Combination of PARP Inhibitor Olaparib, and PD-L1 Inhibitor Durvalumab, in Recurrent Ovarian Cancer: a Proof-of-Concept Phase II Study

Ovarian cancer is the most fatal gynecologic malignancy worldwide (1, 2). The majority of women with epithelial ovarian cancer present at an advanced stage and frequently recur, leading to incurable disease with limited treatment options (1). A critical need remains for new effective therapeutic strategies. Immune checkpoint inhibition, such as programmed death (PD)-1 and PD-ligand 1 (PD-L1) pathway blockade, has led to important clinical advances in various malignancies and has also been tested in recurrent ovarian cancer (3). To date, the monotherapy activity of immune checkpoint inhibitors has been limited in ovarian cancer, leaving opportunity to test combination strategies (3).

An active therapeutic target for combination treatment is the DNA damage response pathway, such as PARP (4). Successful introduction of PARP inhibitors (PARPi) has led to a new treatment paradigm in ovarian cancer, in particular, for patients with BRCA mutation (BRCAm; ref. 4). PARP inhibition has been shown to cause DNA damage via catalytic inhibition of the PARP enzyme and trapping of DNA–PARP complexes, resulting in synthetic lethality in cells deficient in homologous recombination (HR) repair (4).

Emerging data also suggest the efficacy of PARPi may be associated with immunomodulation (5–8). DNA damage by PARPi may enhance tumor mutational load and neoantigen expression, leading to antitumor immune responses (9). Also, high levels of IFNγ increases the cytotoxic effect of PARPi in a BRCA1-deficient ovarian cancer model (10) and increased DNA damage by PARPi activates the stimulator of interferon genes (STING) pathway, resulting in systemic antitumor immunity (5–8). Specifically, the PARPi olaparib promotes accumulation of cytosolic DNA fragments, which enter the cytoplasm and bind to cyclic GMP-AMP synthase (cGAS), leading to upregulation of the cGAS–STING pathway in both BRCA1-deficient and BRCA-proficient ovarian cancer cell lines and mouse models (5, 6). PARPi also upregulates PD-L1 expression through a variety of mechanisms, including IFNγ stimulation, STING pathway activation, or inactivation of glycogen synthase kinase-3β (GSK3β) in ovarian, breast and lung cancer preclinical models (6, 11, 12). Hence, addition of PARPi may complement the clinical activity of immune checkpoint blockade by creating a more immunogenic tumor microenvironment.

There are now multiple clinical trials combining PARPi with immune checkpoint blockade therapy in ovarian cancer (1). However, it is unknown whether PARPi actually induce the immunostimulatory milieu in patients with recurrent ovarian cancer and prime the immune microenvironment for the PD-1/PD-L1 pathway inhibitors. To test this hypothesis, we prospectively designed a proof-of-concept, investigator-initiated phase II study of the PARPi olaparib and the PD-L1 inhibitor durvalumab with collection of paired pre-and on-treatment fresh tissue and blood samples in women with recurrent ovarian cancer. Here we show immunostimulatory changes induced by treatment, including increased expression of IFNγ, CXCL9, and CXCL10, systemic production of IFNγ and TNFα, and tumoral infiltration by lymphocytes. Enhanced plasma IFNγ levels were associated with response and improved progression-free survival (PFS). Our findings also suggest the VEGF/VEGFR pathway may act to counterbalance immunostimulatory changes by PARPi and serve as a target to further improve the efficacy of the PARPi and anti–PD-L1 combination. Overall, our results showed modest clinical activity of this therapeutic combination, but indicate that PARPi creates an immunostimulatory environment that may augment immune responses to anti–PD-L1 among subsets of patients, warranting further investigation.

