Homologous Recombination Status Research Articles

Limitations of homologous recombination status testing and poly (ADP-ribose) polymerase inhibitor treatment in the current management of ovarian cancer

Homologous recombination (HR) is a highly conserved DNA repair system, in which aberrations can lead to the accumulation of DNA damage and genomic scars known as homologous recombination deficiency (HRD). The identification of mutations in key genes (i.e., BRCA1, and BRCA2 (BRCA)) and the quantification of large-scale structural variants (e.g., loss of heterozygosity) are indicators of the HRD phenotype. HRD is a stable biomarker and remains unchanged during recurrence, but fails to reveal the molecular profile of tumor progression. Moreover, interpretation of the current HRD score lacks comprehensiveness, especially for the HR-proficient group. Poly (ADP-ribose) polymerase (PARP) enzymes play an important role in the repair of DNA single-strand breaks, the blockage of which using PARP inhibitors (PARPi) can generate synthetic lethality in cancer cells with HRD. Although numerous studies have demonstrated that the benefit of PARPi is substantial in ovarian cancer (OC) patients, the efficacy is limited by the development of resistance, and seems to be irrespective of HR and/or BRCA mutation status. Moreover, in addition to improving progression-free survival, long-term benefit as overall survival brought by PARPi for advanced, recurrent and refractory OC patients remains unclear. Therefore, further investigations are needed to uncover the role of HR genes beyond BRCA and their interactions with other oncogenic pathways, to determine the value of HRD in the recurrent setting, and to identify alternative strategies for the precise management of advanced, refractory OC patients.

Phase II clinical trial of PLX038 in patients with platinum resistant ovarian, primary peritoneal and fallopian tube cancer.

TPS5630 Background: Platinum-resistant ovarian cancer (PROC) portends a poor prognosis and, although treatments are available, response rates are low. Previous studies have demonstrated that DNA topoisomerase I (TOP1) inhibitors exhibit clinical activity in PROC. Irinotecan also has a 15-25% response rate in some studies of platinum-resistant epithelial ovarian cancer but does not have FDA approval for this indication. Additional studies in preclinical models suggest that TOP1 poisons are more active if administered at low doses on prolonged schedules. PLX038 is a long-acting prodrug of the potent TOP1 inhibitor SN-38. In PLX038, SN-38 is covalently conjugated to circulating polyethylene glycol and is slowly released at a constant rate to provide the active agent with a long half-life, a low Cmax and very high exposure – important facets for optimal safety and efficacy of this drug. Further, in preclinical studies the nanoparticle prodrug accumulates and is retained in tumors, where it slowly releases the SN-38. Methods: This single arm, phase 2 clinical trial is open at Mayo Clinic in Rochester, MN. The primary endpoint is overall response rate and secondary endpoints include progression-free survival and assessment of tolerability of PLX038. Correlative goals include i) measurement of TOP1-DNA covalent complexes in tumor biopsies and circulating tumor cells to assess successful target engagement; ii) assessment of homologous recombination status and SLFN11 expression for correlation with tumor response rate; and iii) pharmacokinetics of SN-38 and SN-38G as well as their association with GI toxicity. Assessment of gut microbiota and association with GI toxicity profile are also being investigated. Eligible patients include women with platinum resistant high grade serous ovarian, primary peritoneal, and fallopian tube cancer who have received fewer than two prior chemotherapy regimens in the platinum-resistant setting. All patients must have an ECOG performance status of 0, 1, or 2 and adequate bone marrow, renal, and hepatic function. As of January 31, 2024, 12 of planned 37 patients have enrolled. Clinical trial information: NCT05465941 .

