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Pembrolizumab in Combination with Bevacizumab and Oral Cyclophosphamide in Heavily Pretreated Platinum-Resistant Ovarian Cancer

Background: Immune checkpoint inhibitors (ICIs) have been widely implemented in the treatment of solid tumors. Although epithelial ovarian carcinoma is considered as scarcely immunogenic, the presence of tumor-inltrating T lymphocytes (TILs) in the ovarian tumor microenvironment (TME) could increase sensitivity to immune checkpoint inhibitors (ICIs). Combinations of ICIs with chemotherapy, anti-VEGF compounds and PARP inhibitors are under evaluation in ovarian cancer. Recently, a Phase II study evaluated the ecacy of Pembrolizumab in Combination with bevacizumab and oral cyclophosphamide in patients with recurrent platinum-sensitive, platinum-resistant, or refractory epithelial ovarian cancer. Methods: Herein, we present a retrospective study of all patients who received pembrolizumab in combination with bevacizumab and oral cyclophosphamide for recurrent platinum-resistant heavily pretreated ovarian cancer in the Oncology Unit of Alexandra University Hospital. Results: Median age at diagnosis was 54.5 years (SD; 8.9; range: 44–72). All patients were diagnosed with high-grade serous ovarian carcinoma (HGSC). Initial disease stage was FIGO IIIC (8/10; 80%), IIIB (1/10; 10%) and IIC (1/10; 10%)). Patients were heavily pretreated with a median of 6 (range: 4–9) prior lines of systemic therapy. All patients have experienced disease progression on rst-line platinum-based chemotherapy and median PFS to rst-line treatment was 20.1 months (95%CI; 11.4 – 28.7). Patients received a median of 4 cycles of pembrolizumab in combination with cyclophosphamide and bevacizumab (range 2-11). ORR was 20% (2/10) with two patients achieving partial response (PR) and two patients achieving stable disease (SD) while disease control rate (DCR) was 40% (4/10). Median PFS was 2.7 months (95%; 0.6 – 4.8) and 6-month PFS rate 20%.


Background
Ovarian cancer is the 8 th most common malignancy in women and the 7 th cause of cancer-related mortality accounting for 207.252 deaths worldwide [1]. Epithelial ovarian cancer (EOC) comprises the majority of malignant ovarian neoplasms (~90%). Although the treatment landscape of ovarian cancer has changed through the years, prognosis remains poor. Around two thirds of ovarian cancer patients are diagnosed at an advanced stage (III/IV) while 5-year survival is as low as 39% and 17% for stage III and IV EOC respectively. Novel treatment strategies have extended the survival, however most patients (65-80%) recur within the rst 5 years after rst-line chemotherapy. Response rates to subsequent chemotherapy remain low, especially in the platinum-resistant population that relapse within 6 months after rst-line therapy (15%) [2]. Recently, 'AURELIA' study established the combination of bevacizumab with chemotherapy by demonstrating an almost doubling of the platinum-free interval (PFI) (3.4 vs. 6.7 months, p < 0.001), but no improvement in overall survival (13.3 vs. 16.6 months) [3]. Further prolongation of progressionfree survival (PFS) was achieved after the introduction of PARP inhibitors as maintenance treatment after the rst-line or subsequent line chemotherapy [4,5].
Immune checkpoint inhibitors (ICIs) have been widely implemented in solid tumors. Anti-programmed death 1(PD1)/programmed cell death ligand 1 (PD-L1) compounds demonstrated only modest activity as monotherapy in advanced recurrent ovarian cancer [6-8]. KEYNOTE-100 Phase II study demonstrated an overall response rate (ORR) of 8% and a median PFS of 2.1 months to pembrolizumab monotherapy in patients with advanced recurrent ovarian carcinoma [6]. Accordingly, JAVELIN study reported a 9.6% ORR to treatment with avelumab in heavily pretreated ovarian cancer patients who had received a median of 3 prior lines of treatment, while it was 15% for nivolumab [7,8]. In order to enhance the antitumor effect of immunotherapy in ovarian cancer, multiple studies were designed to explore combinations of immune checkpoint inhibitors The rationale of immunotherapy administration in ovarian carcinoma is based on the immunogenicity and the presence of tumor-in ltrating T lymphocytes. In 2003, Zhang L et al. associated the presence of intratumoral T cells with a 3.9 -fold PFS and a 2.8 -fold overall survival (OS) compared to tumors that contained no T cells [9]. Immune checkpoint inhibitors could enhance tumor in ltration with immune cells and remove immunosuppressive aspects of the ovarian tumor microenvironment (TME). However, EOC has traditionally been considered as scarcely immunogenic and studies evaluating immune checkpoint inhibitors in platinum-resistant ovarian cancer report contradicting results [10,11]. The addition of anti-VEGF treatment to immunotherapy could prove to be more effective than either treatment regimen alone. Indeed, ovarian TME consists of different types of stromal cells embedded in the omental extracellular matrix like myeloid-derived suppressor cells (MDSCs), tumorassociated macrophages (TAMs), adipocytes, CAFs (cancer activated broblasts) and resident and in ltrating immune cells, including regulatory T cells (T reg ) [12]. VEGF and other angiogenic factors like angiopoietin 2 (ANG2) that seep from the TME facilitate the extravasation and dissemination of cancer cells [13]. Apart from enabling metastasis, VEGF enhances the suppressive function of regulatory T (Treg) cells, inhibits the antigen presentation by dendritic cells (DCs) and consequently the activation of cytotoxic T lymphocytes (CTLs) [13]. Overall, the consequence of vascular abnormality is a compromised CTL-mediated anticancer immune response and an immunosuppressive TME. Of note, PD-1/PD-L1 pathway is often upregulated both on tumor cells and on tumor-in ltrating CTLs rendering them dysfunctional or 'exhausted'. These interactions of PD-1/PD-L1 and VEGF pathways provide the rationale for combining anti-VEGF regimens with immune checkpoint inhibitors.
A Phase II study by Zsiros E et al. was recently published exploring the e cacy of pembrolizumab combined with bevacizumab and cyclophosphamide in recurrent ovarian cancer [14]. We have applied this treatment regimen in heavily pretreated patients with platinum-resistant recurrent ovarian carcinoma treated in our institution. Herein, we retrospectively analyze these patients and we report the e cacy data of this combination in the real-world setting. According to our knowledge, apart from the aforementioned Phase II study with 40 patients, there is no other published data on this regimen.

