Ad hoc Analysis of the Phase III ENGOT-OV16/NOVA Study: Niraparib Efficacy in Germline BRCA Wild-type Recurrent Ovarian Cancer with Homologous Recombination Repair Defects

In this analysis, we examined the relationship between progression-free survival (PFS) and mutation status of 18 homologous recombination repair (HRR) genes in patients in the non-germline BRCA-mutated (non-gBRCAm) cohort of the ENGOT-OV16/NOVA trial (NCT01847274), which evaluated niraparib maintenance therapy for patients with recurrent ovarian cancer. This post hoc exploratory biomarker analysis was performed using tumor samples collected from 331 patients enrolled in the phase III ENGOT-OV16/NOVA trial's non-gBRCAm cohort. Niraparib demonstrated PFS benefit in patients with either somatic BRCA-mutated (sBRCAm; HR, 0.27; 95% confidence interval, CI, 0.08–0.88) or BRCA wild-type (BRCAwt; HR, 0.47; 95% CI, 0.34–0.64) tumors. Patients with BRCAwt tumors with other non-BRCA HRR mutations also derived benefit from niraparib (HR, 0.31; 95% CI, 0.13–0.77), as did patients with BRCAwt/HRRwt (HRR wild-type) tumors (HR, 0.49; 95% CI, 0.35–0.70). When patients with BRCAwt/HRRwt tumors were further categorized by genomic instability score (GIS), clinical benefit was observed in patients with homologous recombination–deficient (GIS ≥ 42; HR, 0.33; 95% CI, 0.18–0.61) and in patients with homologous recombination–proficient (HRp; GIS < 42; HR, 0.60; 95% CI, 0.36–0.99) disease. Although patients with sBRCAm, other non-BRCA HRR mutations, or GIS ≥ 42 benefited the most from niraparib treatment, PFS benefit was also seen in HRp (GIS < 42) patients without HRR mutations. These results support the use of niraparib in patients with recurrent ovarian cancer regardless of BRCA/HRR mutation status or myChoice CDx GIS. Significance: We retrospectively evaluated the mutational profile of HRR genes in tumor samples from 331 patients from the non-germline BRCA-mutated cohort of the phase III NOVA trial of patients with platinum-sensitive high-grade serous ovarian cancer. Patients with non-BRCA HRR mutations generally benefited from second-line maintenance treatment with niraparib compared with placebo.


Introduction
The PARP family of nuclear proteins is recruited to DNA repair complexes and activated on sensing DNA single-strand breaks (SSB), playing a crucial role in SSB repair (1). In the presence of a PARP inhibitor (PARPi), unrepaired SSBs lead to stalled replication forks and accumulation of DNA double-strand breaks (DSB; ref. 2). In normal cells, DSBs are repaired effectively by a high-fidelity, error-free DNA repair process called homologous recombination repair (HRR; ref. 3). In cells with faulty HRR, called homologous recombination deficient (HRd), such as those bearing BRCA mutations (BRCAm), PARP inhibition induces accumulation of DNA DSBs, leading to the activation of nonhomologous end-joining pathway, an error-prone process to repair DNA DSBs; this process results in chromosomal instability, cell-cycle arrest, and subsequent apoptotic cell death (3). PARP inhibition also results in PARP-DNA complexes by trapping PARP1/2 protein on the DNA, which will further intensify the DNA replication fork damage. This synthetic lethality between PARP inhibition and homologous recombination defects has served as the basis of PARPi therapy development in multiple solid tumors and is the best-characterized mechanism of action for these agents (4,5). In addition, preclinical and clinical studies have demonstrated that tumor cells that are homologous recombination proficient (HRp) may also respond to PARPi, suggesting the utility of PARPi beyond HRd tumors (6)(7)(8)(9).
In addition to BRCA and BRCA, other genes play critical roles in orchestrating the HRR process, including genes involved in DNA DSB recognition (7), initiation of HRR (7), DNA resection (10), RAD51 filament strand invasion (11,12), DNA synthesis (12), and Holliday junction resolution (13). Defects in expressing these HRR genes will impair the integrity of HRR and may confer sensitivity to PARP inhibition. The sensitivity of tumors with HRR mutations (HRRm), including BRCA, BRCA, PALB, and RADC, to PARPi has been reported in preclinical research (14), as well as clinically (15,16), across tumor types (Supplementary Table S1). In ovarian cancer, analysis of samples from the ARIEL2 trial of rucaparib maintenance therapy found RADC and RADD mutations as well as high-level BRCA promoter methylation to be associated with PARPi sensitivity (17). However, the reported clinical evidence in ovarian maintenance with niraparib is limited.
Whereas the NOVA study used the BRACAnalysis assay (Myriad Genetics, Inc.) to determine gBRCAm status for patient enrollment, the tissue-based myChoice CDx (Myriad Genetics, Inc.) was used to determine tumor, or somatic, BRCAm status and genomic instability score (GIS). Patient tumors were identified as HRd [somatic BRCAm (sBRCAm) and/or GIS ≥ 42] or HRp (non-sBRCAm and GIS < 42). Patients whose tumors were identified as HRd in the non-gBRCAm cohort had longer mPFS in the niraparib arm than in the placebo arm [12.9 vs. 3.8 months (HR, 0.38; 95% CI, 0.24-0.59)]. In addition, patients in the niraparib arm who were identified as HRp had a significantly longer mPFS than those in the placebo arm [6.9 vs. 3.8 months (HR, 0.58; 95% CI, 0.36-0.92)] (7). These results potentially reflect the limitations of current tests to reliably capture patients with genomic scarring and HR-deficient tumors who could potentially benefit from therapy. In addition, they also suggest that mechanisms independent of BRCAm or HRd may confer clinical benefit with niraparib PARPi in ovarian cancer.
We performed a comprehensive retrospective analysis using tumor samples collected from the non-gBRCAm cohort in the ENGOT-OV16/NOVA trial to explore additional biomarkers or mechanisms that may predict sensitivity to niraparib. The mutation status of the 18 HRR genes, including BRCA/, within the Myriad HRD research assay (Myriad Genetics, Inc.) was evaluated as a biomarker for niraparib.

