Background: Arsenic Trioxide (ATO) demonstrated prolonged survival in a transplanted rodent glioblastoma (GBM) model. Given the lack of effective therapies before temozolomide was available, the NCI funded NABTT Brain Tumor Consortium initiated a Phase I study of ATO with radiation in patients with newly diagnosed glioblastoma. Dose escalations using two different schedules of ATO were studied. Methods: Patients with newly diagnosed GBM (MGMT and IDH mutation status unknown) were assigned to receive ATO once or twice weekly during the 6-week course of radiation. The starting ATO dose was 0.25 mg/kg. This was escalated by 0.05 mg/kg until an MTD was reached. Patients did not receive ATO or other adjuvant therapy once the radiation was completed. As ATO affected tumor vasculature and blood flow in experimental imaging studies, perfusion MRIs were obtained before ATO administration and one and six weeks later. Survival was a secondary endpoint. Results: Thirty-one patients were enrolled: median age 54.9 years, 68% male, 77% patients KPS >90, 77% had tumor resection. The MTD was determined to be 0.4mg/kg on the weekly schedule and 0.3mg/kg on the biweekly schedule. ATO was well tolerated with 81% of patients completing the 6 weeks combined treatment. Major dose limiting toxicities were increased aspartate and alanine aminotransferases and one case of hyperkalemia. For all patients the median overall survival (mOS) was 17.7 months and the median progression-free survival (mPFS) was 5.4 months. However, there was a significant survival difference between the two ATO dose schedules across all doses with a mOS of 22.8 months in the biweekly schedule, vs 12.1 months in the weekly schedule (P = 0.039). There was a similar difference in mPFS (10.2 months vs 3.2 months, respectively, P = 0.004). Similarly, there were significant differences in perfusion imaging between the two ATO dose schedules with relative cerebral blood flow showing a significant decrease after one week of ATO treatment on the biweekly schedule (p = 0.007). Conclusions: TheMTDs of ATO in patients with GBM administered once or twice weekly for six weeks with standard radiation were determined. This regimen was well tolerated. Survival in patients on the biweekly schedule was much better (22.8 months) than expected in patients not receiving temozolomide. In addition, patients on biweekly ATO also had significant changes on MRI perfusion studies suggesting a pharmacodynamic effect of this agent. These finding should be explored further in patients with newly diagnosed MGMT unmethylated GBM where temozolomide has very limited activity. Clinical trial information: NCT00045565.