This trial was designed as a proof-of-concept, signal-seeking phase II study with four independent cohorts: ovarian, triple-negative breast, prostate, and lung cancers. This report describes the ovarian cancer cohort. Eligible patients were aged ≥18 years and had histologically confirmed recurrent or metastatic ovarian, fallopian tube, or primary peritoneal cancer. Patients must have had measurable disease based on response evaluation criteria in solid tumors (RECIST) v1.1 criterion and at least one lesion safely accessible for a mandatory percutaneous baseline biopsy. Documentation of germline BRCAm status was requested at enrollment. Patients may have received any number of other systemic therapies including prior PARPi. Other key inclusion criteria included Eastern Cooperative Oncology Group performance status 0–2, adequate organ and marrow function, demonstrated by absolute neutrophil count ≥1,500/mcL; platelets ≥100,000/mcL; hemoglobin ≥ 9 gm/dL; total bilirubin ≤1.5 times the institutional upper limit of normal (ULN); aspartate aminotransferase and alanine aminotransferase ≤2.5 times ULN; creatinine ≤ULN or a creatinine clearance ≥50 mL/minute/1.73 m² (Supplementary Material). Key study exclusion criteria included concurrent anticancer therapy, prior immune checkpoint inhibitors, any investigational anticancer therapy ≤ 3 weeks before first doses of study drugs; central nervous system metastases ≤ 1 year prior to enrollment; severe prior immune-related adverse events (AEs) requiring steroid maintenance, or active or prior documented inflammatory bowel disease; and/or, baseline features suggestive of myelodysplastic syndrome or acute myelogenous leukemia (Supplementary Material).

All patients provided written informed consent before enrollment. The trial was approved by the Institutional Review Board of the Center for Cancer Research (CCR), National Cancer Institute (NCI). The study has been conducted in accordance with ethical principles that have their origin in the Declaration of Helsinki and are consistent with the International Council on Harmonization guidelines on Good Clinical Practice, all applicable laws and regulatory requirements, and all conditions required by a regulatory authority and/or institutional review board. ClinicalTrials.gov identifier: NCT02484404.

Treatment consisted of olaparib 300 mg twice daily and durvalumab 1,500 mg by intravenous infusion every 4 weeks (−4 to +8 days; one cycle is defined as 28 days) until radiologic progression or unacceptable toxicity (Supplementary Fig. S1, Supplementary material). Laboratory assessments (including hematology, fasting serum chemistry, endocrine function and urinalysis) were done before each cycle. Clinical response was assessed every two cycles by imaging using RECISTv1.1 guidelines. Patients were evaluated for toxicity per Common Terminology Criteria for Adverse Events version (CTCAE) v4.0. Study treatment was discontinued for progression of disease, intercurrent illness, AEs not recovering to ≤ grade 1 within 14 days, or patient withdrawal of consent.

For correlative studies, we collected pretreatment fresh-frozen core biopsies and paired blood samples (at baseline and on cycle 1 day 15; Supplementary Material). A pretreatment fresh core biopsy was mandatory for all patients and second biopsy on cycle 1 day 15 was optional because of patient's refusal or safety concerns. Mutations in DNA repair genes were identified by targeted sequencing of tumor DNA with a BROCA-HR sequencing assay (13) on pre-treatment tissue samples for 29 patients without gBRCAm. Homologous recombination deficiency (HRD) was defined on the basis of the published literature (14, 15) as a deleterious germline or somatic mutation identified by BROCA-HR sequencing present in >10% of the neoplastic fraction in one of the following genes: ATM, BARD1, BRCA1, BRCA2, BRIP1, CDK12, NBN, PALB2, RAD51C, RAD51D (13). RNA-sequencing (RNA-seq) was performed using a HiSeq3000 sequencing system (Illumina) at the CCR sequencing facility, NCI (Supplementary Material). A multiplex ELISA angiome assay for 33 cytokines was performed on plasma samples (Supplementary material). Immunohistochemistry (IHC) was used for PD-L1 expression (clone SP142) and tumor-infiltrating lymphocyte (TIL) analysis using standard procedure (ref. 16; Supplementary Material), and for STING pathway expression (ab92605; Supplementary material). Whole-exome sequencing on DNA samples were performed on the Novaseq6000-S2 system at the CCR sequencing facility, NCI (Supplementary Material).