CK2–HTATSF1–TOPBP1 signaling axis modulates tumor chemotherapy response

Homologous recombination (HR) plays a key role in maintaining genomic stability, and the efficiency of the HR system is closely associated with tumor response to chemotherapy. Our previous work reported that CK2 kinase phosphorylates HTATSF1 Ser748 (pS748) to facilitate HTATSF1 interaction with TOPBP1, which in turn, promotes RAD51 recruitment and HR repair. However, the clinical implication of the CK2-HTATSF1-TOPBP1 pathway in tumorigenesis and chemotherapeutic response remains to be elucidated. Here, we report that the CK2-HTATSF1-TOPBP1 axis is generally hyperactivated in multiple malignancies and renders breast tumors less responsive to chemotherapy. In contrast, deletion mutations of each gene in this axis, which also occur in breast and lung tumor samples, predict higher HR deficiency (HRD) scores, and tumor cells bearing a loss-of-function mutation of HTATSF1 are vulnerable to PARP inhibitors (PARPis) or platinum drugs. Taken together, our study suggests that the integrity of the CK2-HTATSF1-TOPBP1 axis is closely linked to tumorigenesis, and serves as an indicator of tumor HR status and modulates chemotherapy response.

Senaparib as first-line maintenance therapy in advanced ovarian cancer: a randomized phase 3 trial.

Poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors as maintenance therapy after first-line chemotherapy have improved progression-free survival in women with advanced ovarian cancer; however, not all PARP inhibitors can provide benefit for a biomarker-unselected population. Senaparib is a PARP inhibitor that demonstrated antitumor activity in patients with solid tumors, including ovarian cancer, in phase 1 studies. The multicenter, double-blind, phase 3 trial FLAMES randomized (2:1) 404 females with advanced ovarian cancer (International Federation of Gynecology and Obstetrics stage III-IV) and response to first-line platinum-based chemotherapy to senaparib 100 mg (n = 271) or placebo (n = 133) orally once daily for up to 2 years. The primary endpoint was progression-free survival assessed by blinded independent central review. At the prespecified interim analysis, the median progression-free survival was not reached with senaparib and was 13.6 months with placebo (hazard ratio 0.43, 95% confidence interval 0.32-0.58; P < 0.0001). The benefit with senaparib over placebo was consistent in the subgroups defined by BRCA1 and BRCA2 mutation or homologous recombination status. Grade ≥3 treatment-emergent adverse events occurred in 179 (66%) and 27 (20%) patients, respectively. Senaparib significantly improved progression-free survival versus placebo in patients with advanced ovarian cancer after response to first-line platinum-based chemotherapy, irrespective of BRCA1 and BRCA2 mutation status and with consistent benefits observed between homologous recombination subgroups, and was well tolerated. These results support senaparib as a maintenance treatment for patients with advanced ovarian cancer after a response to first-line chemotherapy. ClinicalTrials.gov identifier: NCT04169997 .

Abstract 5170: Novel dSTRIDE-HR assays can accurately assess DNA repair defects in human cancers and distinguish HR status of tumors

Abstract Accurate patient stratification is crucial for the successful application of targeted anti-cancer therapeutic approaches. It has been shown that PARP inhibitors are synthetic lethal with defective homologous recombination (HR) repair, with genes such as BRCA1 and BRCA2 identified as conferring therapy resistance or sensitivity, depending on their mutational status. Moreover, there is evidence of patient benefit of carboplatin-based therapy in HR deficient (HRD) tumors. With a growing emphasis on HRD in cancer treatment, efforts have been made to define the HRD phenotype. Nonetheless, the current approaches employed in the clinic, such as Next Generation Sequencing (NGS), HRD score or RAD51 assays have significant limitations and their interpretation can be unclear. To answer the need for a functional HR-specific biomarker, we have recently developed and introduced two new assays, called collectively dSTRIDE-HR. The assays are based on the STRIDE platform technology that enables direct and sensitive detection of single- or double-strand DNA breaks in situ in fixed cells. Novel dSTRIDE-HR explicitly detect double-strand DNA breaks localized in close proximity to RPA (dSTRIDE-RPA) or RAD51 (dSTRIDE-RAD51) proteins. The assays have been shown to be reliable reporters of HR activation in cellular models after exogenous DNA damage induction, yet their potential to predict HR status in an in vivo setting has not been tested so far. To assess the suitability of the dSTRIDE-HR assays for determining the HR status of patients’ tumor cells, we applied these assays in FFPE sections from biopsies derived from breast cancer patients with a different mutational status of BRCA1 and BRCA2 genes. The assays showed low dSTRIDE-RPA and dSTRIDE-RAD51 readouts in BRCA1/2 mutated tumors in comparison to wild type tumors, as well as a great concordance of readouts within a given genetic background. Additionally, the cell cycle analysis proved nearly twofold increase in foci numbers of dSTRIDE-RPA and dSTRIDE-RAD51 in G2/S phase. Our study unveils a potential of dSTRIDE-HR assays to become predictive biomarkers reporting on the status of HR repair in breast tumors, although more patient samples need to be tested to confirm initial findings. Citation Format: Monika Jarosz, Zsombor Prucsi, Franek Sierpowski, Karolina Stepien, Sophie Ostrowska-Paton, Szymon Koman, Nadeem Riaz, Simon Powell, Fresia Pareja, Edaise da Silva, Magdalena Kordon-Kiszala, Kamil Solarczyk. Novel dSTRIDE-HR assays can accurately assess DNA repair defects in human cancers and distinguish HR status of tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5170.

