Purpose: Deficiency in homologous recombination (HR) repair of DNA damage is characteristic of many high-grade serous ovarian cancers (HGSC). It is imperative to identify patients with homologous recombination deficient (HRD) tumors as they are most likely to benefit from platinum-based chemotherapy and PARP inhibitors (PARPi). Existing methods measure historical, not necessarily current HRD, and/or require high tumor cell content which is not achievable for many patients. We set out to develop a clinically feasible assay for identifying functionally HRD tumors that can predict clinical outcomes. Experimental Design: We quantified RAD51, a key HR protein, in immunostained FFPE tumor samples obtained from both chemotherapy-naïve and neoadjuvant chemotherapy (NACT) treated HGSC patients. We defined cut-offs for functional HRD separately for these sample types, classified the patients accordingly as HR-deficient or HR-proficient, and analyzed correlations with clinical outcomes. From the same specimens, genomics-based HRD estimates (HR gene mutations, genomic signatures and genomic scars) were also determined, and compared with functional HR status. Results: Functional HR status significantly predicted several clinical outcomes, including progression-free survival (PFS) and overall survival (OS), when determined from chemo-naïve (PFS p<0.0001; OS p<0.0001) as well as NACT-treated (PFS p<0.0001; OS p=0.0033) tumor specimens. The functional HR test also identified as HRD those PARPi-at-recurrence -treated patients with longer OS (p=0.0188). Conclusions: We developed a functional HR assay performed on routine FFPE specimens, obtained from either chemo-naïve or NACT-treated HGSC patients, that can significantly predict real-world platinum-based chemotherapy and PARPi response.
ObjectiveA major challenge in the treatment of platinum-resistant high-grade serous ovarian cancer (HGSOC) is lack of effective therapies. Much of ongoing research on drug candidates relies on HGSOC cell lines that are poorly documented. The goal of this study was to screen for effective, state-of-the-art drug candidates using primary HGSOC cells. In addition, our aim was to dissect the inhibitory activities of Wee1 inhibitor adavosertib on primary and conventional HGSOC cell lines.MethodsA comprehensive drug sensitivity and resistance testing (DSRT) on 306 drug compounds was performed on three patient-derived genetically unique HGSOC cell lines and two commonly used ovarian cancer cell lines. The effect of adavosertib on the cell lines was tested in several assays, including cell-cycle analysis, apoptosis induction, proliferation, wound healing, DNA damage, and effect on nuclear integrity.ResultsSeveral compounds exerted cytotoxic activity toward all cell lines, when tested in both adherent and spheroid conditions. In further cytotoxicity tests, adavosertib exerted the most consistent cytotoxic activity. Adavosertib affected cell-cycle control in patient-derived and conventional HGSOC cells, inducing G2/M accumulation and reducing cyclin B1 levels. It induced apoptosis and inhibited proliferation and migration in all cell lines. Furthermore, the DNA damage marker γH2AX and the number of abnormal cell nuclei were clearly increased following adavosertib treatment. Based on the homologous recombination (HR) signature and functional HR assays of the cell lines, the effects of adavosertib were independent of the cells' HR status.ConclusionOur study indicates that Wee1 inhibitor adavosertib affects several critical functions related to proliferation, cell cycle and division, apoptosis, and invasion. Importantly, the effects are consistent in all tested cell lines, including primary HGSOC cells, and independent of the HR status of the cells. Wee1 inhibition may thus provide treatment opportunities especially for patients, whose cancer has acquired resistance to platinum-based chemotherapy or PARP inhibitors.
<div>AbstractPurpose:<p>Deficiency in homologous recombination (HR) repair of DNA damage is characteristic of many high-grade serous ovarian cancers (HGSC). It is imperative to identify patients with homologous recombination–deficient (HRD) tumors as they are most likely to benefit from platinum-based chemotherapy and PARP inhibitors (PARPi). Existing methods measure historical, not necessarily current HRD and/or require high tumor cell content, which is not achievable for many patients. We set out to develop a clinically feasible assay for identifying functionally HRD tumors that can predict clinical outcomes.</p>Experimental Design:<p>We quantified RAD51, a key HR protein, in immunostained formalin-fixed, paraffin-embedded (FFPE) tumor samples obtained from chemotherapy-naïve and neoadjuvant chemotherapy (NACT)-treated HGSC patients. We defined cutoffs for functional HRD separately for these sample types, classified the patients accordingly as HRD or HR-proficient, and analyzed correlations with clinical outcomes. From the same specimens, genomics-based HRD estimates (HR gene mutations, genomic signatures, and genomic scars) were also determined, and compared with functional HR (fHR) status.</p>Results:<p>fHR status significantly predicted several clinical outcomes, including progression-free survival (PFS) and overall survival (OS), when determined from chemo-naïve (PFS, <i>P</i> < 0.0001; OS, <i>P</i> < 0.0001) as well as NACT-treated (PFS, <i>P</i> < 0.0001; OS, <i>P</i> = 0.0033) tumor specimens. The fHR test also identified as HRD those PARPi-at-recurrence–treated patients with longer OS (<i>P</i> = 0.0188).</p>Conclusions:<p>We developed an fHR assay performed on routine FFPE specimens, obtained from either chemo-naïve or NACT-treated HGSC patients, that can significantly predict real-world platinum-based chemotherapy and PARPi response.</p></div>
<p>HRR gene mutations found in WGS/WES or clinical BRCA1/2 mutational testing.</p>
<p>A. PFS-based survival curves shown separately for discovery and validation cohorts. B. Response to second-line platinum therapy. C. PFS-based survival curves excluding BRCAmut fHRD patients.</p>
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