The primary objective was overall response rate (ORR) by RECISTv1.1 in evaluable patients who had undergone CT imaging at baseline and at least one protocol-specified follow-up timepoint. Secondary objectives included safety evaluation and PFS. Prespecified exploratory objectives were to investigate potential predictive biomarkers described in the correlative studies.

The study was conducted using Simon optimal two-stage phase II design to rule out a 10% ORR in favor of a 30% ORR, with α = 0.10 and β = 0.10. These parameters were chosen for this single arm, signal-seeking study to minimize the number of women exposed to a potentially inactive combination and to target a sufficiently high ORR to support moving into a definitive trial should this trial be positive. The null hypothesis of 10% was selected to accommodate the inclusion of heavily pretreated platinum-resistant BRCAwt patients. A response in two of the first 12 patients sufficed to move to the second stage of accrual, adding another 23 patients. The regimen would be considered sufficiently interesting if ≥ 6/35 patients had a complete or partial response (CR/PR). The probability of early termination was 65.9% under the null hypothesis. PFS was estimated using the Kaplan–Meier method beginning at the on-study date and continuing until progression or death without progression. Patients who have not progressed had their follow-up censored at February 12, 2019 for this evaluation. Differences between Kaplan–Meier curves tests were determined using a log-rank test. Evaluations for the effect of cytokine parameters on PFS were determined using a Cox proportional hazards model after adjusting for standard clinical factors including BRCAm positivity, platinum sensitivity, previous lines of therapy, and previous bevacizumab. Safety evaluation included all enrolled patients. Patients considered nonevaluable had either no post-baseline CT scan or discontinued after less than 8 weeks without documented progression.

Between February 2016 and April 2018, 35 patients were enrolled and received at least one full cycle of treatment (Supplementary Fig. S1A). One patient with BRCA2m platinum-sensitive disease was receiving treatment at the time of data cutoff (February 12, 2019), at >21-month continuous treatment. Clinical characteristics of patients are summarized in Table 1. Patients were heavily pretreated with a median of four prior therapies (IQR, 2–5.5) and all were immune checkpoint inhibitor–naïve. The majority of patients [86% (30/35)] had platinum-resistant disease and six (17%) had known germline BRCAm (gBRCAm) confirmed by commercial BRCA testing prior to enrollment. Six of 29 germline BRCA wild-type (BRCAwt) patients were found to have somatic DNA repair pathway mutations suggestive of HRD including BRCA1 (1), BRCA2 (1), BRIP1 (2), PALB2 (1), CDK12 (1; Supplementary Table S1). Higher coverage BROCA-HR sequencing did not identify BRCA reversion mutations in six gBRCAm carriers who had prior PARPi (rucaparib, n = 1), and platinum-based therapy (all).

Changes in tumor size from baseline and duration on study are shown in Fig. 1. Among 35 evaluable patients, five attained a PR; ORR, 14%, 95% CI, 4.8%–30.3%) with a median duration on study of 17.2 months (IQR, 8.6–21); two gBRCAm/one somatic BRCAm (sBRCAm)/2 BRCAwt and non-HRD. The overall median PFS was 3.9 months (IQR, 2–7.25, Supplementary Fig S1B). This study thus did not meet the prespecified primary endpoint of ≥17.1% ORR (six or more complete or PRs among 35 patients). However, the disease control rate [DCR = PR+stable disease (SD)] was 71% (25/35; 95% CI, 53.7%–85.4%). Twelve of 35 (34%; 95% CI, 19.1%–52.2%) had clinical benefit (defined as PR+SD ≥6 months), including 10 of 30 (33.3%) heavily pretreated platinum-resistant patients. Three of 30 (10%) platinum-resistant patients attained a PR with a median duration on study of 17.2 months (IQR, 12.5–20.9) and 7 (23.3%) achieved disease stabilization lasting at least 6 months (median 7.3 months on study, IQR, 7.25–9.4). This indicates combination treatment may provide durable clinical benefit (≥6 months) in subsets of heavily pretreated patients for whom either PARPi or immune checkpoint blockade monotherapy have shown limited activity (1, 17).