Abstract B114: Effect of spatial and temporal inter-tumoral heterogeneity on homologous recombination deficiency scores in high grade serous ovarian cancer

Abstract High-grade serous ovarian cancer (HGSC) is the most common and lethal subtype of ovarian cancer. Extensive genomic instability and heterogeneity are characteristics of HGSC, with near-ubiquitous TP53 loss-of-function mutations, defects in homologous recombination (HR) repair, and extensive copy number aberrations contributing to the vast genomic heterogeneity observed. Most patients relapse and acquire resistance to platinum or PARP inhibitor (PARPi)-based therapy. Diverse mechanisms leading to therapy resistance and lack of predictive biomarkers means that matching the best treatment options to patients is difficult. Testing tumors for HR-Deficiency (HRD) status is now a clinical test to predict PARPi sensitivity in HGSC. We aimed to determine the influence of spatial and temporal heterogeneity on HRD status in HGSC. Patients (n=59) at Hammersmith Hospital (HH) underwent maximal-effort upfront cytoreduction for advanced HGSC, had their tumor dissemination mapped and tumor biopsies collected (range 4-15). Paired relapse samples were collected for 10 patients. Tumor DNA was extracted (n=5 tumors per case, plus relapse), Illumina Human OmniExpress genotyping performed for 59 cases and whole genome sequencing (WGS; 30x depth) performed for 21 cases. Allele-specific copy number (CN) was quantified using ASCAT. Multi-site CN data was also accessed from two cohorts (GSE38787, GSE40546). Tumors that failed QC or had an aberrant cell fraction &amp;lt;0.3 were excluded. HR scores were estimated in each cohort using different genomic instability score (GIS) or mutational signature-based algorithms and relevant cut-offs were applied to determine HRD and HR-Proficient (HRP) tumors. Variations in HR scores were observed across the cohorts with a proportion of patients presenting with a mixed HR score, displaying both HRD and HRP scores in their tumors: HH cohort (20%, 11/54 patients); GSE38787 (17%, 4/24 patients); and GSE40546 (28%, 4/14 patients) using a scarHRD algorithm. In the HH cohort (n=45), we examined if mixed HR status was associated with survival and demonstrated that patients with HR-mixed or all HRP tumors had poorer progression-free (p=0.0052) and overall survival (p=0.00092) than patients with all HRD tumors. Applying different approaches to the HH WGS data (scarHRD, CHORD, &amp;gt;63 score cut-off), approximately 20% of patients demonstrated HR-mixed scores in their tumors in each approach. However, depending on the approach chosen to define HRD, different cases showed HR-mixed scores, 2/21 mixed cases overlapped between the different algorithms applied with tumors changing from HRD to HRP and vice versa depending on the approach applied. Differences in HR-deficiency/proficiency scores suggests that HR status may not be consistent across advanced disseminated HGSC, indicating that a single tumor biopsy may not accurately depict spatial HGSC tumor biology. HR score variations in different methods (GIS or mutational signatures) indicates that these tests are not analogous for defining HRD and thus PARPi suitability. Citation Format: Elizabeth L. Christie, Marc Lorentzen, Maiqi Liu, Nikki Burdett, Edward Curry, Chun Hei Kwok, Ahwan Pandey, Katherine Nixon, Jennifer Ploski, James J. Clark, Iain McNeish, David D.L. Bowtell, Jonathan Krell, Christina Fotopoulou, Paula Cunnea. Effect of spatial and temporal inter-tumoral heterogeneity on homologous recombination deficiency scores in high grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B114.

