K, was not changed. Among the CLL patients tested (n = 37), we did not find any difference in sensitivity to MK2206 induced apoptosis based on critical prognostic factors of CD38, ZAP-70, IGHV and del(17p) status. Importantly, we detected synergistic or additive activity between MK2206 and Bendamustine in 11 tested CLL samples when these combinations were used to treat CLL cells in vitro for 72hrs. Thus the median CI value for this group of patients was 0.8 (0.1 – 1.1). Six were found to have CI ≤ 0.8 and five fell within the additive CI values (0.8 – 1.2). Production of immune or chemotactic cytokines (e.g. CCL3, CCL4, MCP-1, IL-1Ra, IL-8 and IL-2R) at 24 hour incubation increased significantly above baseline when CLL cells were stimulated anti-IgM. Akt inhibition with MK2206 selectively abrogated upregulation of CCL3, CCL4, MCP-1 and IL-2R production, but not for IL-8 or IL-1Ra secretion. MK2206 also abolished BCR mediated Akt activation and decreased Erk activation. Conclusion: MK2206, a robust and selective Akt inhibitor, induced significant in vitro apoptosis of CLL B-cells in vitro. Preclinical evidence of a synergistic effect between MK2206 and Bendamustine was also observed independent of prognostic risk. MK2206 abolished BCR mediated Akt activation and selectively abrogates BCR mediated production of cytokines that may promote apoptotic resistance. These findings support the use of MK2206 in treating CLL and indeed we have initiated a phase I/II trial of MK2206 in combination with Bendamustine and Rituximab for relapsed CLL patients(N1087, October 2011). Acknowledgments: This study was funded by the NCI-K23, NCCTG and CLL Global Foundation. Disclosures: Shanafelt: Cephalon: Research Funding; Genentech: Research Funding. Kumar:Genzyme: Research Funding; Novartis: Research Funding; Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Honoraria. Kay:Celgene: Research Funding.

Abstract 76 Background: We measured proliferation in MM cells transfected with 13,984 small interfering RNAs in the absence/presence of increasing concentrations of bortezomib (BTZ) and identified 37 non lethal genes as reproducible BTZ sensitizers. The strongest hit was CDK5 a gene expressed at high levels in MM and neural tissues with relatively low expression in other organs suggesting a large therapeutic window. The small molecule CDK5 inhibitor Dinaciclib is a novel, potent, small molecule inhibitor of cyclin dependent kinases (CDK). It selectively inhibits CDK1, CDK 2, CDK 5 and CDK9 with 50% inhibitory concentrations (IC50) in the low nanomolar concentration. Based on these data, we undertook the current trial to evaluate the maximally tolerated dose of Dinaciclib in this patient population as well as assess the efficacy of single agent Dinaciclib in relapsed MM. Patients and Methods: Patients with relapsed MM and measurable disease were enrolled on this phase 1/2 trial provided they had not more than 4 prior lines of therapy for MM, had adequate performance status and organ and hematological function. Patients receiving strong inhibitors/inducers of CYP3A4 were not allowed unless the drugs could be discontinued. The primary objectives were to (i) to determine the MTD of dinaciclib in subjects with relapsed MM (phase 1) and (ii) to evaluate the confirmed response rate with dinaciclib in patients with relapsed MM (phase 2). Dinaciclib was administered on day 1 of a 21-day cycle at doses of 30–50 mg/m2. Dose escalation was done using a 3+3 design and MTD was defined as one dose level below that resulted in >=2 DLTs among 6 patients. Results: Overall, twenty-nine patients were accrued to this study (19 phase I, 10 phase II) from July 2009 to December 2011. Two patients were replaced for study violations and are not included in analysis. The Phase II analysis included the 6 patients treated at the Phase I dose level 50mg/m2 as well as the first 9 patients accrued to the Phase II portion (15 evaluable patients). The median age of the 27 evaluable patients was 66 (range, 49–81); 52% were male. The median duration from diagnosis was 42 months (range, 7–207 months); median number of prior therapies was 4 (range, 1–5). Overall, 11 (41%) patients were considered high-risk by FISH and 56% were ISS stage III at study entry. Patients have received a median of 2 cycles (range: 1–12) and all patients have discontinued treatment for disease progression (18), alternative Treatment (2), adverse Event (4), or other reasons (3). One dose limiting toxicity was seen over all dose levels. One patient at 40 mg/m2 experienced grade 3 constipation possibly related to treatment. The dose level of 50 mg/m2 was