Treatment with durvalumab and olaparib was overall well-tolerated. The most common treatment-related grade 3 or 4 AE was hematologic toxicity, predominantly anemia. We found approximately 31% of patients (11/35; 95% CI, 17%–49%) had grade ≥3 anemia, which was a higher frequency than reported (10%–20%) in other PARPi and immune checkpoint blockade combination studies (18, 19). Clinical workup determined that none of the anemia cases were immune-related. We speculate that the higher frequency of grade ≥3 anemia is likely due to small sample size and the heavily pretreated population in our cohort with a median of three prior cytotoxic chemotherapy regimens (IQR, 2–4.5). All treated patients had at least one any grade treatment-associated AE, summarized in Table 2.

Baseline tissue samples were collected in 32 of 35 patients because three patients had the pretreatment biopsy procedure aborted for safety concerns (Supplementary Material). Twenty-two patients (seven BRCAm, one BRCAwt, and HRD positive (PALB2 mutation), 14 BRCAwt, and non-HRD] underwent the second biopsy on cycle 1 day 15. Paired blood samples were collected in all patients. A summary of the baseline biomarker endpoints with clinical response can be found in Fig. 2. Changes in biomarker expression after treatment are summarized in Supplementary Fig. 2.

Exploratory analysis of RNA-seq data from 20 paired fresh-frozen biopsy samples showed increased expression of IFNγ (median fold change, 2.31; IQR, 0.86–4.72, P = 0.029; Fig. 3A) and IFNγ-induced chemokines CXCL9 (median fold change, 2.14; IQR, 1.54-5.64, P = 0.0001; Fig. 3B) and CXCL10 (median fold change, 2.10; IQR, 1.13–3.92, P = 0.01; Fig. 3C) after treatment, suggesting the PARPi and anti–PD-L1 combination therapy induces an immune-inflamed tumor microenvironment. There was no association between high levels of local IFNγ with clinical benefit. Among eight evaluable patients achieving clinical benefit, seven had a positive baseline IFNγ signature previously determined by Ayers and colleagues (Fig. 3D) and five had a positive baseline expanded 18-gene immune signature (20), although there was no statistical association with clinical response. Finally, we evaluated whether a predefined high-grade serous ovarian cancer (HGSOC) immunoreactive (C2) subtype by transcriptomic analysis (21) was associated with clinical benefit. Of the eight evaluable patients deriving clinical benefit, all expressed moderate to high levels of the pretreatment immunoreactive signature. In contrast, patients with no clinical benefit had either low levels (n = 4) or did not express immunoreactive signature (n = 8) at baseline (P = 0.0047, Fig. 3E).

Using paired plasma samples from 32 patients, we identified systemic upregulation of several immunostimulatory cytokines after treatment such as IFNγ (median fold change, 2.31; IQR, 1.41–3.52) and TNFα (median fold change, 1.27; IQR, 1.11–1.46; P < 0.0001 for both after adjusting for multiple comparisons, Fig. 4A). A greater fold change increase in IFNγ was observed in patients deriving clinical benefit (n = 12) compared with those who did not (n = 20; median fold-change 3.17 vs 1.97, P = 0.029). In addition, there was an association between higher levels of IFNγ after treatment and improved PFS [hazard ratio (HR), 0.37 (95% CI, 0.16–0.87), P = 0.023] using a univariate Cox proportional hazards model. This effect was maintained when clinical factors (BRCAm positivity, platinum sensitivity, previous lines of therapy, and previous bevacizumab) were included in a multivariable Cox proportional hazards model [HR, 0.27 (95% CI, 0.10 – 0.72), P = 0.0086]. An increase in the circulating angiogenic factors, for example, VEGFR3 (median fold-change, 1.09; IQR, 1.05–1.19) and placental growth factor (PlGF; median fold change, 2.52; IQR, 1.59–3.06; P < 0.0001 for both, Fig. 4B), was also observed with treatment. A multivariable Cox proportional hazards model demonstrated increased levels of VEGFR3 over baseline were associated with worse PFS [HR, 3.22 (95% CI, 1.23–8.40], P = 0.017). No tumoral increase in expression of VEGFR3 or PlGF was observed by RNA-seq analysis. Together, our findings suggest olaparib and durvalumab combination therapy induces systemic immune activation as well as a possible compensatory angiogenic response.