Functional Homologous Recombination (HR) Screening Shows the Majority of BRCA1/2-Mutant Breast and Ovarian Cancer Cell Lines Are HR-Proficient.

Tumors with a pathogenic BRCA1/2 mutation are homologous recombination (HR)-deficient (HRD) and consequently sensitive to platinum-based chemotherapy and Poly-[ADP-Ribose]-Polymerase inhibitors (PARPi). We hypothesized that functional HR status better reflects real-time HR status than BRCA1/2 mutation status. Therefore, we determined the functional HR status of 53 breast cancer (BC) and 38 ovarian cancer (OC) cell lines by measuring the formation of RAD51 foci after irradiation. Discrepancies between functional HR and BRCA1/2 mutation status were investigated using exome sequencing, methylation and gene expression data from 50 HR-related genes. A pathogenic BRCA1/2 mutation was found in 10/53 (18.9%) of BC and 7/38 (18.4%) of OC cell lines. Among BRCA1/2-mutant cell lines, 14/17 (82.4%) were HR-proficient (HRP), while 1/74 (1.4%) wild-type cell lines was HRD. For most (80%) cell lines, we explained the discrepancy between functional HR and BRCA1/2 mutation status. Importantly, 12/14 (85.7%) BRCA1/2-mutant HRP cell lines were explained by mechanisms directly acting on BRCA1/2. Finally, functional HR status was strongly associated with COSMIC single base substitution signature 3, but not BRCA1/2 mutation status. Thus, the majority of BRCA1/2-mutant cell lines do not represent a suitable model for HRD. Moreover, exclusively determining BRCA1/2 mutation status may not suffice for platinum-based chemotherapy or PARPi patient selection.

Niraparib maintenance in newly diagnosed advanced ovarian cancer — review and case series

Introducing PARP inhibitors maintenance therapy into clinical practice significantly improved treatment outcomes in patients with high-grade platinum-sensitive advanced ovarian cancer, the most lethal gynecological malignancy. Niraparib is a potent PARP inhibitor whose safety and efficacy were assessed in the placebo-controlled, randomized clinical trial PRIMA. Niraparib significantly prolonged progression-free survival in the overall population of high-grade advanced ovarian cancer regardless of BRCA and homologous deficiency status compared to placebo. However, the most significant benefit was observed in BRCA mutated and homologous recombination deficient subgroups. Niraparib has a manageable toxicity profile and is well-tolerated by patients. Most common toxicities are hematological and can be managed with drug interruption and dose reduction that do not decrease efficacy. Niraparib is recommended for patients who responded to the first-line chemotherapy with platinum compound regardless of homologous recombination status. This review will discuss the use of niraparib in newly diagnosed advanced ovarian cancer patients focusing on its efficacy and tolerability. Additionally, a case series will be presented to further discuss this drug use in clinical practice in Poland.

An activity-based functional test for identifying homologous recombination deficiencies across cancer types in real time

Homologous recombination (HR)-mediated DNA repair is a prerequisite for maintaining genome stability. Cancer cells displaying HR deficiency (HRD) are selectively eliminated by poly(ADP-ribose) polymerase inhibitors (PARPis). To date, sequencing of HR-associated genes and analyzing genome instability have been used as clinical predictions for PARPi therapy. However, these genetic tests cannot reflect dynamic changes in the HR status. Here, we have developed a virus- and activity-based functional assay to quantify real-time HR activity directly. Instead of focusing on a few HR-associated genes, our functional assay detects endpoint HR activity and establishes an activity threshold for identifying HRD across cancer types, validated by PARPi sensitivity and BRCA status. Notably, this fluorescence-based assay can be applied to primary ovarian cancer cells from patients to reflect their level of HRD, which is associated with survival benefits. Thus, our work provides a functional test to predict the response of primary cancer cells to PARPis.

