Shannon Grabosch

PD-L1 biology in response to chemotherapy in vitro and in vivo in ovarian cancer

Meeting abstracts PD-L1 is an immune checkpoint molecule expressed by a variety of tumors, including ovarian, which binds to circulating PD-1 expressing effector T cells allowing for tumor escape from the immune system. PD-L1 blockade prevents PD-L1/PD-1 interaction and is currently explored as

Multiplex profiling identifies distinct local and systemic alterations during intraperitoneal chemotherapy for ovarian cancer: An NRG Oncology/Gynecologic Oncology Group Study

OBJECTIVES Ovarian cancer leads to abdominal carcinomatosis and late stage (III/IV) diagnosis in 75% of patients. Three randomized phase III trials have demonstrated that intraperitoneal (IP) chemotherapy improves outcomes in epithelial ovarian cancer. While IP treatment is validated by clinical trials, there is a poor understanding of the mechanism(s) leading to the survival advantage other than the increased concentration of cytotoxic drugs within the tumor microenvironment. A better understanding of this process through analysis of dynamic biomarkers should promote novel approaches that may enhance tumor clearance. We propose this pilot study to confirm the feasibility of collecting serial peritoneal samples from implanted catheters in women receiving IP chemotherapy. We believe these specimens may be used for multiplex analysis to reveal unique biomarker fluctuations when compared to peripheral blood. METHODS From 13 women participating on GOG 252, 30 whole blood, 12 peritoneal fluid (PF), and 20 peritoneal wash (PW) with 30mL saline were obtained. Samples were requested prior to the first three chemotherapy cycles. Samples were assessed for volume, cell populations, protein, RNA, and miRNA content changes. RESULTS Median volume for PF was 1.6mL and 3.1mL for PW. PW is a dilution of PF capable of capturing measurable biomarkers. Peritoneal aspirates contain a unique profile of biomarkers distinct from blood. miRNA undergo earlier alteration with chemotherapy than genes. Flow cytometry does not adequately capture biomarker fluctuations. CONCLUSIONS As a proof of principle study, this trial provides evidence that sampling the peritoneal cavity can be adapted for biomarker analysis.

Abstract A59: PD-L1 biology in response to chemotherapy in vitro and in vivo in ovarian cancer.

Objective: PD-L1 is an immune checkpoint molecule expressed by a variety of tumors, including ovarian, which binds to circulating PD-1 expressing effector T cells allowing for tumor escape from the immune system. PD-L1 blockade prevents PD-L1/PD-1 interaction and is currently explored as therapy of solid tumors. Ovarian cancer patients receive combination cisplatin/taxane chemotherapy as standard of care. Chemo-induced effects on tumor PD-L1 expression have been only partially addressed. We studied here the effect of platinum/taxane exposure on PD-L1 expression in vitro and in vivo. Methods: Human (OVCA 420 and OVCA432) and mouse (2F8) ovarian cancer cell lines were exposed to increasing doses of cisplatin and paclitaxel for different time periods. PD-L1 expression was analyzed with flow cytometry and western blot. Through continuous exposure in vitro of mouse 2F8 ovarian cancer cells to increasing doses of cisplatin we have derived a new cisplatin-resistant line (2F8-Cis). In vivo, we have challenged n=37 mice IP with 0.8 million 2F8 cells . Tumor-bearing mice were treated with cisplatin, anti-PD-L1 antibody, both drugs, or isotype control every two weeks for three doses starting at day 14 post-inoculation. Tumor- and ascites-derived cancer cells were analyzed with flow cytometry. Result: Exposure of OVCA420 and OVCA432 to cytotoxic doses of cisplatin or paclitaxel trigger PD-L1 up-regulation. Similarly, 2F8-Cis cells show increased cell surface PD-L1 compared to parental 2F8 cells, providing the rationale for combination therapy with PD-L1 blockade. In vivo treatment of mice with aggressive 2F8 tumors respond well to cisplatin and anti-PD-L1 individually with increased survival (median 45 days versus 24 days for isotype control, p=0011). At necropsy, anti-PD-L1 therapy significantly reduced tumor burden (1.48 g versus 0.25 g, p=0.0294). Tumor cells cultured from cisplatin-only treated mice expressed higher levels of PD-L1, in line with our in vitro results. A higher percentage of PD-1 expressing cells were found amongst the tumor cells in these cultures versus cisplatin/anti-PD-L1 treated mice. Although high dose anti-PD-L1 immediately following cisplatin administration can control tumor burden (0.48 g), it does not significantly prolong survival (median 29 days). We are currently testing an alternative therapeutic schema exploring a lower anti-PD-L1 dose and a different timing post-chemo. Conclusion: Tumor cells upregulate PD-L1 in response to chemotherapy exposure and combination PD-L1 blockade in conjunction with chemotherapy effectively controls tumor burden. Optimization of timing and dosage for this combination therapy will likely increase its therapeutic benefit. Citation Format: Shannon Grabosch, Feitianzhi Zeng, Lixin Zhang, Mary Strange, Joan Brozick, Robert P. Edwards, Anda Vlad. PD-L1 biology in response to chemotherapy in vitro and in vivo in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A59.