determined to be the MTD for the Phase II portion. The overall confirmed response rate was 3 of 27 (11%); including two patients at 40 mg/m2 dose (1 VGPR, 1 PR) and one patient at 50 mg/m2 dose (1 VGPR) with a PR or better. In addition, two patients at 50 mg/mg2 dose have achieved an MR; translating to an overall response rate of 18.5% (5 of 27). Figure 1 shows a waterfall plot of the M protein responses among patients with a measurable M spike. Overall, 21 patients had disease progression and 14 patients have died. Median follow-up for patients still alive is 11.8 months (range: 6.5–19.3). Leukopenia and thrombocytopenia were the most common hematological AEs, and gastrointestinal symptoms, alopecia, and fatigue, were the most common non-hematological AEs seen in the study. Conclusions: The current study demonstrates potential single agent anti-myeloma activity of dinaciclib in a relapsed MM patient population with 2 patients achieving a deep response (VGPR) and many patients obtaining some degree of M protein stabilization or decrease. Ongoing studies are examining dinaciclib in combination with proteasome inhibitors, exploring both weekly and every three-week dosing schedules. Disclosures: Zonder: Millennium Pharmaceuticals, Inc: Consultancy, Research Funding; Celgene: Research Funding.

Abstract 2862 Follicular lymphoma (FL) is a B-cell malignancy that exhibits significant intratumoral infiltration by non-malignant T lymphocytes. The pathophysiological significance of infiltrating T cells is poorly understood but recent studies have suggested that CD4+CD25+ regulatory T (Treg) cells are highly represented in lymph nodes involved by FL. These Treg cells display the ability to suppress the proliferation and cytokine production of other tumor-infiltrating T cells and migrate to areas of B-cell lymphoma in response to chemotactic signals provided by the malignant B-cells. Denileukin diftitox, a chimeric immunotoxin composed of the modified cytotoxic domain of diphtheria toxin and human interleukin-2 (IL-2) protein, targets cells expressing CD25 and has proven efficacy in patients with relapsed B-cell lymphoma. In this study, we combined denileukin diftitox with rituximab in a cohort of previously untreated, advanced-stage follicular lymphoma patients. Our hypothesis was that denileukin diftitox would deplete the Treg cells, thereby removing the inhibition of the immune response, and rituximab would deplete the B-cells thereby preventing further recruitment of Treg cells to the areas of lymphoma. Between August 2008 and March 2010, twenty-four patients with stage III and IV follicular grade 1 or 2 non-Hodgkin lymphoma were accrued to the study. One patient died before treatment was given and is not included in the analysis. The median age was 60 years (range: 27 – 79), 12 (52%) of the patients were male, 19 (83%) had a PS of 0 and 4 (17%) had a PS of 1. Based on the Follicular Lymphoma International Prognostic Index (FLIPI), 3 (13%) were low risk, 14 (61%) were intermediate risk and 6 (26%) were high risk. Patients received rituximab 375 mg/m2 on days 1, 8, 15 and 22 and denileukin diftitox 18 mcg/kg/day on days 1–5 every 3 weeks for 4 cycles. A median of 4 cycles of therapy was given (range: 1 – 4). Thirteen patients completed treatment per protocol (57%), however 5 patients discontinued treatment due to adverse events (22%), 2 refused further treatment (9%) and 1 discontinued due to disease progression (4%). Nine of the 23 patients (39%; 95% CI: 21–61%) responded to treatment, 3 (13%) had a complete response and 6 (26%) had a partial response. Twenty-one patients (91%) are alive with a median follow-up of 8.7 months (range: 3.4–19.5). Seven (30%) patients have progressed and two (8.7%) has died. The median time to progression is 13.4 months (95% CI: 10.4 – NA). The combination, however, was associated with significant toxicity. Thirteen patients (57%) experienced grade 3 or greater adverse events. Six patients (26%) had symptoms of capillary leak syndrome, 1 of whom died. In correlative studies performed on the peripheral blood, the number of CD25+ T-cells decreased after treatment when compared to pretreatment numbers (median 24%; range: 8–44%). We conclude that while the addition of denileukin diftitox to rituximab decreased the numbers of CD25+ T-cells, denileukin diftitox contributed significantly to the toxicity of the combination. Furthermore, the overall response rate and time to progression in this study were no better than what would be expected in follicular lymphoma patients treated with rituximab alone. Disclosures: No relevant conflicts of interest to declare.