Performance of a RAD51-based functional HRD test on paraffin-embedded breast cancer tissue

PurposeBRCA-deficient breast cancers (BC) are highly sensitive to platinum-based chemotherapy and PARP inhibitors due to their deficiency in the homologous recombination (HR) pathway. However, HR deficiency (HRD) extends beyond BRCA-associated BC, highlighting the need for a sensitive method to enrich for HRD tumors in an alternative way. A promising approach is the use of functional HRD tests which evaluate the HR capability of tumor cells by measuring RAD51 protein accumulation at DNA damage sites. This study aims to evaluate the performance of a functional RAD51-based HRD test for the identification of HRD BC.MethodsThe functional HR status of 63 diagnostic formalin-fixed paraffin-embedded (FFPE) BC samples was determined by applying the RAD51-FFPE test. Samples were screened for the presence of (epi)genetic defects in HR and matching tumor samples were analyzed with the RECAP test, which requires ex vivo irradiated fresh tumor tissue on the premise that the HRD status as determined by the RECAP test faithfully represented the functional HR status.ResultsThe RAD51-FFPE test identified 23 (37%) of the tumors as HRD, including three tumors with pathogenic variants in BRCA1/2. The RAD51-FFPE test showed a sensitivity of 88% and a specificity of 76% in determining the HR-class as defined by the RECAP test.ConclusionGiven its high sensitivity and compatibility with FFPE samples, the RAD51-FFPE test holds great potential to enrich for HRD tumors, including those associated with BRCA-deficiency. This potential extends to situations where DNA-based testing may be challenging or not easily accessible in routine clinical practice. This is particularly important considering the potential implications for treatment decisions and patient stratification.

First-line maintenance therapy with niraparib in advanced platinum-sensitive ovarian cancer: two 'long responder' case reports and review of the current literature.

The standard of care for newly diagnosed advanced ovarian cancer is surgical cytoreduction plus platinum-based chemotherapy; however, recurrent disease frequently occurs after treatment. Poly(ADP-ribose) polymerase (PARP) inhibitors as first-line maintenance therapy have been demonstrated to significantly reduce the risk of disease progression or death in patients with advanced ovarian cancer who have a complete or partial response to first-line platinum-based chemotherapy. Niraparib is the only PARP inhibitor that offers a significant progression-free survival benefit compared with placebo in this patient population regardless of the homologous recombination status. However, predictive factors for treatment responses and approaches to dose optimization remain to be investigated. In this study, two Chinese patients with newly diagnosed advanced ovarian cancer exhibited long-term responses to niraparib treatment, and hematological toxicity was successfully managed by dose adjustment. The literature on clinical trials and real-world experience on the efficacy, tolerability, and dose individualization of niraparib treatment in Western and Chinese patients was also reviewed. Future research is warranted to identify the characteristics of 'long responders' to niraparib treatment.

The multifaceted functions of homologous recombination in dealing with replication-associated DNA damages

The perturbation of DNA replication, a phenomena termed “replication stress”, is a driving force of genome instability and a hallmark of cancer cells. Among the DNA repair mechanisms that contribute to tolerating replication stress, the homologous recombination pathway is central to the alteration of replication fork progression. In many organisms, defects in the homologous recombination machinery result in increased cell sensitivity to replication-blocking agents and a higher risk of cancer in humans. Moreover, the status of homologous recombination in cancer cells often correlates with the efficacy of anti-cancer treatment. In this review, we discuss our current understanding of the different functions of homologous recombination in fixing replication-associated DNA damage and contributing to complete genome duplication. We also examine which functions are pivotal in preventing cancer and genome instability.

The role of Chemotherapy Response Score (CRS) in the decision making process for patients with advanced high grade serous ovarian cancer undergoing neoadjuvant chemotherapy.