We next tested the hypothesis that PARPi could induce immune cell infiltration and PD-L1 upregulation as preclinically demonstrated in BRCAm ovarian tumors (6). TILs and PD-L1 expression were analyzed in 22 paired paraffin-embedded biopsy samples (Supplementary Material). There was an overall significant increase in TILs (median 5% pre vs 10% on-therapy, P = 0.035), with higher %TILs observed in 11 of 15 (73%) evaluable on-treatment biopsy samples (Supplementary Fig. S3A). Change in TILs was not associated with clinical benefit, BRCAm status, or immunoreactive subtype. However, higher TILs at baseline was associated with clinical benefit (Supplementary Fig. S3B–S3E; Supplementary Table S2), as seen in other solid tumor reports (22). The majority of patients (9/14 evaluable samples, 64%) contained PD-L1–positive carcinoma cells after treatment, defined as >1% PD-L1 staining (Supplementary Table S2). Seven (58%) carcinomas of 12 evaluable pairs gained PD-L1 expression (median 5% increase, IQR, 5.0–16.25%; representative patient in Supplementary Fig. S3F–S3I). No tumors completely lost PD-L1 expression (positive to negative). In addition, neither baseline nor changes in PD-L1 expression were predictive of treatment response (Supplementary Table S2) consistent with other reports for immune checkpoint blockade combination treatments (23).

To test whether PARPi leads to STING pathway activation clinically as has been shown in a BRCA1-deficient syngeneic ovarian cancer mouse model (6), STING expression was evaluated in 14 of 22 paired paraffin-embedded biopsy samples [four BRCAm, one BRCAwt, and HRD positive (PALB2), and nine BRCAwt and non-HRD]. Most patients had either decreased or unchanged STING expression after treatment (Supplementary Fig. S4). Only four (29%) patients had increased STING expression after treatment [median 0.26% (pre) vs 2.6% (on-therapy), P = 0.125], all who either had SD or progressive disease (PD). Overall, there was no association between the change in STING expression and BRCAm or clinical response. Baseline STING expression was not associated with clinical benefit. We also evaluated RNA-seq data for STING pathway-related gene levels, for example, STING, interferon regulatory factor 3 (IRF3) and TANK-binding kinase 1 (TBK1), and found no significant changes in pre- versus on-treatment samples. Furthermore, among the four patients with increased STING expression by IHC, none showed an increase in STING RNA expression. Although levels of IFNβ were undetectable, increased expression of inflammatory chemokines activated downstream of type I IFNs was observed (24). Specifically, increased expression of CCL5 (median fold change, 3.30; IQR, 1.48–4.43; P = 0.008) and CCL4 (median fold change, 2.12; IQR, 1.50–2.84; P = 0.01) was seen among those attaining clinical benefit (Supplementary Fig. S5A and S5B). In addition, increased expression of CXCL10, induced by both IFNγ and type I IFNs (25), was observed as described above (Fig. 3C). Together, the data suggests that STING pathway activation is unlikely to be a predominant mechanism driving enhanced response to treatment although the increased expression of chemokines downstream of type I IFNs warrants further validation.

We observed no significant changes in tumor mutational burden (TMB; Fig. 5A) and all remained below five somatic mutations/Mb after treatment. In addition, we evaluated whether baseline TMB was associated with response to treatment as has been shown with immune checkpoint inhibition in lung and urothelial cancer (26). We first observed that all 32 baseline tumors had less than five somatic mutations/Mb, consistent with the modest mutational load described in ovarian cancer (ref. 26; Fig. 5B). We noted no difference in baseline mutational load between BRCAm and BRCAwt patients and no correlation of TMB either at baseline or after treatment was seen with clinical response (Fig. 5C and 5).

