Primary cytoreductive surgery for advanced stage ovarian cancer harbors a survival adavantage if tumor burden can be decreased to optimal status, defined as nodules smaller than 1.0 cm3 (3). Complete cytoreduction with elimination of all macroscopic disease adds further survival benefit over optimal cytoreduction. In patients with apparent unresectable disease at presentation or who have significant baseline morbidities, NACT can be administered followed by interval cytoreductive surgery, and additional post-operaative consolidation chemotherapy (3).
Unfortunately, the majority of patients with advanced ovarian malignancies will experience a recurrence and ultimately tumors that initially were platinum sensitive, will become platinum resistant. It remains controversial as to whether NACT augments the emergence of chemoresistant tumor cells by selectively eliminating only chemosensitive tumor cells, allowing a greater proportion of chemoresistant cells available to expand (30). Furthermore, while clinical studies indicate that a shorter interval to resumption of chemotherapy may improve survival (31–35), it is less clear whether heated intraperitoneal chemotherapy (HIPEC) at the time of surgery is beneficial. Furthermore, perturbation of the immune system resulting from surgical wounding may impact residual disease progression. Recent studies have shown that altered myloid cell differentiation resulting in an immunosuppressive state can result from surgery (36) or myocardial infarction (37) to promote the outgrowth of breast cancer cells.
In this study, we developed a mouse model of NACT using syngeneic murine ID8 ovarian cancer cells. In an orthotopic model, these cells were shown to create primary tumors morphologically similar to papillary serous high-grade carcinoma and to produce diffuse peritoneal seeding and ascites production similar to the clinical presentation of high-grade serous ovarian cancer (24). Given the increasing use of a combination of NACT and interval surgery, the development of reliable animal models will be important to further understanding of the impact NACT has on tumor biology.
Our group previously examined the role for earlier administration of adjuvant chemotherapy using a murine ovarian cancer primary surgery model (17). In that study, we found that surgery accelerated residual tumor growth and that this effect was minimized through the administration of earlier cisplatin on post-operative day 7 (38); however, in this model, surgery consisted of wounding without extirpation of tumor cells. In this model, we also found that surgical wounding impaired the efficacy of intraperitoneal cisplatin treatment administered on the day of wounding. In NACT, cells are exposed to platinum based-chemotherapy for several cycles and thus the impact of surgery and timing of cisplatin may differ from that of chemo-naïve cells in primary surgery.
When compared to animals that had not received ICS and tumor cytoreduction, ICS did not appear to reduce tumor burden appreciably, although it reduced the number of animals that developed massive ascites, as did peri- or post-surgical cisplatin treatment. Surprisingly, intraperitoneal cisplatin treatment on the day of ICS increased tumor burden measured 6 weeks later. This may be attributable to the surgical procedure as the tumor burden was significantly greater than that measured in animals treated similarly but that had not undergone ICS. Moreover, the increase in tumor burden was prevented when delaying cisplatin treatment by 7 days. Importantly, surgical wounding induces a cascade of cytokines and growth factors, many of which overlap with cellular processes related to cancer cell growth and metastasis, including angiogenesis (39). Our observation of improved control of ascites in animals who undergo wounding compared to control animals suggests there may a relationship to those growth factors recruited for wound healing and the membrane stabilization of neovascularization within residual tumor deposits following sub-optimal cytoreduction in our model. The burden of ascites was least in those animals who received cisplatin on POD0 suggesting a possible synergistic effect of platinum and surgical wounding that is not fully understood. It may be that those factors related to vascular permeability are blocked while permitting the recruitment of pathways related endothelial stabilization. In a trial of adding bevacizumab to chemotherapy and continuing it as maintenance therapy, women who have stage IV disease and sub-optimal cytoreduction experience the greatest benefit from the addition of bevacizumab, a mono-clonal antibody targeting vascular endothelial growth factor which regulates angiogenesis (40). It is feasible that the combination of interval surgical wounding and peri-operative cisplatin in this model of sub-optimal cytoreduction affects antiangiogenic pathways contributing to improved control of ascites. Further work is needed to explore this relationship of angiogenic pathways at interval surgery in those who have received extensive chemotherapy pre-operatively.
