Citations

2031 Background: The mammalian target of rapamycin (mTOR) functions within the PI3K/Akt pathway as a critical modulator of cell survival. Preclinical studies in GBM indicate that the combination of mTOR inhibitors, such as everolimus (RAD001), with either radiation therapy (RT) or temozolomide (TMZ) provide increased tumor cell killing. Methods: Newly diagnosed GBM pts were eligible for the study. RAD001 was dosed orally at 70 mg/week weekly, starting 1 week prior to RT/TMZ, and continued throughout RT/TMZ, adjuvant TMZ and then until progression. This was a single arm phase II design powered to detect a true overall survival at 12 months (OS12) of 73% (vs 58% in historical controls). Secondary endpoints were toxicity, response rate, and time to progression (TTP). A subgroup of patients with measurable residual disease were eligible for the PET imaging component of the study, consisting of an 18FLT-PET/CT scan performed before and after the initial two doses of RAD001 but before the first dose of RT or TMZ. Results: 103 patients were accrued to phase II of which 100 were evaluable. The median age was 60.5 years (23-81), median ECOG PS was 1, 46 patients had GTR, 33 STR, and the remainder had biopsy at diagnosis. Treatment tolerance was acceptable: 17% patient had at least one grade 3 hematologic toxicity; 14% had at least one grade 4 hematologic toxicity, 42% had at least one grade 3 non-heme toxicity, while 12% had at least one grade 4 non-heme toxicity. No increased incidence of infectious complications was observed and there were no treatment related deaths. Median PFS was 5.3 months (1.3-21.4), with 22 patients progression free. Mature OS data will be available at the meeting. MGMT methylation status analysis is ongoing. Of the pts who had evaluable FLT-PET data, three of eight (37.5%) had a drop in SUVmax >25% after two treatments of RAD001. Conclusions: RAD001 with standard of care chemoradiation had moderate toxicity. Serial 18FLT-PET was feasible for evaluating drug-induced changes in tumor proliferation following RAD001. Final outcome data and association of MGMT status with outcome will be reported at the meeting.

2046 Background: Vorinostat (VOR) is a histone deacetylase inhibitor that represents a rational targeted agent in GBM treatment. Given its single-agent activity in recurrent disease (Galanis,et al, 2009) and radiosensitizing properties, this phase I/II trial was designed to test the addition of VOR to standard chemoradiation in newly diagnosed GBM patients (pts): the phase I portion of the trial is the focus of this report. Methods: A standard cohorts of three design was used to assess the safety of VOR in combination with RT and concomitant TMZ and establish the phase II dose of the combination. VOR was given orally days 1 - 5 every wk beginning with the first dose of RT (total dose 60 Gy) and (75mg/m²/day). Following a 4 - 6 week rest, pts received up to 12 cycles of standard adjuvant TMZ in combination with VOR on days 1-7 and 15 – 21 of each cycle; dose was based on NABTT trial 04-03 (Lee, et al, 2012). Results: The phase I component is complete with 15 pts, 12 pts at dose level 0 (VOR 300 mg/day days 1 - 5, weekly x 6 wks), and 3 pts at dose level 1 (VOR 400 mg/day, days 1 – 5 weekly x 6 wks) in combination with RT/TMZ. Dose limiting toxicity (DLT) in dose level 1 included grade 3 fatigue in 2 pts, grade 3 wound dehiscence in 1 pt, and grade 4 neutropenia and thrombocytopenia in 1 pt. In dose level 0, 1/6 pts had DLT (gr 3 dyspnea). An MTD expansion cohort of 6 additional patients was added to dose level 0; one patient experienced grade 4 thrombocytopenia and grade 3 fatigue, and 1 patient experienced grade 3 febrile neutropenia. In the 12 pts treated in the phase II dose, most common toxicities were hematologic, including lymphopenia (gr 3/4 in 66.7%), thrombocytopenia (gr 3 in 16.7%, gr 4 in 16.7%) and neutropenia (gr 3 in 16.7%, gr 4 in 8.3%). Grade 3 fatigue was observed in 8.3% of the pts. Conclusions: MTD for VOR in combination with TMZ/RT in newly diagnosed GBM patients is 300 mg/d, days 1 - 5 weekly during RT. Toxicity was primarily hematologic. This dose was used in the recently completed phase II trial of the combination (110 pts). RNA expression profiling in patient samples is in process to assess vorinostat responsive signatures observed in preclinical models. Clinical trial information: NCT00731731.