Finally, we examined baseline tumor biopsies for somatic JAK1/2 mutations as they have been shown to confer resistance to checkpoint blockade in melanoma models via impaired IFNγ signaling and downregulation of PD-L1 expression (27). Four of 23 patients who had no clinical benefit were found to have at least one somatic JAK1/2 missense mutation (c.1976G>A; c.2371G>A; c.1789G>T; c.86C>T; and c.397C>T), while no patients with clinical benefit had a JAK mutation. However, we did not see an association between JAK mutations and tumoral IFNγ gene expression, PD-L1 staining, or levels of IL6, which drives the JAK/STAT signaling pathway (28). TGFβ signaling in the tumor microenvironment has also been associated with poor response to PD-1/PD-L1 blockade (29). Consistent with this, RNA-seq analysis showed pretreatment TGFB1 expression levels were significantly higher in nonresponders (n = 12) than responders (n = 8; median z-score 0.48 vs -0.43, P = 0.017), while other pathway genes, including TGFB2 and TGFBR2, showed no significant difference.

Immunotherapy represents a paradigm shift in the treatment of various cancers although it has demonstrated modest activity in ovarian carcinoma (1). Preclinical studies have suggested that PARP inhibition may increase mutational burden (9) or activate the STING pathway (5–8). The STING pathway is a potent activator of type I IFNs and elicits antitumor immune responses (5–8, 11), including increased activation of intratumoral CD4⁺ and CD8⁺ T cells, production of immune-stimulatory cytokines, and recruitment of antigen-presenting dendritic cells, thus making PARPi an attractive combination treatment strategy for the immune checkpoint inhibitors. However, in our patients with recurrent ovarian cancer, we observed no significant changes in TMB or STING expression, independent of BRCAm status. We report a 14% ORR and 71% DCR, and found that the PARPi and anti–PD-L1 combination creates an immunostimulatory environment that may enhance durable antitumor immune responses to immune checkpoint blockade in subsets of patients.

The PARPi and anti–PD-L1 combination therapy did not show significant improvement in clinical efficacy per RECIST criteria. However, we noted one third of our patients received clinical benefit lasting longer than 6 months although they were mostly platinum-resistant and heavily pretreated, with over half having received four or more previous treatment regimens. Therefore, these findings are encouraging and suggest that improved patient selection may further enhance PARPi and anti–PD-L1 efficacy in this difficult-to-treat population, as nearly all patients with recurrent ovarian cancer ultimately develop platinum resistance and are left with limited treatment options (30).

There are several ongoing clinical trials combining the PD1/PD-L1 inhibitor and PARPi or other DNA-damaging agents in recurrent ovarian cancer. The results of those studies reported to date are consistent with our findings in predominantly platinum-resistant patients. In the phase I/II TOPACIO study, the PARPi niraparib and anti-PD-1 pembrolizumab yielded an 18% ORR and 65% DCR independent of BRCAm and HRD status (18) and the phase III JAEVELIN Ovarian 200 study of the combination of avelumab and pegylated liposomal doxorubicin showed a 13% RR (31). The 72% RR observed in the phase II MEDIOLA study of olaparib and durvalumab likely reflects their cohort of exclusively platinum-sensitive, PARPi-naïve BRCAm ovarian cancer patients (32). Unlike the MEDIOLA study, our trial did not include an olaparib lead-in period given our predominantly BRCAwt, platinum-resistant cohort for whom PARPi monotherapy RRs are <10% and may rapidly progress while on PARPi treatment alone (33). Importantly, no comprehensive biomarker studies using paired fresh tissue and blood samples have been reported to date.

Our data indicate that PARPi does not significantly increase TMB in patients with ovarian cancer, regardless of BRCAm status, consistent with preclinical findings that long-term treatment with the PARPi niraparib did not increase the mutational load in BRCA1m breast carcinoma cells (34). This may be due to a short exposure to PARPi and also less mutagenic effects by PARPi. Moreover, neither baseline nor change in TMB after treatment were associated with clinical response, which likely reflects the overall low mutational load in ovarian cancer (26). Most trials that report a survival benefit use a threshold of approximately 10 or more somatic mutations/Mb to define high TMB (26) while all patients remained <5 mutations/Mb in our study.