Abstract 4954 Background Multiple Myeloma (MM) remains incurable with current approaches and newer therapies targeting the abnormal disease biology need to be developed. The malignant plasma cell is highly dependent on the marrow microenvironment, which promotes tumor cell proliferation as well as survival. There is increased bone marrow vascular density in patients with MM and the degree of neo-angiogenesis appear to be prognostic in this disease. Studies have also shown that vascular endothelial growth factor (VEGF) is intricately involved in the interaction between the marrow microenvironment and the myeloma cell. These findings suggested that the VEGF pathway may be a valuable target for therapeutic approach in this disease. Patients and Methods We designed a phase 2 trial of Sunitinib in patients with relapsed multiple myeloma who had received less than three prior therapies. Sunitinib malate (SU11248; Sutent) is a novel, multi-targeted, small molecule inhibitor of the receptor tyrosine kinases (RTKs), including vascular endothelial growth factor receptor-1 (VEGFR-1), -2, and -3, and platelet-derived growth factor receptor (PDGFR) -α and -β. The treatment consisted of Sunitinib 37.5 mg given daily in a continuous fashion in six-week cycles. Toxicities were graded according to CTCAE response version 3.0 and responses were assessed according to the International Myeloma Working Group consensus criteria. Response rate of single agent Sunitinib was the primary endpoint of the study. Results 13 patients were accrued between July of 2007 and April 2009 before the study was closed. The median age of the study population was 61 years (range, 53 - 84), and included nine females (69%). The median time from diagnosis to registration was 39.2 months (range, 4.5 - 88) and 10 patients had received previous stem cell transplant. Previous therapy included lenalidomide in 7 patients, thalidomide in 5 patients and bortezomib in 2 patients. All 13 patients were alive at last follow up with a median follow-up of 11.2 months (range, 1.3 -21.2). All patients have gone off study, disease progression being the most common cause (9 patients), followed by adverse events (3 pts) and alternate treatment in the remaining patient. A hematological grade 3 or higher adverse event was documented in 6 patients and a grade 3 or higher non-hematological adverse event was seen in 8 patients. A grade 3 or higher neutropenia was observed in 5 pts and thrombocytopenia in 2 pts. Fatigue was common and was seen in 11 pts. The median number of cycles administered was one (range, 1-5) and the total number of cycles across 13 patients was 22. Treatment delays or interruptions were seen in five and six patients respectively. The most common reason for treatment delays was hematologic adverse events. No responses (PR or better) were seen, and stable disease was seen in eight of the 13 patients as the best response. The median time to progression was 2.9 months. Conclusions Single agent Sunitinib has no clinically relevant efficacy in this group of patients with relapsed myeloma with one or two previous therapies. Hematological toxicity was higher than anticipated. Whereas these results indicate that VEGFR TK inhibitors are not active in previously treated myeloma, it does not preclude the possibility that VEGF targeting strategies may enhance the efficacy of other therapies. Disclosures Kumar: CELGENE: Research Funding; MILLENNIUM: Research Funding; BAYER: Research Funding; GENZYME: Research Funding; NOVARTIS: Research Funding. Gertz:celgene: Honoraria; millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees. Dispenzieri:Celgene: Research Funding.

