We have previously characterized the reproducibility of brain tumor relative cerebral blood volume (rCBV) using a dynamic susceptibility contrast magnetic resonance imaging digital reference object across 12 sites using a range of imaging protocols and software platforms. As expected, reproducibility was highest when imaging protocols and software were consistent, but decreased when they were variable. Our goal in this study was to determine the impact of rCBV reproducibility for tumor grade and treatment response classification. We found that varying imaging protocols and software platforms produced a range of optimal thresholds for both tumor grading and treatment response, but the performance of these thresholds was similar. These findings further underscore the importance of standardizing acquisition and analysis protocols across sites and software benchmarking.

The use of rCBV as a response metric in clinical trials has been hampered, in part, due to variations in the biomarker consistency and associated interpretation across sites, stemming from differences in image acquisition and post-processing methods. This study leveraged a dynamic susceptibility contrast magnetic resonance imaging digital reference object to characterize rCBV consistency across 12 sites participating in the Quantitative Imaging Network (QIN), specifically focusing on differences in site-specific imaging protocols (IPs; n = 17), and PMs (n = 19) and differences due to site-specific IPs and PMs (n = 25). Thus, high agreement across sites occurs when 1 managing center processes rCBV despite slight variations in the IP. This result is most likely supported by current initiatives to standardize IPs. However, marked intersite disagreement was observed when site-specific software was applied for rCBV measurements. This study's results have important implications for comparing rCBV values across sites and trials, where variability in PMs could confound the comparison of therapeutic effectiveness and/or any attempts to establish thresholds for categorical response to therapy. To overcome these challenges and ensure the successful use of rCBV as a clinical trial biomarker, we recommend the establishment of qualifying and validating site- and trial-specific criteria for scanners and acquisition methods (eg, using a validated phantom) and the software tools used for dynamic susceptibility contrast magnetic resonance imaging analysis (eg, using a digital reference object where the ground truth is known).

BackgroundAnti-angiogenic therapy is known to induce a greater degree of hypoxia, including in glioblastoma (GBM). Evofosfamide (Evo) is a hypoxia-activated prodrug which is reduced, leading to the release of the alkylating agent bromo-isophosphoramide mustard. We assessed the safety, tolerability, preliminary efficacy, and biomarkers of Evo plus bevacizumab (Bev) in Bev-refractory GBM.MethodsTwenty-eight patients with Bev-refractory GBM were enrolled in a dose escalation study receiving from 240 mg/m2 (cohort 1) to 670 mg/m2 (cohort 4) of Evo every 2 weeks in combination with Bev. Patients deemed surgical candidates underwent a single dose of Evo or placebo with pimonidazole immediately prior to surgery for biomarker evaluation, followed by dose escalation upon recovery. Assessments included adverse events, response, and survival.ResultsEvo plus Bev was well tolerated up to and including the maximum dose of 670 mg/m2, which was determined to be the recommended phase II dose. Overall response rate was 17.4%, with disease control (complete response, partial response, and stable disease) observed in 14 (60.9%) of the 23 patients. The ratio of enhancement to non-enhancement was significant on log-rank analysis with time to progression (P = 0.023), with patients having a ratio of less than 0.37 showing a median progression-free survival of 98 days versus 56 days for those with more enhancement.ConclusionsEvo plus Bev was well tolerated in patients with Bev-refractory GBM, with preliminary evidence of activity that merits further investigation.

Glioblastoma (GBM, WHO grade IV) is the most common and aggressive of the primary brain tumors in adults. As part of an ongoing clinical trial with a hypoxia activated prodrug, we sought to explore the correlation between abnormal tumor vasculature and hypoxia in GBM following bevacizumab (BEV) failure. MRI and 18F-FMISO PET scans were acquired at study entry. Associations between baseline hypoxia volume (HV), SUV peak, as measured by FMISO PET and vascular parameters including rCBV, TTP, enhancing and non-enhancing tumor volumes as measured by dynamic susceptibility contrast imaging (DSC), T1 post contrast and FLAIR was determined using Pearson correlation. Each marker was modeled with a univariate cox regression model for progression free survival (PFS) time. 8 patients from University of Texas Health Science center and 14 patients from Dana-Farber Cancer Institute had evaluable MR and PET imaging. Significant positive correlations were found between HV and enhancing volume (R=0.6884, p<0.0001) as well as volume ratio and srCBV (R=0.6032, p<0.0001). Moderate positive correlation were found between volume ratio and HV (R=0.5049, p=0.0165) as well as volume ratio and SUVpeak (R=0.5669, p=0.0059). Nonenhancing volume is associated with HV (R=0.2602, p=0.0153) so is SUVpeak and srCBV (R=0.3253, p=0.0056). In the univariate cox model, bigger enhancing (p=0.043), nonenhancing (p=0.0061) and HV (p=0.0172) were significantly associated with shorter progression time. The hypoxic volume following bevacizumab failure correlates with both the volume of enhancement and the fraction of enhancement within the mass. Findings from the full 23 patient cohorts and the association with other biomarkers and response to treatment is being assessed and will be presented.