2014 Background: PFS6 is low (2.3%) and med OS brief (4 mos) for recurrent GBM pts who progress after prior RT/TMZ and bevacizumab (Bev), and then receive non-Bev containing regimens (Reardon et al, Br J Canc 2012). Following VEGFi exposure, tumor cell resistance potentially may result from activation of Ras/Raf/MEK/ERK (MAPK) and PI3K/Akt/mTOR signaling pathways. Temsirolimus (mTOR inhibitor) and sorafenib (Raf, PDGFR, VEGFR inhibitor) have previously shown limited single agent activity in GBM. Methods: Recurrent GBM pts who showed radiographic progression despite prior surgery, RT + temozolomide, and VEGFi (initially or at recurrence) received sorafenib 200 mg PO bid and temsirolimus 20 mg IV weekly (the NCCTG N0572 phase I MTD) until progression. All pts had received < 2 prior chemo regimens. A two-stage design was used, with 90% power to detect an increase in PFS6 from 8% to 23%, requiring 41 evaluable pts. The primary endpoint was PFS6; secondary endpoints included OS, TTP, and ORR. Toxicity (TOX) was assessed using CTC ver 3.0. Results: 44 evaluable pts were accrued. Four of the first 40 (10%) reached PFS6, but this did not meet the pre-study threshold for success. Median TTP was 1.8 mos. (40 events, 95% CI: 1.5 – 2.3); median OS was 5 mos. (36 events, 95% CI: 6.6 – 3.1), and no objective responses were reported. Eight pts are alive; 4 have not progressed (median F/U 3.1 mos.; max, 12.2 mos.). Grade 3+ non-heme AE were observed in 51% (23/45; fatigue-5, hypophosphatemia-7, hypercholesterolemia-5, hypertriglyceridemia-5). One grade 4 hematologic AE (thrombocytopenia) occurred. 13.3% (6/45 pts) went off due to TOX, and 71.1% (32/45) due to disease progression. Conclusions: Sorafenib plus temsirolimus was tolerable, but the primary endpoint threshold for success (PFS6) was not met in post-VEGFi recurrent GBM patients. Nevertheless, PFS6 (10%) and OS (5 mos.) compared favorably to contemporary series of Bev-refractory, recurrent GBM pts who are subsequently treated with a non-Bev containing regimen. Supported by NCI CA-25224. Clinical trial information: NCT00329719.

7073 Background: In preclinical studies, combinations of bortezomib and RT result in synergistic tumor killing (Cancer Chemother Pharmacol. 2007;59:207-15). Our group reported the results from the phase I portion of this study previously (J Clin Oncol. 200;28 (suppl; abstr 7085)). Based on these data, we undertook a phase II study of bortezomib in combination with paclitaxel/CBCDA and RT. Methods: Based on results from our previously reported phase I trial, systemic therapy included bortezomib (1.2 mg/m² IV days 1,4,8,11), paclitaxel (175 mg/m² IV day 2), and carboplatin (CBCDA; AUC=6 day 2) given every 3 weeks for 2 cycles. Thoracic radiotherapy (RT) included 60Gy/30 daily fractions starting day 1. The primary endpoint was the 12-month survival rate. If 41 or more of these 60 patients (68%) were alive at least 12 months after study entry, the trial would be deemed as positive and further study would be warranted. Results: 27 pts were enrolled to the phase II portion: M/F=17/10; stage IIIA/IIIB=13/14; ECOG PS 0/1=9/18; and median age: 58 (range 43-79). 18 pts (67%) completed therapy per protocol. At a planned interim analysis, 23 of 26 evaluable patients (88.5%, 95% CI: 70-98%) survived for at least 6 months, which exceeded the boundary of 21 or more needed to continue to full accrual. This trial could have continued per protocol, but was closed early due to slow accrual. With a median follow-up of 15.0 months in the 17 patients still alive, the 12-month survival rate was 71% (95% CI: 49 – 85). The median OS was 19 months (95% CI: 11 – no upper). Grade 3 or higher adverse events (regardless of attribution) were reported in 22 (82%) patients. Grade 4/5 adverse events were reported in 15 (56%) patients. There was one grade 5 event (pneumonitis). The most common (5 or more patients) grade 3/4 adverse events were fatigue (22%), leukopenia (63%), neutropenia (67%), and thrombocytopenia (44%). Conclusions: The addition of bortezomib resulted in high levels of severe hematologic toxicity, but also demonstrated evidence of activity with a 12-month survival rate of 71% and a median OS of 19 months. Further testing of this regimen in a larger study appears warranted.

