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Objective: Ovarian cancer often presents with advanced stage disease. Despite surgery and chemotherapy, recurrence remains inevitable and prognosis poor. Advances in immunotherapy including programmed cell death-1 (PD-1) inhibition show promise, however clinical trials show modest response rates as a single agent therapy in ovarian cancer patients. Radiation therapy is an attractive strategy to enhance the efficacy of immune checkpoint blockade in ovarian cancer due to its ability to increase tumor-infiltrating lymphocytes and enhance immune activation. While ovarian cancer is known to be radiosensitive, total abdominal radiation is not routinely utilized due to high abdominopelvic toxicity. We have recently shown that ultra-high dose rate FLASH irradiation reduces intestinal radiation-induced toxicity. Importantly, we demonstrated efficient tumor control in preclinical models of ovarian cancer peritoneal metastasis. Here we sought to determine the efficacy and safety of FLASH irradiation combined with PD-1 inhibition in preclinical models of ovarian cancer. Methods: Female C57BL/6 mice were intraperitoneally inoculated with ID8-Asc or UPK10 ovarian cancer cells. Total abdominal FLASH irradiation at 14 Gy was administered 10 days post-inoculation. Anti-PD-1 and isotype control antibodies were injected on days 7, 10, and 13 post-inoculation. Mice were sacrificed 22-27 days post-inoculation for tumor burden and immune cell analysis. Stool count, stool weight, complete blood count, and survival were evaluated to compare acute and chronic toxicity as well as efficacy in the treatment arms. Results: Four cohorts of mice were analyzed: Isotype control antibody (IgG), anti-PD-1 (aPD-1), FLASH + IgG, and FLASH + aPD-1. In the ID8-Asc model, irradiated mice showed a 44% decrease in solid stool production with subsequent recovery to baseline by 14 days post-treatment. There was no evidence of hematopoietic toxicity in any of the treatment arms. Survival analysis is ongoing. Exploratory necropsy demonstrated significant tumor burden control in the FLASH cohorts, and in particular, a 25-fold decrease in tumor number in the FLASH + aPD-1 cohort compared to control (mean tumor weight 1.82 mg vs. 357.2 mg, respectively). FLASH + aPD-1 increased intratumoral CD4+ and CD8+ T cells and correlated with enhanced tumor control compared to FLASH or aPD-1 single agent treatments. Similar results were observed in the UPK10 model. Conclusion: Combination FLASH and PD-1 inhibition enhanced tumor control and recruitment of tumor infiltrating lymphocytes compared to IgG, aPD-1, and FLASH treatments alone, while demonstrating safety and tolerability. Our data identify a new opportunity for FLASH and checkpoint inhibition in the treatment of ovarian cancer. Citation Format: Stephanie Chow, Joshua T. Eggold, Karen Levy, Jinghui Wang, Rakesh Manjappa, Dylan Y. Breitkreutz, Amy S. Yu, Karl Bush, Oliver Dorigo, Billy W. Loo, Erinn B. Rankin. Total abdominal ultra-rapid FLASH irradiation enhances the efficacy of PD-1 inhibition in preclinical models of ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5357.

Radiation therapy is the most effective cytotoxic cancer therapy available for the treatment of localized tumors. However, radiation-induced toxicity to normal tissues limits the radiation dose and therefore the curative potential of radiotherapy. In particular, the highly radiosensitive intestine greatly limits the use of radiation for patients with intra-abdominal tumor diseases including women with ovarian cancer. Here we sought to investigate the safety and efficacy of FLASH radiation therapy in the treatment of widespread ovarian cancer peritoneal metastases. We performed abdominal irradiation on healthy and ovarian tumor-bearing mice at conventional (CONV, (0.07 Gy/sec)) or FLASH (200 Gy/sec) dose rates and examined gut function by stool counts, DNA damage in crypt cells by γ-H2AX staining, cell death and proliferation by TUNEL/ caspase-3 staining and BrdU immunohistochemistry respectively. We report that ultrahigh dose rate FLASH irradiation causes significantly less radiation-induced intestinal injury in both healthy and tumor-bearing mice compared to CONV dose rate irradiation. Abdominal FLASH reduced the mortality from gastrointestinal syndrome, preserved gut function and epithelial integrity as reflected by their stool counts and FITC-Dextran analysis. In addition, we found decreased cell death and enhanced proliferation of crypt base columnar cells (CBCs) following FLASH irradiation in comparison to CONV irradiation. We also detected reduced number of γ-H2AX foci in crypt cells indicating less DNA damage and/or increased DNA repair after FLASH compared to CONV irradiation. Importantly, FLASH and CONV irradiation have similar efficacy in the reduction of ovarian cancer peritoneal metastases. These findings suggest that FLASH irradiation has biological advantages compared to conventional dose rate irradiation in reducing radiation-induced intestinal injury within the irradiation field and therefore may be an effective strategy to enhance the therapeutic index of radiotherapy for the treatment of abdominal and pelvic tumor disease. Citation Format: Suchitra Natarajan, Karen Levy, Jinghui Wang, Stephanie Chow, Joshua Eggold, Phoebe Loo, Rakesh Manjappa, Frederick M. Lartey, Emil Schüler, Lawrie Skinner, Marjan Rafat, Ryan Ko, Anna Kim, Duaa Al Rawi, Rie von Eyben, Oliver Dorigo, Kerriann M. Casey, Edward E. Graves, Karl Bush, Amy S. Yu, Albert C. Koong, Peter G. Maxim, Billy W. Loo, Erinn B. Rankin. FLASH irradiation enhances the therapeutic index of abdominal radiotherapy in mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5351.