We found that the PARPi and anti–PD-L1 combination induces systemic immune activation, possibly STING-independent, including increased expression of IFNγ and related immunostimulatory chemokines, systemic production of TNFα and IFNγ, and tumor infiltration by lymphocytes in patients with ovarian cancer. These data were not unanticipated as Fenerty and colleagues also reported olaparib significantly increases tumor cell sensitivity to NK-cell killing and antibody-dependent cellular cytotoxicity in both BRCAwt and BRCAm prostate carcinoma cells, independent of STING expression or modulation (35). Furthermore, our findings are consistent with studies of DNA-damaging chemotherapy showing apoptotic ovarian cancer cells induced by carboplatin and paclitaxel are immunogenic and enhance T-cell IFNγ secretion (36). IFNγ causes tumor inflammation and is associated with antitumor activity, including inhibiting tumor cell proliferation and promoting apoptosis (37, 38), potentially allowing for more effective immunotherapy combinations. Although IFNγ RNA expression levels were not associated with clinical benefit possibly due to small sample size, an increase in IFNγ plasma levels after treatment was associated with improved response and PFS, supporting the hypothesis that immune activation following treatment may contribute to the observed clinical benefit. Moreover, the immunoreactive molecular subtype, characterized by enhanced cytokine expression, T-cell activation, and TILs (21), was associated with clinical benefit, suggesting the immune-inflamed microenvironment induced by PARPi and anti–PD-L1 combination therapy may particularly benefit patients with immunoreactive tumors at baseline.

We also speculate that immunostimulatory effects may have been negated by various resistance mechanisms, for example, JAK1/2 mutations, TGFβ signaling, or an increase in counter-regulatory angiogenic factors like VEGFR3, resulting in modest clinical activity. JAK1/2 mutations result in loss of the antitumor effects of IFNγ via lack of expression of downstream signaling receptors and have been associated with primary resistance to immunotherapy due to the absence of reactive PD-L1 expression (27) while TGFβ signaling counteracts antitumor immunity by restricting T-cell infiltration (29). Consistent with our multivariable Cox proportional hazards model demonstrating increased posttreatment levels of VEGFR3 were associated with worse PFS, Chen and colleagues reported patients with melanoma who had no response to immune checkpoint inhibitors had higher on-treatment RNA expression of VEGFA thus implicating angiogenesis as a potential mechanism of resistance to immunotherapy (39). Also, overexpression of angiogenic factors in the tumor microenvironment has been shown preclinically to promote immunosuppression and facilitate cancer growth and metastases (40). As such, tumorigenic factors, for example, the VEGF/VEGFR pathway may act to counterbalance the immunostimulatory changes and serve as a target to further modulate the immunosuppressive microenvironment in ovarian cancer for improved efficacy of the PARPi and PD-L1 blockade combination. We are testing this hypothesis in an ongoing phase II trial of durvalumab in combination with olaparib and the VEGFR1-3 inhibitor cediranib in ovarian cancer (NCT02484404).

Here, we conducted the first clinical investigation into the immunomodulatory effects of olaparib and durvalumab in patients with heavily pretreated recurrent ovarian cancer. Our results show the combination induces antitumor immune responses in subsets of patients via an IFNγ-induced inflamed immune microenvironment. Limitations of our study include its small size and the heterogeneous patient population, including platinum-sensitive and resistant patients, BRCAm and BRCAwt patients, although a majority of patients had BRCAwt and platinum-resistant disease. We also acknowledge that the data on STING are suggestive but not definitive as activation of STING is not dependent on increased STING levels but rather on STING function. Furthermore, only a mandatory baseline and optional cycle 1 day 15 biopsy were collected, which may not capture changes in tumoral biology that occurred at other timepoints. Finally, based on our patient population, we did not include an olaparib lead-in period and are therefore unable to definitively differentiate the immunomodulatory effects of PARPi alone versus combination therapy.

In summary, we found subsets of heavily pretreated platinum-resistant patients, for whom either PARPi or immune checkpoint blockade monotherapy have shown limited activity, had a long duration of response although this study did not meet the prespecified ORR primary endpoint. Moreover, our data indicate combination may be best suited for the treatment of patients with recurrent ovarian cancer with immunoreactive subtype and addition of VEGF/VEFGR pathway blockade may be necessary to improve the efficacy of the PARPi and PD-L1 blockade combination, warranting further investigation.