2032 Background: Dasatinib is a potent oral ATP competitive multi-targeted kinase inhibitor of multiple members of the Src kinase family, known to be involved in gliomagenesis and invasion. N0877 is a phase I/randomized phase II trial evaluating the combination of dasatinib, radiation (RT) and temozolomide (TMZ) in newly diagnosed GBM. The phase I portion has been completed and is the focus of this report. Methods: A cohorts-of-3 design was used to assess the safety of dasatinib in combination with RT and concomitant TMZ, and establish the phase II dose of the combination. Dasatinib was given orally for 42 days, beginning with the first day of RT (total dose 60 Gy) and first dose of TMZ (75 mg/m2/d). A 24 - 42 day rest (cycle 2) followed the RT/TMZ/dasatinib. Patients (pts) were observed for DLT to the end of cycle 2. Patients then received 6 cycles (28 day cycles) of dasatinib (once daily) and TMZ (days 1-5). At the completion of 6 cycles of TMZ + dasatinib, pts stay on dasatinib only (28 day cycles) until progressive disease. Results: Phase I component is complete with 13 patients (3 at dose level 0, 3 at dose level 0-A, 7 at dose level 1). One patient in dose level 1 had to be replaced due to the development of unrelated medical issues. One DLT (grade 4 pancytopenia) was observed in 1 patient at dose level 0 (50mg bid) and one DLT (grade 3 rash) was observed in 1 patient at dose level 1 (150mg qd). MTD of dasatinib was determined to be 150mg daily. Most common adverse events throughout the entire study period were hematologic with the most common toxicity being lymphopenia (grade 3 in 69% of patients, grade 4 in 8%). Other toxicities attributed to treatment and occurring in > 10% of patients included anemia (31%), neutropenia (15%), and fatigue(15%). Best response was stable disease in 10 patients, progressive disease in 1 patient, and not evaluable in 2. Conclusions: MTD for dasatinib in combination with TMZ and RT in newly diagnosed GBM patients is 150mg daily. Toxicity was primarily hematologic with minimal non-hematologic events. This dose is currently being used in the ongoing placebo-controlled, randomized phase II trial.

1509 Background: Lack of MGMT methylation in GBM tumors is associated with a worse outcome with combined radiation (RT) and temozolomide (TMZ) therapy, and it is unclear whether non-methylated tumors benefit from TMZ treatment. Methods: The potential benefits of concomitant or adjuvant TMZ relative to methylation status were evaluated in 10 GBM tumors established from patient samples and maintained by serial passage in mice. For each tumor line, 32 mice with established orthotopic xenografts were randomized to A) placebo, B) TMZ PO daily × 5 (120 mg/kg or 66 mg/kg, as indicated) C) RT, 2 Gy bid × 10 fx., D) combined TMZ and RT, and mice were followed until moribund. Efficacy was evaluated by regression modeling and Kaplan-Meier statistical analyses. Results: Six lines with non-methylated MGMT (GBM10, 14, 26, 28, 36, 43) and 4 with methylated MGMT (GBM8, 12, 16, 34) were evaluated, and complete results are available in 6 lines. In all tumor lines and at both dose levels, there was a significant benefit for TMZ relative to placebo (p<0.003). Both methylated tumors (GBM8 and 12) and one non-methylated tumor (GBM14) were markedly sensitive to TMZ with greater than a 50 day extension in survival with monotherapy. Individually and in a pooled analysis, there was no evidence for a synergistic interaction between TMZ and RT in the non-methylated tumors (p=0.91 for pooled analysis). In contrast, there was a striking degree of synergy in GBM12 (p<0.001) and at least additive effects of combined treatment in GBM8 (5 of 8 mice still surviving in the RT/TMZ arm). In a pooled analysis of these 2 MGMT methylated tumors, there was a trend towards synergy (p=0.11). Conclusions: TMZ treatment did not sensitize MGMT non-methylated tumors to radiation, and with one exception, these non-methylated tumors were only modestly sensitive to TMZ alone. In contrast, treatment with TMZ appears to sensitize MGMT-methylated tumors to radiation and monotherapy is associated with marked and significant benefit. [Table: see text] No significant financial relationships to disclose.