5591 Background: The role of chemotherapy response score (CRS) in predicting PARP inhibitors (PARPi) response for high grade serous Advanced Ovarian Cancer (AOC) patients undergoing Neoadjuvant Chemotherapy (NACT) has never been investigated. Also, the correlation between CRS and Homologous Recombination status (HRs) is unclear. Methods: In this observational retrospective study, we collected data from patients with high grade serous AOC, FIGO stage III-IV, undergoing NACT and subsequent Interval Debulking Surgery (IDS) from 2017 to 2021. KELIM score, CRS and BRCA status were considered; additionally, HRs (Foundation test) was reported when available. Primary endpoint was Progression Free Survival (PFS) according to CRS (CRS 1/2 vs 3) in patients receiving PARPi. Results: We enrolled 309 patients; 102 had CRS3 and 193 had CRS1/2 (14 were undetermined). Patients with CRS3 and PARPi maintenance showed the best prognosis, compared to all other subgroups (median PFS Not Reached, p=0.014); additionally, in the BRCA wild type population, CRS3 was predictive of longer survival (median PFS for the CRS3+PARPi BRCAwt population 24 months compared with 15 months for the CRS1/2+PARPi BRCAwt patients, p=0.041). Furthermore, among the 44 patients with known HRs, 59.1% of HR Deficiency (HRD) positive patients had CRS3, conversely 22.7% of HRD negative ones had a CRS3 ( p=0.048) Finally, we found out that a favorable KELIM was significantly associated with CRS3 in the all-comers patients ( p=0.007) and in the BRCAwt ( p=0.04), but it was not predictive of PARPi response. Conclusions: Our findings suggested that the higher the CRS at the interval cytoreduction, the better the subsequent PARPi response. CRS correlates with PARPi response, as the HRD test does in other clinical trials and it may be considered as a surrogate of HRD status. We have planned to retrospectively perform HR test on a total sample size of 77 patients who received PARPi to corroborate this hypothesis. [Table: see text]

A Retrospective Study Comparing Olaparib and Bevacizumab as a Maintenance Therapy for Platinum-Sensitive Recurrent Ovarian Cancer: Impact on Recurrence-Free Survival in Japanese and Asian Populations.

The use of angiogenesis inhibitors and poly ADP-ribose polymerase inhibitors following multi-agent chemotherapy, including platinum-based agents, has become the standard treatment for platinum-sensitive recurrent ovarian cancer (PSROC). However, the optimal maintenance therapy and selection criteria for these patients remain unclear. Thus, this study aimed to optimize the treatment options and selection criteria for patients with PSROC. The clinical data of 51 patients with PSROC admitted to Nippon Medical School Chiba Hokusoh Hospital and Nippon Medical School Hospital were retrospectively collected. The log-rank test was used for the survival analysis, and Cox proportional hazard regression analysis was used for the multivariate survival analysis. Of the 51 patients, 17 received maintenance therapy with bevacizumab (Bev), and 34 received olaparib (Ola). Recurrence-free survival (RFS) was significantly prolonged in the Ola group (27 months; 95% confidence interval (CI), 19-NA months) compared with that in the Bev group (9 months; 95% CI, 5-22 months; p = 0.000103). The efficacy of Ola was independent of background factors, including response to previous chemotherapy, homologous recombination status, histological type, or laboratory data. Ola is superior to Bev as PSROC maintenance therapy, especially in Japanese and Asian populations.

A ribosomal gene panel predicting a novel synthetic lethality in non-BRCAness tumors

Poly (ADP-ribose) polymerase (PARP) inhibitors are one of the most exciting classes of targeted therapy agents for cancers with homologous recombination (HR) deficiency. However, many patients without apparent HR defects also respond well to PARP inhibitors/cisplatin. The biomarker responsible for this mechanism remains unclear. Here, we identified a set of ribosomal genes that predict response to PARP inhibitors/cisplatin in HR-proficient patients. PARP inhibitor/cisplatin selectively eliminates cells with high expression of the eight genes in the identified panel via DNA damage (ATM) signaling-induced pro-apoptotic ribosomal stress, which along with ATM signaling-induced pro-survival HR repair constitutes a new model to balance the cell fate in response to DNA damage. Therefore, the combined examination of the gene panel along with HR status would allow for more precise predictions of clinical response to PARP inhibitor/cisplatin. The gene panel as an independent biomarker was validated by multiple published clinical datasets, as well as by an ovarian cancer organoids library we established. More importantly, its predictive value was further verified in a cohort of PARP inhibitor-treated ovarian cancer patients with both RNA-seq and WGS data. Furthermore, we identified several marketed drugs capable of upregulating the expression of the genes in the panel without causing HR deficiency in PARP inhibitor/cisplatin-resistant cell lines. These drugs enhance PARP inhibitor/cisplatin sensitivity in both intrinsically resistant organoids and cell lines with acquired resistance. Together, our study identifies a marker gene panel for HR-proficient patients and reveals a broader application of PARP inhibitor/cisplatin in cancer therapy.