Patients and Samples
Of the 553 patients enrolled in the ENGOT-OV16/NOVA trial, 350 patients were in the non-gBRCAm cohort as determined by BRACAnalysis (Myriad Genetics, Inc.), which uses blood or saliva samples to test for the presence of deleterious or suspected deleterious germline BRCA/ mutations. Of the 350 patients in the non-gBRCAm cohort, 331 had archival tumor samples available for additional tumor biomarker testing and were included in this analysis.
As part of the ENGOT-OV16/NOVA trial, myChoice CDx was performed on tumor samples prior to database lock. The myChoice CDx test is an integrated next-generation sequencing test assessing sBRCAm status and measuring tumor genomic instability (18). Three algorithms were used to assess genomic instability-loss of heterozygosity (LOH) profiles, telomeric allelic imbalance, and large-scale state transitions-resulting in the myChoice CDx GIS, which is the sum of the three individual scores ( Supplementary Fig. S1). Although the myChoice CDx GISs distribute along a continuum, patients were categorized as either myChoice CDx HRD-positive (now called HRd) or HRD-negative (now called HRp) according to the prespecified cut-off score of 42 and/or sBRCAm presence (19). This analysis excludes patients who were enrolled in the gBRCAm cohort of the trial, as determined by BRACAnalysis, regardless of HR status.

HRR Biomarker Test
The Myriad research-grade assay (Myriad Genetics, Inc.) was performed on the 331 available patient tumor samples from the 350 patients in the non-gBRCAm cohort to determine the mutation status of 18

Statistical Analysis
All statistical analyses in this article were post hoc. An exploratory analysis was performed on the 331 patients from the non-gBRCAm cohort with available tumor samples to determine HRR gene mutation status. For the subgroup analyses, we performed a two-sided log-rank test using the stratification factors from randomization (best response to last platinum-based therapy, HRD status, and time from penultimate platinum-based therapy to progression) to analyze PFS, which was summarized using Kaplan-Meier methods. Patients were censored according to the methods used in the primary analysis (7). We estimated HRs with two-sided 95% CIs using a stratified Cox proportional hazards model with the stratification factors used in randomization.
HRs refer to the comparison of the niraparib arm with the placebo control arm. Formal P-value correction for multiple testing was not applied, but the multiplicity was accounted for in the interpretation of results. This approach was considered the most suitable given the exploratory nature of the analyses and that the measured biomarkers (BRCAm, HRRm, and myChoice CDx GIS) were selected on the basis of biological relevance.

Data Availability
All patients provided written informed consent as approved by an Institutional Review Board, in accordance with ethical guidelines as described in the U.S.

