Epithelial ovarian cancer (EOC) is the most deadly gynecologic malignancy, responsible for an estimated 13,940 deaths annually in the United States [1
]. Historically, EOC has been clinically managed as one homogeneous entity with a combination of surgical cytoreduction and platinum-taxane chemotherapy [2
]. However, histologic subtypes of EOC demonstrate significant biologic and genetic differences that impact their susceptibility to cytotoxic chemotherapy and targeted agents [2
]. The most common EOC histologic subtype is high-grade serous (HGS) carcinoma (70% of EOC cases), with the remainder being low-grade serous, clear cell, endometrioid, and mucinous [5
]. Additionally, in profiling the molecular landscape of ovarian tumors, the Cancer Genome Atlas (TCGA) identified distinct molecular features between histologic subtypes. For example, HGS tumors had near universal TP53 gene mutations (96%), with half containing abnormalities in the homologous recombination (HR) pathway [6
]. Focusing on unique molecular features of EOC can provide the foundation to advance clinical management beyond the historical one-size-fits-all approach and improve long-term clinical outcomes.
Exploiting defects in the HR pathway has transformed clinical management of EOC and has placed emphasis on the development of reliable molecular assays to identify the presence of HR defects. The HR pathway is a set of critical DNA damage response mechanisms that protect genomic stability through high-fidelity repair of double-stranded DNA breaks [5
]. Among a number of HR pathway protein factors, BRCA1 and BRCA2 proteins are critical to the integrity of the HR repair response. BRCA1 is a versatile protein that complexes with a number of proteins in the BRCA1-associated genome surveillance complex in order to link the detection of double-stranded DNA breaks and DNA damage repair effectors [5
]. The main function of the BRCA2 protein is recruitment of RAD51 to regions of double-stranded breaks for HR repair [5
]. With defective HR pathway proteins, cells with homologous recombination deficiency (HRD) are more susceptible to the intra- and interstrand crosslinking action of platinum agents [11
]. Furthermore, HRD has become an attractive target for poly-ADP ribose polymerase inhibitors (PARPi) due to mechanisms of synthetic lethality and/or PARP trapping [5
]. Given the crucial involvement of BRCA1 and BRCA2 proteins in the HR pathway, PARPi treatment in patients with BRCA1/2 mutations has significantly improved progression-free survival (PFS), thereby dramatically changing the landscape for EOC management [16
]. Given this clinical benefit, expanding the scope of PARPi use through the identification of other biomarkers of HRD has been a particular focus of research [5
]. In addition to germline and somatic BRCA1/2 mutations, other HRD markers include BRCA1 promoter methylation and other HR-related gene mutations (e.g., ATM, BARD1, BRIP1, CHEK2, NBN, PALB2, RAD51C, and RAD51D) [5
]. Another assay that has garnered strong interest is the combined homologous recombination deficiency score (HRD score) [19
]. HRD score is an unweighted sum of three independent DNA-based measures of genomic instability (loss of heterozygosity, telomeric allelic imbalance, and large-scale transitions) in the tumor [19
]. A high HRD score (≥42) has been shown to be predictive of clinical benefit with PARPi therapy, independent of BRCA1/2 status [26
Immunotherapy also holds promise to revolutionize management of EOC and improve clinical outcomes, but again, requires robust and clinically feasible molecular assays to define key features and predict treatment response. Tumor molecular features, such as microsatellite instability (MSI), increase tumor immunogenicity and are strongly predictive of response to immune checkpoint inhibitors [28
]. Microsatellites are repeated segments of DNA that are prone to DNA replication errors and these errors are typically corrected by DNA mismatch repair (MMR) proteins [29
]. Nonfunctional or absent MMR proteins result in a substantial increase in mutations in the microsatellite regions, a condition called microsatellite instability [29
]. Given this genetic hypermutable state, MSI or MMR-deficient tumors produce a greater neoantigen load and elicit a stronger antitumor immune response compared to microsatellite stable or MMR-proficient tumors [29
]. Marabelle et al. reported improved objective response rates among patients with recurrent MSI or MMR-deficient EOC tumors treated with pembrolizumab [30
]. Given this clinical benefit, pembrolizumab was approved for use in MSI or MMR-deficient solid tumors in 2017, signifying the first tissue-agnostic indication for a drug. Furthermore, high tumor mutational burden (TMB), a measure of the number of gene mutations within the tumor, has been cited as a predictive biomarker for response to immune checkpoint inhibitors [32
Given the clinical importance of molecular profiling for HRD, MSI, and TMB, we developed a consecutive series of cases of EOC to conduct both molecular characterization and collect extensive clinical follow-up data. With this powerful cohort and molecular assays, we evaluated the association of these biomarkers and other clinical prognostic factors with survival outcomes.