Abstract 6108: Novel functional dSTRIDE-HR assays to report on the status of homologous recombination repair in cancer cells

Abstract Since the early days of the synthetic lethality concept in DNA Damage Response the status of homologous recombination (HR) repair in cancer cells have been the focus of attention of researchers and clinicians. While different approaches exist, such as the RAD51 immunofluorescence (IF) or HRD genomic assays, functional biomarkers that can assess HR proficiency are missing. We report here the development, optimization and validation of two complementary, HR-specific functional assays. The assays, which are based on the STRIDE platform technology, detect double-strand DNA breaks localized in close proximity to RPA or RAD51 proteins. The optimization phase of assay development was performed in U2OS cells. First, repeatability (intra-run variation) and reproducibility (inter-run variation) of the assays were measured in untreated cells. Then, a series of technical negative controls was performed which have shown that the number of false-positive readouts is below 10% of the total number of signals. Finally, treatment of cells with compounds known to induce double-strand DNA breaks (etoposide and cisplatin) resulted in statistically significant increase in the number of detected dSTRIDE-RAD51 and dSTRIDE-RPA foci when compared to untreated controls. The assays were further validated in NCI-H661 (BRCA2 wild-type) and NCI-H169 (BRCA2 KO) cell line pair. The cells were treated with two concentrations of etoposide and the readouts from dSTRIDE, detecting the total pool of DSBs and dSTRIDE-HR assays were compared. In NCI-H661 cells, treatment with etoposide resulted in an increase in the number of double-strand breaks detected by dSTRIDE and as expected, more DSBs were formed after treatment with the higher concentration. dSTRIDE-HR assays confirmed that approximately 15% and 10% of these DSBs contain RPA and RAD51 proteins, respectively. In NCI-H169 cells etoposide produced a stronger reaction with even more DSBs detected by dSTRIDE, but importantly, no increase in the number of dSTRIDE-RAD51 foci was observed. dSTRIDE-RPA foci increased after treatment hinting that this step of HR remains unperturbed. Interestingly, the number of dSTRIDE-RAD51 foci in untreated cells was comparable between the two cell lines. In conclusion, we show here that two newly developed dSTRIDE-HR assays are well validated and can be successfully applied to report on the status of homologous recombination repair in different cell models. Citation Format: Kamil Solarczyk, Agnieszka Waligórska, Karolina Uznańska, Zsombor Prucsi, Olga Wójcikowska, Ewelina Matuszyk, Magdalena Bartyńska, Agata Kitlińska, Aleksandra Bober, Franek Sierpowski, Maja Białecka, Monika Jarosz, Malgorzata Szczygiel, Szymon Koman, Karolina Korpanty, Lukasz Beben, Lukasz Bandzarewicz, Przemyslaw Stachura, Magdalena Kordon-Kiszala. Novel functional dSTRIDE-HR assays to report on the status of homologous recombination repair in cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6108.

Functional Homologous Recombination Assay on FFPE Specimens of Advanced High-Grade Serous Ovarian Cancer Predicts Clinical Outcomes.

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. 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. fHR 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 fHR test also identified as HRD those PARPi-at-recurrence-treated patients with longer OS (P = 0.0188). 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. See related commentary by Garg and Oza, p. 2957.

Development of Homologous Recombination Functional Assays for Targeting the DDR.