Post hoc Classification of HRR Gene Mutation Status Among Patients in the Non-gBRCAm Cohort of the Phase III ENGOT-OV16/NOVA Trial
Baseline characteristics of the 331 patients from non-gBRCAm cohort with known HRR status were included in this analysis are shown in Supplementary Table S2. Demographic and clinical characteristics were well balanced in the niraparib and placebo arms. For exploratory purposes, patients whose

Relationships Between GIS, BRCA Mutation Status, and Non-BRCA HRRm Status
The GIS histogram of the non-gBRCAm cohort showed a bimodal distribution, and the current Myriad GIS cutoff of 42 separated the two modes   (Fig. 4D). Because non-BRCA HRR genes function differently within the HRR pathway, niraparib treatment response was also evaluated in patients with tumor mutations in the more well-characterized HRR genes known to contribute to HRD and PARPi sensitivity (i.e., RADC, RADD, BRIP, and PALB) and patients with other, less well-studied HRR genes. Although the data should be approached with caution because of the small sample size, the benefit of niraparib treatment was more apparent in patients with mutations in well-known HRR genes than in the other group (Supplementary Table S3).  Table S4).

Discussion
We conducted a comprehensive retrospective analysis of the phase III NOVA/ENGOT-OV16 non-gBRCAm cohort, evaluating niraparib efficacy in subgroups of patients across the spectrum of biomarker status, including all combinations of sBRCA status, with or without a HRRm, and myChoice CDxidentified GIS status. We observed a statistically significant and clinically meaningful benefit of niraparib treatment in the broad NOVA patient population regardless of the biomarker status. These results are consistent with results from the NOVA clinical trial and demonstrate a continuum of benefits across biomarkers, with the highest sensitivity in patients with deleterious BRCAm, followed by those with myChoice CDx HRd tumors, and those with myChoice CDx HRp tumors (7,20). Although HRRm in BRCAwt tumors was associated with higher sensitivity to niraparib, we were unable to determine any biomarker that could identify a patient subset that did not show a clinical benefit. Given these results, although the myChoice CDx GIS may help to estimate the magnitude of benefit from maintenance treatment with niraparib, the benefit-risk ratio of this testing is low in this patient population, especially when the failure rate of the test is high (17% in the ENGOT-OV16/NOVA trial). RADC are frequently methylated in ovarian cancer, which results in a "BR-CAness" phenotype that confers sensitivity to PARPis. BRCA and RADC promoter methylations are often reported to be mutually exclusive with mutation events (22). Alternatively, the efficacy of niraparib could be explained via a DNA repair-independent effect. PARP1/2 are known to have pleotropic effects that extend beyond DNA repair, and there is growing evidence that PARP-mediated actions impact tumor cell proliferation and viability via alternative mechanisms of action such as PARP-regulated gene transcription (23), ribosome biogenesis (24), and immune activation (25). The efficacy seen with niraparib in BRCAwt/HRRwt/HRp tumors could be the result of a functional HR deficiency not identified by either the BRCA/HRR mutational analysis or the myChoice CDx HRD test. The myChoice CDx assay is based on capturing genomic scarring resulting from past HR deficiency events, which may not always reflect the current homologous recombination status of the tumor (26). In this analysis, the inability to discern between past genomic scarring and current functional homologous recombination deficiency status could also have been exacerbated by the archival nature of the tumor samples used for testing. The archival samples may not have been reflective of the homologous recombination status of the tumor at the time of niraparib treatment. Moreover, myChoice CDx genomic scarring is based on large structural chromosomal instabilities that do not include the additional genomic features associated with homologous recombination deficiency, such as mutational signatures and microhomology deletion.
When the NOVA trial was designed, the myChoice CDx test was not an approved diagnostic test, and BRACAnalysis was used to assign cohorts during randomization. A key difference between BRACAnalysis and myChoice CDx is that BRACAnalysis only detects germline mutations in BRCA, whereas the BRCA portion of myChoice CDx detects both germline and somatic mutations (in addition to the genomic scarring score). Therefore, it was not surprising that  conclusions on this topic. All HRR gene mutations are not expected to contribute equally to HR pathway deficiency. However, because the number of patients with any given HRR gene mutation was small, it was not feasible to assess the potential impact of each gene individually. The heterogeneous nature of non-BRCA HRR genes also may have contributed to the large 95% CI observed for HRs for patients with non-BRCA HRRm tumors. In addition, the myChoice CDx also has its limitations and may have failed to identify all patients with HR-deficient status. Small sample sizes must also be taken into consideration for the different subgroup analyses. Because the number of patients in each subgroup was small and not always evenly distributed between treatment arms, the 95% CIs for the HRs for several subgroups were quite large. Accordingly, caution should be used when extrapolating results to other patient populations. In addition, NOVA only enrolled patients who were platinum sensitive to their penultimate platinum-containing regimen. Because the platinum-free treatment interval following first-line treatment is an important predictor of responsiveness to subsequent treatment (27,28), the selection of likely responders in NOVA may limit the generalizability of the HRR findings. Future studies to prospectively test the impact of HRR gene mutations on PARPi efficacy (generally and on a per-gene basis) will be important to validate these results.