TH-302 (evophosphamide) is a hypoxia-activated prodrug that, when activated in hypoxic conditions (<0.5% O2), releases the bis-alkylating agent bromo-isophosphoramide mustard (Br-IPM), which can then act as a DNA crosslinking agent. In vivo, TH-302 has shown to potentiate the anti-tumor efficacy of other antiangiogenic agents. Pur prior phase 1 study with dose expansion showed preliminary activity with a 24% objective response rate and a 26% PFS rate at 4 months. A multicenter, single-arm, two-stage prospective study, non-blinded with combination therapy with bevacizumab at 10 mg/kg intravenously (IV) every 2 weeks and TH-302 at 670 mg/m2 IV every 2 weeks, in 6 week cycles, until disease progression. The primary endpoint is progression free survival at 4 months. Patients underwent baseline assessment for hypoxic burden by 18F-misonidazole PET, dynamic susceptibility contrast (DSC) perfusion imaging, and serum sampling for biomarker analysis. Accrual is complete with 36 patients receving study drug. Treatment was well tolerated, with adverse events as expected and the most common toxicity rash along the perineum. While 6 patients remain on treatment, 6 of the required 7 PFS4 events have occured with one more needed to meet the designated positive endpoint. Correlation with imaging and serum biomarkers is ongoing and may help further define the subset of patients with the greatest benefit. Evofosfamide appears to have clinically meaningful activity in bevacizumab refractory glioblatoma. Results will be updated.

2045 Background: Glioblastoma (GBM) is the most common malignant brain tumor in adults, and is characterized by poor survival and marked resistance to treatment. Hypoxic volume is directly correlated with poor outcome in GBM, with structural and functional tumor vasculature changes regarded as a primary driver of tumor hypoxia. As part of an ongoing clinical trial with a hypoxia activated prodrug, we sought to explore the correlation between abnormal tumor vasculature and hypoxia in bevacizumab (BEV) resistant GBM. Methods: MRI and 18F-FMISO PET scans were acquired at study entry. The MRI protocols include standard anatomical sequences as well as Dynamic Susceptibility Contrast (DSC/perfusion) imaging. Enhancing tumor ROIs were determined from Delta T1 maps, non-enhancing tumor ROIs were obtained from FLAIR images, ratio between enhancing and non-enhancing volumes were calculated, values from standardized rCBV maps were extracted from corresponding enhancing ROIs. Decay correction was applied to18F-FMISO images and converted to SUV units. After registration to MR images, cerebellar regions were used as surrogates for blood activity. The FMISO images were then normalized to generate tissue to blood ratio (TB). A threshold of TB > 1.2 was used in discriminating the hypoxia volume (HV). Correlation between parameters was assessed using Pearson correlation. Results: 7 patients from University of Texas Health Science center and 7 patients from Dana-Farber Cancer Institute had evaluable MR and PET imaging. We compared MRI parameters (enhancing and non-enhancing tumor volumes, srCBV) with hypoxia (HV and maxSUV). There was a positive correlation between HV and enhancing volume (R = 0.732, p = 0.0029) as well as between HV and ratio (R = 0.644, p = 0.013. The correlation between other pairs were not statistically significant (HV vs srCBV: p = 0.862, T1 vs maxSUV p = 0.492, srCBV vs maxSUV: p = 0.393, ratio vs maxSUV = 0.178). Conclusions: The hypoxic volume following bevacizumab failure correlates with both the volume of enhancement and the fraction of enhancement within the mass. The clinical implications of this is being assessed in an ongoing phase 2 study. Clinical trial information: NCT02342379.

VB-111 (Ofranergene Obadenovec) is non-integrating, non-replicating, Adeno 5 vector which targets the vasculature through induction of endothelial apoptosis. Our prior phase 2 study has shown a nearly doubling of overall survival (OS) in recurrent glioblastoma (rGBM) (59v32 weeks), and our pivotal phase 3 GLOBE study is fully accrued. To better understand any predictors of benefit and better define alternative mechanisms of action we assess both molecular and radiographic biomarkers of response. Tumor volumes were determined from T1-post and FLAIR T2 weighted images, and values extracted from ROIs of ADC, MTT, standardized rCBV maps. Baseline serum and 6h post infusion underwent cytokine profiling using a luminex array. To delineate the source of cytokines orthotopic U251 tumors were treated with VB-111 or control, resected, microglia (CD11b) and T cells (CD3) sorted, and individually cocultured with activated splenocytes. In the univariate cox analysis, baseline srCBVs for both VB-111 and combination groups demonstrated statistically significant association with OS (p=0.0198 and p=0.0458 respectively). In the VB-111 group, post-treatment srCBV also demonstrated significant association with OS (p=0.024). Whereas, in combination group, baseline MTT is significantly associated with OS (p=0.0323). Several cytokines 6h post VB-111 infusion correlated with overall survival including IL-1Rα, IL-17α, MIP1α, and TNFα (p<0.05). When U251 tumor microglia and T cells were cocultured with splenocytes and activated with LPS (72h), several cytokines including MIP1α, MIP1β, IL-10 and RANTES were significant higher from mice administered VB-111 (p<0.05). Conversely, using CD3/CD28 for activation, a decrease in immunosuppressive cytokines was observed including IL-10, 4, 3, and 5. These results suggested that tumoral microglia are responsible for cytokine release with a profile that promotes a cytotoxic T cell response. Improved survival in VB-111 treated patients correlates with both vascular imaging parameters and cytokine response, supporting both immune and anti-angiogenic mechanisms of this anti-cancer virotherapy.