BACKGROUND: Green tea has long been touted as a health promoting substance. The active chemical compounds in green tea are called polyphenols or catechins. Epigallocatechin gallate(EGCG) is the major catechin in green tea. We previously reported the in vitro ability of EGCG to induce apoptotic cell death in chronic lymphocytic leukemia(CLL) B-cells in vitro (Blood 104:788). After publication of our findings, clinical activity in individuals using over the counter green tea extracts were reported (Leuk Res 30:707). Based on this information, we opened a phase I/II trial of green tea extracts for patients with asymptomatic, early stage CLL in fall of 2005. METHODS: The purpose of the phase I portion of this trial was to determine the optimal dose of EGCG in the Polyphenon E preparation for chronic daily administration and define tolerability in CLL patients. Previously untreated patients with asymptomatic, Rai stage 0–II CLL not currently meeting National Cancer Institute(NCI) Working Group(WG) Criteria for treatment were eligible for participation. Polyphenon E with a standardized dose of EGCG was obtained from NCI. The phase I portion of the trial was designed with 8 dose levels(range 400–2000 mg orally BID) using the standard 3 patient per dose level design. Patients remained on study up to 6 months. Grade 2 adverse events attributed to study treatment that did not respond to supportive care were considered dose limiting toxicity. The trial was designed to administer Polyphenon E in the fasting state. After accrual to dose levels 1 and 2, the U.S. FDA mandated all U.S. trials of Polyphenon E administer drug in the fed state. Accordingly, drug was administered in the fed state for dose levels 3–8. Trough plasma EGCG levels were measured 1 month after initiation of therapy. Response was classified using the NCI WG Criteria. RESULTS: As of August 2007, 33 patients have been accrued to dose levels 1–8. The maximum tolerated dose(MTD) has not been reached. Side effects have generally been mild. The most common toxicities were nausea(grade 1: 42%; grade 2: 3%), elevation in SGOT (42%; all grade 1), and abdominal pain (36%; all grade 1). To date, no patient has had a sustained 50% reduction in both absolute lymphocyte count (ALC) and lymphadenopathy that would meet the NCI WG criteria for partial response. A majority of patients have had a reduction in ALC(Table). Among the 10 patients who had palpable adenopathy at study enrollment, 7 patients experienced at least a 50% reduction in the sum of the products of all nodal areas at some point during treatment. Trough plasma EGCG levels after 1 month of treatment ranged from 2.93974 ng/mL(median 40.5 ng/mL). Plasma levels did not clearly relate to the degree of reduction in ALC suggesting sensitivity to Polyphenon E may relate more to characteristics of the leukemic clone than plasma EGCG levels. CONCLUSION: Daily oral EGCG in the Polyphenon E preparation was well tolerated by CLL patients in this phase I trial. The MTD has not been reached. As classified by the NCI WG criteria, no partial or complete remissions have been observed to date, however declines in ALC and lymphadenopathy have been observed in the majority of patients. The phase II portion of this trial will open at Mayo Clinic Fall 2007. Best reduction in ALC n % of patients At least 10% decline 25 76% At least 20% decline 14 42% At least 30% decline 8 24% At least 40% decline 4 12% At least 50% decline 2 6%