A. Cimino-Mathews is an employee/paid consultant for and reports receiving commercial research grants from Bristol-Myers Squibb and reports receiving speakers bureau honoraria from Roche Diagnostics. A.B. Nixon is an employee/paid consultant for Eli Lilly, Kanghong Pharma, GlaxoSmithKline, and Promega, and reports receiving commercial research grants from Acceleron Pharma, Amgen, AstraZeneca/MedImmune, Eureka Therapeutics, Genentech, HTG Molecular Diagnostics, Leadiant Biosciences, MedPacto Inc., Novartis, Tracon Pharma. J.M. Taube is an employee/paid consultant for Bristol-Myers Squibb and Akoya Biosciences, and reports receiving commercial research grants from Bristol-Myers Squibb, Akoya Biosciences, AstraZeneca, Merck, and Compugen. J-M. Lee is an advisory board member/unpaid consultant for AstraZeneca. C.M. Annunziata received research funding from Precision Biologics. J.-M. Lee received research funding from AstraZeneca and Eli Lilly. No potential conflicts of interest were disclosed by the other authors.

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

Conception and design: J.-M. Lee

Development of methodology: A. Cimino-Mathews, J.R. Nair, J.W. Hodge, A.B. Nixon, J.M. Taube, J.-M. Lee

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): E.J. Lampert, A.S. Zimmer, A. Cimino-Mathews, E.M. Swisher, J.W. Hodge, A.B. Nixon, M.H. Bagheri, E. Levy, M.R. Radke, S. Lipkowitz, C.M. Annunziata, J.M. Taube, J.-M. Lee

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): E.J. Lampert, A.S. Zimmer, A. Cimino-Mathews, J.R. Nair, Y. Liu, E.M. Swisher, J.W. Hodge, A.B. Nixon, M.H. Bagheri, M.R. Radke, S.M. Steinberg, J.-M. Lee

Writing, review, and/or revision of the manuscript: E.J. Lampert, A.S. Zimmer, A. Cimino-Mathews, J.R. Nair, Y. Liu, E.M. Swisher, A.B. Nixon, S. Lipkowitz, C.M. Annunziata, J.M. Taube, S.M. Steinberg, J.-M. Lee

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): E.J. Lampert, A.S. Zimmer

Study supervision: J.-M. Lee

Other (research nurse): E. Nichols

We thank Drs. Elise C. Kohn, Bernard Parker, Lori Minasian and Farah Zia, Irene Ekwede RN, Nicole Houston RN, Kathryn Trewhitt NP, and Ms. Mireya Gomez for their contributions in clinic. We thank Bao Tran, Maggie Cam, and Xiongfong Chen for their expertise in generating the RNA and WES data used in this study. We also thank Drs. Kohn and Steeg for their invaluable comments concerning the manuscript. This research was supported by the Intramural Research Program of the NCI, CCR (grant number: #ZIA BC011525; to J.-M. Lee). Durvalumab and olaparib were supplied to the CCR, NCI under a Cooperative Research and Development Agreement between the CCR/NCI and AstraZeneca. Research funding was also provided by AstraZeneca and Stand Up To Cancer-Ovarian Cancer Research Fund Alliance-National Ovarian Cancer Coalition Dream Team Translational Research Grant (grant number: SU2C-AACR-DT16-15; to E. Swisher). Stand Up to Cancer is a division of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the scientific partner of Stand Up To Cancer. This research was made possible through the NIH Medical Research Scholars Program, a public-private partnership supported jointly by the NIH and contributions to the Foundation for the NIH from the Doris Duke Charitable Foundation (DDCF grant #2014194), the American Association for Dental Research, the Colgate-Palmolive Company, Genentech, Elsevier, and other private donors (to E.J. Lampert). This project has also been funded in part with federal funds from the NCI, NIH, under contract no. HHSN261200800001E (to E. Nichols).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.