Phospho-RPA2 predicts response to platinum and PARP inhibitors in homologous recombination-proficient ovarian cancer

Abstract

BACKGROUNDTreatment of tubo-ovarian high-grade serous carcinoma (HGSC) includes cytoreductive surgery, platinum-based chemotherapy, and often poly(ADP-ribose) polymerase (PARP) inhibitors. While homologous recombination (HR) deficiency is a well-established predictor of therapy sensitivity, over 50% of HR-proficient HGSCs also exhibit sensitivity. Currently, there are no biomarkers to identify which HR-proficient HGSCs will be sensitive to standard-of-care therapy. Replication stress may serve as a key determinant of response.METHODSWe evaluated phospho-RPA2-T21 (p-RPA2) foci via immunofluorescence as a biomarker of replication stress in formalin-fixed, paraffin-embedded HGSC samples collected at diagnosis from patients treated with platinum chemotherapy (discovery cohort, n = 31; validation cohort, n = 244) or PARP inhibitors (n = 63). Recurrent HGSCs (n = 38) were also analyzed. p-RPA2 score was calculated using automated imaging analysis.RESULTSSamples were defined as p-RPA2-high if more than 16% of cells had ≥2 p-RPA2 foci on automated analysis. In the discovery cohort, HR-proficient, p-RPA2-high HGSCs demonstrated significantly higher rates of a chemotherapy response score of 3 to platinum chemotherapy than HR-proficient, p-RPA2-low HGSCs. In the validation cohort, patients with HR-proficient, p-RPA2-high HGSCs had significantly longer survival after platinum treatment than those with HR-proficient, p-RPA2-low HGSCs. Additionally, the p-RPA2 assay effectively predicted survival outcomes in patients treated with PARP inhibitors and in recurrent HGSC samples.CONCLUSIONOur study underscores the importance of considering replication stress markers, such as p-RPA2, alongside HR status in therapeutic planning. This approach has the potential to increase the number of patients receiving effective therapy while reducing unnecessary toxicity.FUNDINGThe Reproductive Scientist Development Program, GOG Foundation, Pilot Translational and Clinical Studies function of the Washington University Institute of Clinical and Translational Sciences, the Foundation for Barnes-Jewish Hospital, Washington University School of Medicine Dean's Scholar Program, The Cancer Biology Pathway Training Grant (5T32CA113275-17), The Lucy, Anarcha, and Betsey (L.A.B.) Award from the Department of Obstetrics and Gynecology at Washington University School of Medicine, and Veterans Affairs Office of Research and Development (I01BX006020).

Keywords: Clinical Research; DNA repair; Molecular diagnosis; Obstetrics/gynecology; Oncology.

Figure 1. p-RPA2 score predicts therapy response and survival in patients with RAD51-high HGSCs treated with platinum chemotherapy in a discovery cohort.

(A) RAD51 score, p-RPA2 score, chemotherapy response score (CRS), and platinum sensitivity in patients with HGSCs before neoadjuvant chemotherapy. p-RPA2 score is defined as the percentage of cells having ≥2 p-RPA2 foci. Dashed black line indicates manual quantification 20% cutoff, which delineates p-RPA2-high and p-RPA2-low HGSCs. All foci were counted in n > 100 cells per experiment. Technical replicates were performed for 30% of samples. (B) Representative images of DAPI and p-RPA2 and overlay of DAPI/p-RPA2 in patient-derived FFPE HGSC samples; images taken at ×63 original magnification. (C) Kaplan-Meier curves evaluating progression-free survival in patients with HGSCs stratified by p-RPA2 score (n = 27, hazard ratio 0.7, 95% CI 0.3–1.7, P = 0.4). (D) Kaplan-Meier curves evaluating progression-free survival in patients with HGSCs stratified by RAD51 and p-RPA2 scores. (E) Proportion of RAD51-high HGSCs that had a CRS of 3 stratified by p-RPA2 score (P = 0.03). (F) Kaplan-Meier curves evaluating overall survival in patients with RAD51-high HGSCs stratified by p-RPA2 score (n = 19, hazard ratio 0.08, 95% CI 0.01–0.64, P = 0.02). *P < 0.05, by Student’s 2-tailed t test.