The proposed immune-mediated mechanisms of action of Rituximab (ADCC or “vaccinal” effect) suggest that the efficacy of rituximab therapy depends on the immunocompetency of the host. Therapies, such as IL-12, that stimulate the immune system may increase the efficacy of rituximab. Recently, we reported that absolute lymphocyte count (ALC), as a surrogate maker of immune recovery, is a survival prognostic factor in autologous stem cell transplantation for non-Hodgkin’s lymphoma (NHL). We hypothesize host immune competence (i.e, normal ALC counts) would predict a superior survival in NHL patients treated with rituximab-based regimens. The primary end-point of the study was to assess whether the ALC count prior to rituximab therapy predicted progression-free survival (PFS) and the second end-point was to identify which lymphocyte subset was responsible for affecting PFS. We studied 41 NHL patients that participated in our Phase I interleukin-12 plus rituximab study. These patients were selected because the Phase I study required baseline ALC count and flow cytometry analysis providing lymphocyte subset data prior to treatment. There were 20 females and 21 males in the study. The median age of the cohort was 53 years (range: 34–84). PFS was assessed from the initial date of treatment. The median follow-up was 42 months (1–84 months). Patients with an ALC ≥ 0.9 × 109/l experienced a superior PFS versus an ALC < 0.9 × 109/l prior to rituximab therapy (31 months vs 4 months, p < 0.0001). The relapse rate in the ALC ≥ 0.9 × 109/l group was 55% (11/20) versus 95 % (20/21) in the ALC < 0.9 × 109/l (p < 0.0016). Patients with a CD4 count ≥ 500 cells/μl also experienced a superior PFS versus a CD4 count < 500 cells/μl (21 months vs 9 months, p < 0.0062). The relapse rate in the CD4 count ≥ 500 cells/μl group was 56% (9/16) versus 88 % (22/25) in the CD4 count < 500 cells/μl (p < 0.0216). No association with PFS was identified for CD3, CD8, CD19, CD16/56, the use of IL-12, or lymphoma type by WHO classification. CD4 count was found to be an independent prognostic factor for PFS. In the subgroup of patients with follicular lymphoma, patients with an ALC ≥ 0.9 × 109/l experienced a superior PFS versus an ALC < 0.9 × 109/l prior to rituximab therapy (35 months vs 9 months, p < 0.0076). The relapse rate in the ALC ≥ 0.9 × 109/l group was 45% (5/11) versus 90 % (9/10) in the ALC < 0.9 × 109/l (p < 0.0243). Patients with a CD4 count ≥ 500 cells/μl also experienced superior PFS versus a CD4 count < 500 cells/μl (not reached vs 9 months, p < 0.0098). The relapse rate in the CD4 count ≥ 500 cells/μl group was 29% (2/7) versus 86 % (12/14) in the group with a CD4 count < 500 cells/μl (p < 0.0216). CD4 count was found to be an independent prognostic factor for PFS when compared to the Follicular Lymphoma International Prognostic Index at the time of treatment. Our data suggest the significance of an immunocompetent host in predicting the efficacy of rituximab therapy. These data support our previous report that patients with higher number of activated CD4+ cells at the lymphoma site have a better prognosis (Ansell et al, JCO2001; 19: 720–6). These findings warrant further studies to understand the role of ALC (i.e, CD4 count) in the mechanism of action of rituximab. Supported in part by CA69912

Background: Myelofibrosis with myeloid metaplasia (MMM) is a progressive and fatal myeloproliferative disorder with limited therapeutic options. R115777 is a non-peptidomimetic enzyme-specific inhibitor of farnesyl protein transferase (FT) which we recently reported has in vitro activity against aberrant myeloid progenitors from MMM patients (Leukemia2003;17:849). This observation, coupled with the clinical activity demonstrated in high risk acute myeloid leukemia (Blood2001;97:3361) and chronic myeloid disorders (Blood2003;101:1692) led us to perform a Phase II trial in patients (pts) with MMM. Methods: Eligible pts had histologically confirmed MMM and were symptomatic, defined by anemia (hemoglobin Results: 34 pts (median age 65 yrs, range 55–80; 71% male) were enrolled in the trial. All pts displayed symptomatic disease at enrollment, with 10 who were erythrocyte transfusion dependent; and 19 (56%) had a Lille MMM prognostic score ≥1; no pts were significantly thrombocytopenic at entry by enrollment criteria. Median pre-study spleen and liver sizes were 12 cm (range: 0–35) and 2 cm (range: 0–15) below the left and right costal margin, respectively. Median time to discontinuation of therapy was 5.5 months (95% CI:2.9–6.4). Reasons for early termination were progression (7), adverse reactions (5), co-morbidities (2), refusal