With growing clinical need and interest in molecular tumor assays in the management of EOC, we sought to characterize HRD biomarkers, MSI, and TMB and determine their associations with survival outcomes. Given the potential clinical impact of the HRD score assay, evaluation in multiple study populations is essential. We evaluated the impact of lowering the HRD score threshold from 42 to 33, given that retrospective studies have suggested that such a cutoff may improve sensitivity in detecting responses and benefits to platinum-containing agents and/or PARPi [35
]. Both HRD score thresholds of 42 or 33 were associated with improved OS and PFS among patients with HGS tumors in univariable analyses. However, compared to the previously used threshold of 42, the lower threshold of 33 remained significantly associated with improved OS among all patients in univariable and multivariable analyses. Additionally, in multivariable analyses, improved PFS for those with HRD scores ≥33 approached statistical significance.
After lowering the HRD score threshold to 33, there were an additional 8 patients (making a total of 50 patients) who met the criteria of HRD based on HRD score alone, out of a total of 95 with completed HRD score testing. Among the 95 patients with complete HRD score testing, there were also 50 with negative germline and somatic BRCA1/2 results (34 HGS and 16 non-HGS), with 20 having high HRD scores. Thus, based on the findings in this study, HRD score testing could help identify a substantial number of patients with HRD beyond patients that would be identified with BRCA1/2 testing alone. Furthermore, among those with HRD testing, there was interestingly only one patient with an HR defect who did not have a high HRD score ≥33 (a patient with endometrioid histology, ATM germline mutation, and an HRD score of 0). Although ATM plays an early role in the HR pathway, there is evidence that ATM-deficient cells may still have overall functional HR pathways (albeit delayed kinetics) with sensitivity to PARPi because ATM is postulated to also regulate response to double-strand DNA breaks at multiple levels and inhibit error prone nonhomologous end joining [42
]. This mechanism may explain the low HRD score in the aforementioned patient with a germline ATM mutation.
Given the growing importance of immunotherapy in solid tumors and the need to establish biomarkers to predict responses, we evaluated MSI and TMB in the EOC population undergoing PCS. In our study, we observed only one MSI tumor (clear cell histology) out of 107 (0.9%) EOC tumors with successful MSI testing. This finding is consistent with the other studies that have reported an MSI prevalence of approximately 1–2% [44
]. A meta-analysis and systematic review by Pal and colleagues highlighted the wide range in the prevalence of MSI in 977 EOC patients in 18 studies ranging from 0% to 36.7% [46
] and a pooled frequency of 12% (95% CI 8–17%), but the variations in reported prevalence may be related to interstudy differences, including study population heterogeneity, histologic subtypes evaluated, or detection methods [45
]. Additionally, we observed TMB to be low in nearly all our patients except for the same patient with an MSI tumor. Other studies have reported TMB to be lower in EOC compared to other tumors but that it may be increased in tumors in the presence of HR defects [47
]. It is difficult to comment on the relevance of MSI and TMB status on prognosis given that the overwhelming majority of tumors did not demonstrate MSI and had low TMB. In the literature, there have been mixed results regarding MSI and TMB for EOC patients with MSI tumors [49
]. Future studies should investigate MSI and TMB in the non-HGS EOC population.