Figure 2. A functional replication stress assay for predicting response to DNA-damaging therapy in HR-proficient HGSCs.

Schematic of combined RAD51 and p-RPA2 immunofluorescence assay in HGSC FFPE samples including automated quantification. After confirmation of cellularity in FFPE HGSC samples, they were screened for γH2AX. Only samples with 25% or more cells displaying 2 or more γH2AX foci were included in the analysis, ensuring sufficient DNA damage to elicit a DNA damage response. RAD51 foci were then assessed in geminin-positive cells, with cells containing 5 or more RAD51 foci classified as positive. Samples were considered RAD51-high or HR-proficient if more than 6% of the geminin-positive cells were positive for RAD51 on automated analysis. A minimum of 100 cells were assessed per sample. Subsequently, samples were evaluated for p-RPA2 foci. Cells with 2 or more p-RPA2 foci were considered positive, and samples with more than 16% of positive cells on automated analysis were considered p-RPA2-high or replication stress–high.

Figure 3. Automated p-RPA2 score predicts survival in patients with RAD51-high HGSCs treated with platinum chemotherapy in a validation cohort.

(A) RAD51 and p-RPA2 scores in the validation cohort. (B and C) Kaplan-Meier curves evaluating progression-free survival (n = 236, aHR 0.53, 95% CI 0.34–0.84, P = 0.006) (B) and overall survival (n = 240, aHR 0.27, 95% CI 0.14–0.52, P < 0.001) (C) in patients with HGSCs stratified by p-RPA2 score. (D and E) Kaplan-Meier curves evaluating progression-free survival (n = 178, aHR 0.34, 95% CI 0.19–0.61, P < 0.001) (D) and overall survival (n = 183, aHR 0.18, 95% CI 0.09–0.37, P < 0.001) (E) in patients with RAD51-high HGSCs stratified by p-RPA2 score. (F and G) Kaplan-Meier curves evaluating progression-free survival (n = 58, aHR 0.82, 95% CI 0.29–2.39 P = 0.72) (F) and overall survival (n = 57, aHR 0.28, 95% CI 0.06–1.27, P = 0.10) (G) in patients with RAD51-low HGSCs stratified by p-RPA2 score. aHR, adjusted hazard ratio for age, stage, residual disease, and BRCA mutation status.

Figure 4. Automated p-RPA2 score predicts survival in patients with RAD51-high HGSCs treated with PARP inhibitors.

(A) RAD51 and p-RPA2 scores of patients treated with a PARP inhibitor. (B) Pie chart illustrating the timing of PARP inhibitor therapy. (C) Kaplan-Meier curves evaluating progression-free survival in patients with RAD51-high HGSCs who received frontline PARP inhibitor therapy stratified by p-RPA2 score (n = 25, hazard ratio 0.33, 95% CI 0.09–1.16, P = 0.08). (D and E) Kaplan-Meier curves evaluating progression-free survival in patients with RAD51-high, p-RPA2-high HGSCs (n = 74, hazard ratio 0.39, 95% CI 0.15–1.00, P = 0.05) (D) or RAD51-high, p-RPA2-low HGSCs (n = 50, hazard ratio 0.42, 95% CI 0.18–0.97, P = 0.04) (E) treated with or without frontline PARP inhibitor maintenance therapy.

Figure 5. Automated p-RPA2 and RAD51 scores at the time of recurrence predict survival.

(A) RAD51 and p-RPA2 scores in recurrent HGSCs. (B) HGSCs stratified by RAD51 score at time of diagnosis and recurrence (n = 37). (C) Kaplan-Meier curves evaluating overall survival after recurrence in patients with HGSCs stratified by RAD51 score at time of recurrence (n = 38, hazard ratio 0.43, 95% CI 0.17–1.06, P = 0.06). (D) HGSCs stratified by p-RPA2 score at time of diagnosis and recurrence (n = 37). (E) HGSCs stratified by RAD51 and p-RPA2 scores at time of diagnosis and recurrence (n = 37). (F) Kaplan-Meier curves evaluating overall survival after recurrence in patients with HGSCs stratified by RAD51 and p-RPA2 scores at time of recurrence (n = 38, hazard ratio 0.35, 95% CI 0.16–0.78, P = 0.01).