Abstract 3208: Chemo-induced biology of PD-L1 and in vivo combination immune therapy for ovarian cancer

Rationale: Ovarian cancer patients receive platinum/taxane-based chemotherapy as standard of care. Five year survival has remained unchanged for several decades, at less than 45%, pointing to the need for new and improved therapies. We recently reported in vivo anti-tumor efficacy of PD-L1 immune checkpoint blockade in a new transplantable ovarian cancer mouse model. However, chemo-induced effects on ovarian tumor PD-L1 expression have not been addressed. We studied here the effect of platinum/taxane on tumor PD-L1 expression in vitro and in vivo and tested the efficacy of PD-L1 blockade in combination with cisplatin, using several treatment regimens. Methods and Results: Our results demonstrate that in vitro exposure of several human ovarian cancer cell lines, with various baseline susceptibilities to cisplatin and taxol, triggers PD-L1 upregulation. Similar effects were observed when newly tumor-derived, platinum sensitive (2F8) and platinum resistant (2F8cis) murine ovarian cancer cell lines were exposed to chemo drugs in vitro. Based on these findings we postulated that PD-L1 immune checkpoint blockade in combination with cisplatin may provide therapeutic benefit in ovarian cancer. In vivo, we have challenged n = 37 mice IP with 0.8 million 2F8 cells. Tumor-bearing mice were treated with cisplatin, anti-PD-L1 antibody, both drugs or isotype control every two weeks for three doses starting at day 14 post-inoculation. Study endpoint was overall survival. Secondary endpoints were tumor CD8 infiltration, changes in Th1/cytotoxic immunity. Tumors and ascites-derived cancer cells were analyzed with flow cytometry. RNA was extracted from splenocytes and analyzed with Nanostring using probes for n = 511 immune genes. In line with in vitro results, tumor cells isolated ex vivo from cisplatin-treated mice expressed increased PD-L1. Compared to control treated mice, both Cisplatin alone and anti-PD-L1 alone increase overall survival (p = 0.002 and p = 0.02, respectively). No increase in survival was observed in anti-PD-L1/Cisplatin-treated mice, due to an over-responsive immune cascade overwhelming the animal. Addition of celecoxib, a cyclooxygenase-2 inhibitor, to the anti-PD-L1/cisplatin combination was well tolerated and led to improved overall survival. Conclusion: Ovarian cancer cells upregulate PD-L1 in response to chemotherapy exposure in vivo and in vitro. Though effective independently, combination cisplatin and anti-PD-L1 blockade did not improve survival, likely due to cytokine release syndrome. Celecoxib added to cisplatin and anti-PD-L1 improves overall survival. Anti-tumor immunity in responder mice revealed a cytotoxic T cell mediated gene signature. These findings reveal benefits and pitfalls of chemotherapy in combination with immune checkpoint blockade and have high translational potential for ovarian cancer treatment. Citation Format: Shannon Grabosch, Feitianzhi Zeng, Lixin Zhang, Tianzhou Ma, George Tseng, Robert P. Edwards, Anda M. Vlad. Chemo-induced biology of PD-L1 and in vivo combination immune therapy for ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3208.

Abstract 260: In vivo efficacy of intraperitoneal anti-PD-L1 therapy in ovarian cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Objectives: Programmed cell death ligand 1 (PD-L1) is an immune checkpoint cell surface molecule expressed on many types of cancers including ovarian cancer. Its interaction with the programmed death (PD-1) receptor on T cells essentially halts the immune response and allows tumor escape. Monoclonal antibodies to either PD-L1 or PD-1 block this interaction and show efficacy in a variety of cancers, though to a lesser degree in ovarian cancer. We have evaluated the efficacy of intraperitoneal anti-PD-L1 therapy in two ovarian cancer mouse models. Methods: Murine 2F8 tumor cells derived from an orthotopic tumor isolated from a triple transgenic MUC1+/−KrasG12D/+PtenloxP/loxP (MKP) mouse as we recently described. MUC1 transgenic (MUC1.Tg) mice received an intraperitoneal inoculation of 8×105 syngeneic 2F8 cells. Untreated mice develop extensive tumor burden and expire around 29 days. Mice were treated with 200 μg anti-PD-L1 antibody (or rat IgG as control) every two weeks for three doses starting at 21 days post-inoculation, thus corresponding with late tumor stage. C57BL/6 mice were inoculated via intraperitoneal (IP) injection with 1×106 IG10 (spontaneously transformed mouse ovarian surface epithelial) tumor cells. Treatment with anti-PD-L1 (or rat IgG as control) started 22 days following inoculation when the mice began showing ascites. Immunohistochemistry for perforin was performed on tumor tissue sections. Immune gene profiling of spleen cells collected at necropsy was performed via Nanostring, using 561 mouse immunology-related genes. Results: The 2F8 tumors are aggressive with little T cell infiltration. The anti-PD-L1 treated MUC1.Tg mice showed substantial infiltration of perforin positive cells within the tumor and significantly increased survival (p = 0.001) compared to isotype control- treated mice. Splenocyte profiling of 561 immune genes via Nanostring revealed a treatment-induced immune gene signature that points to T cell functions and cytotoxic anti-tumor immune responses. A similar gene signature favoring cytotoxic anti-tumor activity was also revealed in IG10 tumor bearing C57Bl/6 mice treated with anti-PD-L1. Conclusion: These preclinical results from two different ovarian cancer tumor models demonstrate that targeting PD-L1 is a viable therapeutic strategy in ovarian cancer and increases survival despite (1) non-immunogenic tumors, (2) late treatment initiation, (3) low dose and administration frequency. The tumors respond well to anti-PD-L1 blockade, due to increased systemic T cell responses and intratumoral T cell accumulation, without changing tumor-specific antibody levels. Our studies have high translational potential and support IP administration of PD-L1 blockers in ovarian cancer patients. Citation Format: Shannon Grabosch, Jyothi T. Mony, Lixin Zhang, Tianzhou Ma, Tejas Tirodkar, Joan Brozick, George Tseng, Esther Elishaev, Robert P. Edwards, Xin Huang, Anda M. Vlad. In vivo efficacy of intraperitoneal anti-PD-L1 therapy in ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 260. doi:10.1158/1538-7445.AM2015-260