As expected, factors such as younger age, early stage, optimal cytoreduction, or presence of BRCA1/2 mutations were associated with improved OS and PFS (especially for HGS patients) and these prognosticators have been reported in the literature [51
]. We did not observe any association between BRCA1 promoter methylation and survival and it is difficult to assess the impact of BRCA1 promoter methylation in this study given the small number of cases with methylation (n
= 7). Unlike germline or somatic BRCA1/2 mutations, BRCA1 promoter methylation as a prognosticator has been reported with mixed results [53
]. Similar to BRCA1 promoter methylation in this study, there were few cases of germline non-BRCA1/2 HR mutations and this likely impacted the ability to observe a favorable association with survival outcomes. This result is in contrast with the results of molecular analyses of samples from patients enrolled in the larger GOG 218 trial (n
= 307), where the investigators observed non-BRCA1/2 HR-related mutations to be a favorable prognosticator [20
The utilization of HRD score testing in clinical trials evaluating PARPi therapy has highlighted the assay’s potential clinical benefit to EOC patients. In a phase III, double-blind randomized control trial (PRIMA), patients with HRD score ≥42 had improved PFS when treated with niraparib compared to placebo (HR 0.50, 95% CI 0.31–0.83) in the frontline setting [56
]. In a phase II, single-arm QUADRA trial, Moore and colleagues observed an objective response rate of 26% to niraparib for recurrent, platinum-sensitive EOC patients with HRD scores ≥42 (compared to 4% in the HR proficient or unknown group) on subgroup analysis [27
]. The results of the QUADRA trial led to FDA approval of niraparib monotherapy for patients with platinum-sensitive tumors and a high HRD scores in the recurrent setting. Additionally, a phase III PAOLA-1 trial investigated the use of bevacizumab (anti-VEGF monoclonal antibody) and olaparib maintenance compared to placebo/bevacizumab maintenance in advanced stage EOC in the frontline setting [26
]. In a subgroup analysis of patients with an HRD score ≥42, the median PFS was improved in the olaparib/bevacizumab arm compared to the placebo/bevacizumab arm (28.1 vs. 16.6 months; HR 0.31, 95% CI 0.20–0.47) [26
]. Based on these results, the addition of olaparib to bevacizumab maintenance in the frontline setting received FDA approval in May 2020 for patients with advanced EOC and HR deficient tumors that are responsive to platinum-based chemotherapy and bevacizumab. In a phase III VELIA trial, patients with advanced HGS ovarian cancer in the frontline setting were placed in three arms: chemotherapy with placebo and placebo maintenance (control), chemotherapy with velaparib followed by placebo maintenance (velaparib-combination only), and chemotherapy with velaparib followed by velaparib maintenance (velaparib-throughout) [36
]. The trial designated HRD scores ≥33 as HR deficiency, given that retrospective analyses had demonstrated a subset of patients with HRD scores below 42 who derived some PFS benefit when treated with PARPi [35
]. The VELIA investigators demonstrated an HRD score ≥33 was associated with improved PFS (HR 0.80, 95% CI 0.64–0.997) when treated with velaparib-throughout compared to placebo [36
]. These aforementioned clinical trials highlighted the importance of HRD score testing as a tool to identify patients likely to benefit from PARPi therapy. Biomarkers such as germline/somatic BRCA1/2, germline non-BRCA1/2 mutations, and BRCA1 promoter methylation may not fully capture defects in the HR pathway. Thus, genomic signatures captured through HRD assays can potentially expand the benefit of PARPi to more EOC patients.
One of the strengths of this study was the follow-up period for the study population of 79.1 months (range 18.6–114.9 months). A long follow-up period improves the ability to detect differences in OS based on biomarker status. Furthermore, this study evaluated multiple biomarker surrogates for HRD in order to evaluate evidence of HRD. Additionally, this cohort had a larger than usual representation of Hispanic patients (13.3% compared to typically 4%) [20
]. This study has several limitations. Molecular analyses could not be uniformly performed on all cases. When performing molecular analyses, blood or tissue samples were not always sufficient to complete germline non-BRCA1/2 HR-related mutation, BRCA1 promoter methylation, or HRD score testing. In addition, because only pretreatment core biopsies were available for patients in the NACT cohort, these specimens were reserved for clinical care and this research study focused exclusively on the PCS cohort. Furthermore, PCS and NACT populations may vary across institutions due to different practice patterns, which may influence the generalizability of these findings. Additionally, given the longitudinal nature of the study, long-term survival outcomes may also be influenced by differences in treatment regimens following frontline therapy. Furthermore, as discussed earlier, the clinical impact of studied prognosticators may also be modified in the current landscape of greater PARPi use. In addition, there were low numbers of endometrioid, clear cell, and low-grade serous tumors for HRD characterization. This representation was expected given that HGS tumors represent the great majority of EOC. Lastly, while the HRD ≥42 group did not show a statistically significant association with overall survival in the Kaplan–Meier analysis or multivariable models, it is possible that evaluation in a larger cohort of cases would have greater statistical power to determine an association. Nevertheless, it is important to note that the hazard ratios for the HRD ≥33 group showed consistently significant HRD effects compared to the HRD ≥42 group. In the multivariable models, evaluation of these two different cutoffs again showed a significant HRD effect using the HRD ≥33 cutoff but not when using the HRD ≥42 cutoff (Table 5