Methods
We retrospectively identi ed all patients with advanced/metastatic high grade serous ovarian carcinoma (HGSC) treated with the combination of pembrolizumab with bevacizumab and oral cyclophosphamide in the Oncology Unit of Clinical Therapeutics Department of University of Athens in Alexandra General Hospital. The administration of this treatment regimen was approved by the National Organization for Health Care Services (EOPYY). The study has been performed in accordance with the 1964 Helsinki Declaration and has been approved by the Institutional Review Board of Alexandra University Hospital. A written informed consent was obtained from all patients included in this study. Patients received intravenous (IV) pembrolizumab at a dose of 200 mg, IV bevacizumab at a dose of 7.5 mg/kg every 3 weeks and oral cyclophosphamide, 50 mg, once daily during the treatment cycle until disease progression or unacceptable toxicity. Medical records of patients with HGSC who were treated with this combination were retrospectively collected. Clinicopathological characteristics were extracted from the patients' les including: age at diagnosis, FIGO stage at diagnosis, histological subtype, grade, debulking status, performance status (ECOG score), progression-free survival (PFS) after rst-line chemotherapy, number of previous chemotherapy lines, type of maintenance treatments received, BRCA mutation status, number of pembrolizumab/bevacizumab/cyclophosphamide cycles administered, best response to pembrolizumab/bevacizumab/cyclophosphamide, disease progression, PFS at rst-line chemotherapy and at pembrolizumab/bevacizumab/cyclophosphamide and overall survival (OS). Optimal debulking was de ned as maximum residual tumor <1cm in diameter after IDS.

Statistics
Descriptive statistics were used to assess clinicopathological parameters of the patients. Progression Free Survival (PFS) was calculated from the initiation of treatment with pembrolizumab, bevacizumab and cyclophosphamide until disease progression (PD) or last follow-up. Overall survival (OS) was calculated from the initiation of treatment until death or last follow-up. Statistical analysis was performed with SPSS 24.0 statistical software.