2038 The effects of combining histone deacetylase (HDAC) inhibitors and proteasome inhibitors were evaluated in both established glioblastoma multiforme (GBM) cell lines and short-term cultures derived from the Mayo Clinic xenograft GBM panel. Co-exposure of LBH589 (LBH) and botezomib (btzmb) results in a striking induction of apoptosis, at minimally toxic doses of either drug alone, in established U251, U87, and D37 GBM cell lines, as well as in GBM8 (G-8), GBM10 (G-10), GBM12 (G-12), GBM14 (G-14), and GBM56 (G-56) cells, which have been developed from the Mayo xenograft GBM panel and maintained in short-term cultures. Synergism of apoptosis induction was also observed in U251 cells when co-exposing other HDAC inhibitors, including LAQ824 (LAQ) and trichostatin A (TSA), with another proteasome inhibitor MG132, demonstarting a class effect. In U251 cells, btzmb alone or in combination with LBH decreased Raf-1 levels and inactivated Akt and Erk. Either LBH589 or bortezomib alone increased expression of the cell cycle regulators p21 and p27. Additionally, the combination, but not the individual agents, markedly increased JNK activation. In addition, cell death after exposure to LBH589 and bortezomib is associated with cytochrome c release, Bax translocation, as well as caspase and PARP cleavage. Bax translocation may contribute to the synergistic cytotoxicity of the combination of LBH and btzmb in glioma cells since Bax translocation to mitochondria is known to precede cytochrome c release and apoptosis, but other pathways are likely to be involved. This combination regimen warrants further preclinical and possible clinical study for glioma patients. No significant financial relationships to disclose.

OBJECTIVE Convection-enhanced delivery (CED) and osmotic pump delivery both have been promoted as promising techniques to deliver drugs to pediatric diffuse intrinsic pontine gliomas (DIPGs). Correspondingly, the aim of this study was to understand how infusate molecular weight (MW), duration of delivery, and mechanism of delivery (CED or osmotic pump) affect volume of distribution (V d ) in the brainstem, to better inform drug selection and delivery in future DIPG investigations. METHODS A series of in vivo experiments were conducted using rat models. CED and osmotic pump delivery systems were surgically implanted in the brainstem, and different MW fluorescent dextran beads were infused either once (acute) or daily for 5 days (chronic) in a volume infused (V i ). Brainstems were harvested after the last infusion, and V d was quantified using serial sectioning and fluorescence imaging. RESULTS Fluorescence imaging showed infusate uptake within the brainstem for both systems without complication. A significant inverse relationship was observed between infusate MW and V d in all settings, which was distinctly exponential in nature in the setting of acute delivery across the 570-Da to 150-kDa range. Chronic duration and CED technique resulted in significantly greater V d compared to acute duration or osmotic pump delivery, respectively. When accounting for V i , acute infusion yielded significantly greater V d /V i than chronic infusion. The distribution in CED versus osmotic pump delivery was significantly affected by infusate MW at higher weights. CONCLUSIONS Here the authors demonstrate that infusate MW, duration of infusion, and infusion mechanism all impact the V d of an infused agent and should be considered when selecting drugs and infusion parameters for novel investigations to treat DIPGs.

Abstract BACKGROUND Glioblastomas show marked intra- and inter-tumor heterogeneity and are strongly resistant to both radio- and chemo-therapy, which are standard therapeutic modalities for this tumor. MicroRNAs (miRNAs) have the potential to serve as effective therapeutics for glioblastoma as they modulate the activity of multiple signaling pathways. METHODS Glioblastoma cultures were transfected with miR-34a or control miRNA mimics to assess biological function and therapeutic potential in vitro. miR-34a was packaged into bacterially-derived nanocells and administered intravenously for delivery to orthotopic patient-derived glioblastoma xenografts in mice. RESULTS Overexpression of miR-34a strongly reduced the activation status of the three core signaling networks that have been found to be deregulated in the vast majority of glioblastoma tumors, the receptor tyrosine kinase, p53 and Rb networks. miR-34a transfection also inhibited the survival of multiple established glioblastoma cell lines as well as primary patient-derived xenograft cultures representing the proneural, mesenchymal and classical subtypes. Transfection of miR-34a synergized with temozolomide (TMZ) in in vitro cultures of glioblastoma cells with primary TMZ sensitivity, primary TMZ resistance and acquired TMZ resistance. Intravenous administration of bacterially-derived nanocells carrying miR-34a strongly enhanced TMZ sensitivity in an orthotopic patient-derived xenograft mouse model of glioblastoma. CONCLUSIONS miR-34a strongly sensitizes a wide range of glioblastoma cell cultures to TMZ, suggesting that combination therapy of TMZ with miR-34a may serve as a novel therapeutic approach for the treatment of glioblastoma tumors. Bacterially-derived nanocells are an effective vehicle for the delivery of miR-34a to glioblastoma tumors.