Identification of tumours that have homologous recombination deficiency (HRD) has become of increasing interest following the licensing of PARP inhibitors. Potential methods to assess HRD status include; clinical selection for platinum sensitive disease, mutational/methylation status, genomic scars/signature and functional RAD51 assays. Homologous recombination (HR) is a dynamic process with the potential to evolve over a disease course, particularly in relation to previous treatment. This is one of the major drawbacks of genomic scars/signatures, as they only demonstrate historic HR status. Functional HR assays have the benefit of giving a real time HR status readout and therefore have the potential for clearer identification of patients who may benefit from PARP inhibitors at that specific time point. However, the development of RAD51 foci assays ready for clinical practice has been challenging. Pre-clinical considerations have included; controlling for variation in tumour proliferation, tissue type and whether DNA damage induction is required. Furthermore, the assays require correlation with clinical outcomes, an understanding of how they complement current testing modalities and validation of test performance in large cohorts. Despite these challenges, given the profound benefit from PARP inhibitors seen in those with an HRD phenotype to date, the ongoing development and validation of these functional HR assays remains of high clinical importance.

Disparity in the era of personalized medicine for epithelial ovarian cancer.

The treatment of high-grade serous ovarian cancer and high-grade endometrioid ovarian cancer has seen significant improvements in recent years, with BRCA1/2 and homologous recombination status guiding a personalized approach which has resulted in improved patient outcomes. However, for other epithelial ovarian cancer subtypes, first-line treatment remains unchanged from the platinum-paclitaxel trials of the early 2000s. In this review, we explore novel therapeutic approaches being adopted in the treatment of clear cell, mucinous, carcinosarcoma and low-grade serous ovarian cancer and the biological rational behind them. We discuss why such disparities exist, the challenges faced in conducting dedicated trials in these rarer histologies and look towards new approaches being adopted to overcome them.

Concurrent Olaparib and Radiotherapy in Patients With Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) cells are sensitive to poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors used as radiosensitizers. Whether combining PARP inhibitors with radiotherapy in patients with TNBC would enhance the biological effectiveness of the irradiation and improve locoregional control is unclear. To assess the safety and tolerability of PARP inhibition with olaparib used concurrently with radiotherapy in patients with TNBC with residual disease after neoadjuvant chemotherapy. This phase 1 prospective dose-escalation trial (Olaparib and Radiation Therapy for TNBC [RadioPARP] trial) using a time-to-event continual reassessment method was performed from September 2017 to November 2019, with follow-up until November 2021. Participants had an incomplete pathologic response after neoadjuvant chemotherapy or unresectable TNBC despite previous neoadjuvant chemotherapy, an Eastern Cooperative Oncology Group Performance Status score of 0 or 1, and adequate organ functions. Olaparib was administered orally in the form of tablets and given at increasing doses (50 mg, 100 mg, 150 mg, or 200 mg twice daily). Olaparib therapy was started 1 week before radiotherapy and was continued concomitantly with radiotherapy. After breast-conserving surgery, a total dose of 50.4 Gy was delivered to the whole breast, with a 63-Gy simultaneously integrated boost to the tumor bed for patients younger than 60 years. After radical mastectomy or for unresectable tumors despite neoadjuvant chemotherapy, a total dose of 50.0 Gy was delivered to the chest wall (after mastectomy) or to the whole breast (for unresectable tumors). Regional lymph node stations could be treated with a total dose of 50.0 Gy to 50.4 Gy in cases of node-positive disease. Main outcomes were the safety and tolerability of PARP inhibition with radiotherapy for early-stage, high-risk TNBC. Secondary outcomes included overall survival (OS) and event-free survival (EFS). Among the 24 patients included in the trial (100% female; median age, 46 years [range, 25-74 years]), no dose-limiting toxic effects were observed, and olaparib was escalated to 200 mg twice daily without reaching the maximum tolerated dose. No late treatment-related grade 3 or greater toxic effect was observed, and the maximum observed treatment-related toxic effects at the 2-year follow-up were grade 2 breast pain, fibrosis, and deformity in 1 patient (4.2%). Three-year OS and EFS were 83% (95% CI, 70%-100%) and 65% (95% CI, 48%-88%), respectively. Homologous recombination status was not associated with OS or EFS. The findings of this phase 1 dose-escalation trial suggest that PARP inhibition with olaparib concurrently with radiotherapy for early-stage, high-risk TNBC is well tolerated and should continue to be evaluated in further clinical trials. ClinicalTrials.gov Identifier: NCT03109080.