Baseline characteristics
A total of 10 patients received the combination in the Oncology Unit of Alexandra University Hospital. The median age of patients at disease diagnosis was 54.5 years (SD; 8.9; range: 44-72). All patients were diagnosed with high-grade serous ovarian carcinoma. Initial disease stage was FIGO IIIC in eight cases (80%), IIIB in one case (10%) and IIC in one case (10%). All patients underwent primary debulking (90%) except for one case where neoadjuvant chemotherapy and interval debulking surgery was applied. Debulking was optimal in most cases (9/10; 90%). All cases had been previously analyzed for presence of BRCA1/2 mutations. BRCA1 mutations were reported in three cases (30%), a somatic mutation in one case and germline mutations in two cases. MSI status was evaluated only in three cases and all of these tumors were microsatellite stable (MSS).
Patients were heavily pretreated with a median of 6 (range: 4-9) prior lines of systemic therapy. Prior bevacizumab and PARP inhibitor therapy was received by 50% (5/10) and 80% (8/10) of patients, respectively. All patients have experienced disease progression on rst-line platinumbased chemotherapy and median PFS was 20.1 months (95%CI; 11.4 -28.7) to rst line chemotherapy. Only two patients have died. Mean OS was 101 months (95% CI; 80.70 -121.8).  Patients received a median of 4 cycles of pembrolizumab in combination with cyclophosphamide and bevacizumab (range 2-11). Best response was partial response (PR) in two cases, stable disease (SD) in three cases and PD in the remaining cases (5; 50%). ORR was 20% (2/10) while disease control rate (DCR) was 50% (5/10). Median PFS with pembrolizumab, cyclophosphamide and bevacizumab was 2.7 months (95%; 0.6 -4.8) and 6-month PFS rate 20% (Figure 1). Response is ongoing in two patients and duration of treatment is depicted in Figure 2. Interestingly, patients with longer OS also demonstrated a longer PFS with pembrolizumab, cyclophosphamide and bevacizumab.  [14]. In our study, all patients were platinum-resistant and more heavily pretreated with a median of 6 prior lines of treatment. In accordance with our results, median PFS was 7.6 months (90% CI; 5.7-10.3) in patients with platinum-resistant disease in the Phase II study [14]. These survival rates comprise an encouraging alternative for this population that therapeutic alternatives are limited.
Multiple studies were designed to explore the combination of ICIs with chemotherapeutic regimens in ovarian carcinoma. Liao [19], nivolumab with bevacizumab and rucaparib (NCT02873962), weekly paclitaxel with pembrolizumab (NCT02440425), pembrolizumab with bevacizumab and pegylated liposomal doxorubicin [PEMBOV; NCT03596281]. However, the combination of pembrolizumab in combination with bevacizumab and oral cyclophosphamide showed promising activity in this heavily treated population.
Only three patients in our study harbored somatic BRCA1 mutations so a correlation between BRCA status and response to immunotherapy cannot be safely extracted. There is evidence that BRCA-mutated ovarian cancer is associated with higher PD-L1 expression and higher tumor mutational burden (TMB). Indeed, BRCA1/2-mutated demonstrated signi cantly higher neoantigen loads than the HR pro cient tumors (51 vs 37.5 respectively; p = 0.001), higher number of CD3+ TILs and higher PD-1 and PD-L1 expression in intraepithelial and peritumoral lymphocytes [20]. These characteristics may render BRCA-mutated tumors more susceptible to immunotherapy. However, IMagyn050 and JAVELIN 100 trials did not con rm this association [2,10]. Further subanalysis of studies with ICIs should evaluate the contribution of BRCA mutations in sensitivity to immunotherapy.
Our study has some limitations. Firstly, all of the patients enrolled were heavily pretreated platinum-resistant and had received all of the approved standard-of-care treatment lines and represent a group that may be more or less chemoresistant. Response rates are extremely low in this population and achievement of disease control is quite unlikely. Furthermore, patients with platinum-resistant recurrent ovarian carcinoma were considered eligible irrespective of PD-L1 or MSI status. KEYNOTE-028 showed that PD-L1-positive patients have an improved response rate of 11.5% (95% CI, 2.4-30.2%) and a median PFS of 1.9 months (1.8-3.5 months) [21]. KEYNOTE-100 con rmed this increased response rate in patients with PD-L1 CPS ≥ 10 (ORR: 17.1% vs 5% (CPS<1) and 10.1% (CPS ≥1-10) [6]. It is possible that response to immunotherapy may differ between patients with different PD-L1 or MSI status. In addition, this study was a retrospective single-institutional study lacking a comparison arm to compare the e cacy of this new combination versus standard-of-care regimens. Finally, this novel combination has been recently introduced so the sample size is relatively limited. More patients are needed to extract a safe conclusion. In this moment, data on the use of pembrolizumab, bevacizumab and cyclophosphamide combination emerge only from Phase II studies [14]. Phase III trials should be designed to evaluate the clinical e cacy of this combination versus the standard of care treatment regimens in heavily pretreated recurrent ovarian carcinoma.

Conclusion
Overall, we here demonstrate that the combination of pembrolizumab with bevacizumab and oral cyclophosphamide is an effective alternative in heavily pretreated ovarian cancer patients. Phase III trials should be designed to evaluate this combination prospectively in recurrent platinumresistant ovarian cancer.

Declarations
Funding: This research received no external funding.
Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of Alexandra University Hospital.
Informed Consent Statement: Informed consent was obtained from all individual participants included in the study. Written informed consent has been obtained from the patient(s) to publish this paper.
Data Availability Statement: Data presented in our study can be found in the patients' archives that are safely stored in our Institution. The datasets generated during the current study are available from the corresponding author upon request.

Figure 1
Median PFS to treatment with pembrolizumab, cyclophosphamide and bevacizumab.

Figure 2
Swimmer plot of patients enrolled.