Our findings are most intriguing when contrasted with our previous work in surgical wounding of animals in a model of primary surgery for advanced ovarian cancer (17). In animals naïve to cisplatin, surgical wounding resulted in accelerated tumor growth whereas in the current study there was no significant effect of wounding on final tumor burden. There are several proposed mechanisms that could explain the difference. Firstly, it is known that NACT followed by ICS increases platinum-resistant disease (41) or decreases treatment-free interval (42). Chemo-resistant clones may have be less sensitive to the cascade of growth factors at the time of surgical wounding compared to those who are not exposed to pre-operative chemotherapy due to changes in gene expression after treatment. Additionally, animals exposed to pre-operative cisplatin likely have impaired immune system functioning due to the hematologic toxicity of platinum derivatives. The systemic inflammatory response to surgical wounding and growth of metastatic disease has been linked to immune driven processes in animal models of other solid tumors (36). We hypothesize that pre-operative chemotherapy may alter this pathway of accelerated tumor growth as a consequence of immunosuppressive effects from cisplatin.
We expected to observe that excision of the primary ovarian mass would lead to accelerated growth compared to animals that did not undergo surgery but this was not observed. Large primary tumors secrete factors like angiostatin, endostatin and thrombospondin (43) that inhibit growth of distant disease and removing the dominant mass has been shown to remove this inhibition and lead to increase proliferation of distant disease (44). Animal models of breast cancer have shown that primary surgery leads to altered expression of genes related to tumor adhesions, invasion and angiogenesis (45). It will be important to evaluate circulating levels of these factors in clinical specimens of women undergoing surgery for advanced ovarian cancer. Our findings suggest there may be important differences in the systemic response and immune microenvironment of women undergoing primary versus interval cytoreduction and they should be explored to identify potential therapeutic targets that can be exploited to maximize outcomes.
There are several limitations to the current study. The results of animal models are inherently limited in their translation to human outcomes, particularly in the setting of timing of chemotherapy and mimicking clinical conditions. Much work has been done to determine dose equivalences across various species; however, there has been little to no research into timing of chemotherapy in mice compared to humans. Our choice of using post-operative day 28 cisplatin as the standard of care control is based on the clinical condition of resuming adjuvant treatment approximately 4 weeks post-operative; however, these pharmacokinetics do not necessarily translate directly to mice. Unlike in the clinical setting, we only administered a single post-operative dose of cisplatin which may have limited our results. Our primary outcome was burden of disease using bioluminescence as a surrogate which is not in keeping with most clinical studies which assess survival, which is not ethically feasible to assess in animal studies. High burden and distribution of intraperitoneal disease is likely related to survival in human patients and, therefore, we believe bioluminescence is an appropriate outcome for the purposes of this research question. Finally, the contemporary definitions of ‘optimal’ cytoreduction and residual disease have not been quantified in animal models and therefore it is hard to make assumptions regarding the residual disease following resection of the primary tumor in this model. It is not feasible to subject mice to an extensive cytoreductive procedure in this setting and as such we assume that there is macroscopic disease remaining following unilateral oophorectomy and our findings are within the context of ‘sub-optimal’ cytoreduction.
Despite its limitations, this animal model replicates the conditions of advanced high-grade serous ovarian cancer as best as could be achieved in an animal setting and this is one of the first studies to describe animal models of NACT/ICS in ovarian cancer. Our cell lines were sensitive to platinum-based chemotherapy, animals developed diffusely metastatic disease prior to initiating NACT, many developed ascites at recurrence and findings at necropsy mimic the volume and distribution of disease seen in peritoneal malignancies. Additional strengths include the contrast in results to our study in primary surgical wounding, suggesting that there are mechanisms of wound healing and tumor biology unique to animals pre-treated with cisplatin and that future studies should explore these differences as they may identify targets that can be blocked in order to prevent the phenomenon observed at primary surgery.