of further treatment (2), and death on study (1). One pt died of grade 5 pneumonitis two weeks after study enrollment, which was felt to be unrelated to study treatment. Significant toxicities (≥ grade 3) were seen for myelosuppression (n=13), neuropathy (n=2), fatigue (n=1), rash (n=1) and hyponatremia (n=1). Overall, R115777 resulted in little improvement in anemia, with the exception of one anemia-based partial response. However, the myelosuppressive aspects of the agent may have masked improvements in cytopenias. In contrast, R115777 did result in clinically relevant decreases in organomegaly in 11 pts (33%) (splenomegaly in 3 pts, hepatomegaly in 5 pts, and both in an additional 3 pts.) Across all pts who had some reduction in splenomegaly, the median maximal reduction was 42.5% (of palpable component; range: 8–100%). Likewise, all patients with baseline hepatomegaly had resolution of this problem at some point during therapy. Four pts remain on the agent (from 14 to 22 months) for continued and sustained improvement in organomegaly. Responses observed did not significantly correlate with reductions in bone marrow fibrosis, osteosclerosis, neoangiogenesis, or resolution of baseline karyotypic abnormalities. Conclusions: R115777 has demonstrated preliminary activity against MMM associated organomegaly. Whether this latter benefit is independent of R115777 induced myelosuppression is unclear. The myelosuppression observed was generally mild and without clinical consequence, although it was difficult to differentiate between myelosuppression versus disease progression. Pts continue to be followed to evaluate durability of observed responses, long term effects on anemia and intramedullary manifestations of the disease.

Introduction Antisense oligonucleotides are used to inhibit messenger RNA function and inhibit protein translation. Bcl-2 expression proteins have been implicated in mediating resistance to apoptotic cell death in Waldenstrom Macroglobulinemia (WM). Oblimersen sodium is a specific inhibitor of Bcl-2 expression and has shown activity in multiple myeloma and in Waldenstrom cell lines. The in vitro data led to the development of a phase I study for WM patients with relapsed or refractory disease. Materials and Methods Eligible patients had symptomatic WM who had failed prior cytotoxic chemotherapy. All patients had to have one of the following: a hemoglobin Results To date 9 patients have been accrued, six at dose level one and three patients at dose level two. Data is available for the first six entered. Five women and 1 man have been accrued with a median age of 74.5 years, ranging from 58 to 81. Four patients had relapsed from prior therapy, 2 were refractory. Three had a history of transfusions. The median number of prior regimens was 4 (range 2–7). One patient had a dose-limiting toxicity on the first cycle of therapy, which was grade 3 fatigue and anorexia, and the dose for this patient was reduced. Two patients had non-hematologic grade 3 toxicity, possibly related to therapy. Five out of six patients on dose level one had grade 3 or greater hematologic toxicity. These toxicities occurred after cycle 1 was completed, during the toxicity observation period and were not considered dose limiting toxicities influencing dose escalation. Three patients required dose reductions in subsequent cycles. To date one patient has had a partial response with her serum M spike falling from 3.6 to 1.1 g/dL. Her quantitative IgM fell from 6,000 mg/dL to 1,000 mg/dL(figure). Conclusion Oblimersen was well tolerated at the first dose level in patients with WM. Hematologic toxicity is frequent in this cohort with heavily involved bone marrows when the dose was escalated during successive cycles. Dose reductions with subsequent cycles were frequently required. Evaluation of the maximum tolerated dose continues. Data on response for this dose level is promising, where efficacy will be fully evaluated in the phase II portion of this trial. Figure Figure

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