Effects of Wee1 inhibitor adavosertib on patient-derived high-grade serous ovarian cancer cells are multiple and independent of homologous recombination status.

Objective: A 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. Methods: A 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. Results: Several 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. Conclusion: Our 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.

Targeting DNA Homologous Repair Proficiency With Concomitant Topoisomerase II and c-Abl Inhibition.

Critical DNA repair pathways become deranged during cancer development. This vulnerability may be exploited with DNA-targeting chemotherapy. Topoisomerase II inhibitors induce double-strand breaks which, if not repaired, are detrimental to the cell. This repair process requires high-fidelity functional homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). If either of these pathways is defective, a compensatory pathway may rescue the cells and induce treatment resistance. Consistently, HR proficiency, either inherent or acquired during the course of the disease, enables tumor cells competent to repair the DNA damage, which is a major problem for chemotherapy in general. In this context, c-Abl is a protein tyrosine kinase that is involved in DNA damage-induced stress. We used a low-dose topoisomerase II inhibitor mitoxantrone to induce DNA damage which caused a transient cell cycle delay but allowed eventual passage through this checkpoint in most cells. We show that the percentage of HR and NHEJ efficient HeLa cells decreased more than 50% by combining c-Abl inhibitor imatinib with mitoxantrone. This inhibition of DNA repair caused more than 87% of cells in G2/M arrest and a significant increase in apoptosis. To validate the effect of the combination treatment, we tested it on commercial and patient-derived cell lines in high-grade serous ovarian cancer (HGSOC), where chemotherapy resistance correlates with HR proficiency and is a major clinical problem. Results obtained with HR-proficient and deficient HGSOC cell lines show a 50-85% increase of sensitivity by the combination treatment. Our data raise the possibility of successful targeting of treatment-resistant HR-proficient cancers.

ALDH1A1-related stemness in high-grade serous ovarian cancer is a negative prognostic indicator but potentially targetable by EGFR/mTOR-PI3K/aurora kinase inhibitors.

Poor chemotherapy response remains a major treatment challenge for high-grade serous ovarian cancer (HGSC). Cancer stem cells are the major contributors to relapse and treatment failure as they can survive conventional therapy. Our objectives were to characterise stemness features in primary patient-derived cell lines, correlate stemness markers with clinical outcome and test the response of our cells to both conventional and exploratory drugs. Tissue and ascites samples, treatment-naive and/or after neoadjuvant chemotherapy, were prospectively collected. Primary cancer cells, cultured under conditions favouring either adherent or spheroid growth, were tested for stemness markers; the same markers were analysed in tissue and correlated with chemotherapy response and survival. Drug sensitivity and resistance testing was performed with 306 oncology compounds. Spheroid growth condition HGSC cells showed increased stemness marker expression (including aldehyde dehydrogenase isoform I; ALDH1A1) as compared with adherent growth condition cells, and increased resistance to platinum and taxane. A set of eight stemness markers separated treatment-naive tumours into two clusters and identified a distinct subgroup of HGSC with enriched stemness features. Expression of ALDH1A1, but not most other stemness markers, was increased after neoadjuvant chemotherapy and its expression in treatment-naive tumours correlated with chemoresistance and reduced survival. In drug sensitivity and resistance testing, five compounds, including two PI3K-mTOR inhibitors, demonstrated significant activity in both cell culture conditions. Thirteen compounds, including EGFR, PI3K-mTOR and aurora kinase inhibitors, were more toxic to spheroid cells than adherent cells. Our results identify stemness markers in HGSC that are associated with a decreased response to conventional chemotherapy and reduced survival if expressed by treatment-naive tumours. EGFR, mTOR-PI3K and aurora kinase inhibitors are candidates for targeting this cell population. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Open Source Infrastructure for Health Care Data Integration and Machine Learning Analyses.

PURPOSE: We have created a cloud-based machine learning system (CLOBNET) that is an open-source, lean infrastructure for electronic health record (EHR) data integration and is capable of extract, transform, and load (ETL) processing. CLOBNET enables comprehensive analysis and visualization of structured EHR data. We demonstrate the utility of CLOBNET by predicting primary therapy outcomes of patients with high-grade serous ovarian cancer (HGSOC) on the basis of EHR data. MATERIALS AND METHODS: CLOBNET is built using open-source software to make data preprocessing, analysis, and model training user friendly. The source code of CLOBNET is available in GitHub. The HGSOC data set was based on a prospective cohort of 208 patients with HGSOC who were treated at Turku University Hospital, Finland, from 2009 to 2019 for whom comprehensive clinical and EHR data were available. RESULTS: We trained machine learning (ML) models using clinical data, including a herein developed dissemination score that quantifies the disease burden at the time of diagnosis, to identify patients with progressive disease (PD) or a complete response (CR) on the basis of RECIST (version 1.1). The best performance was achieved with a logistic regression model, which resulted in an area under receiver operating characteristic curve (AUROC) of 0.86, with a specificity of 73% and a sensitivity of 89%, when it classified between patients who experienced PD and CR. CONCLUSION: We have developed an open-source computational infrastructure, CLOBNET, that enables effective and rapid analysis of EHR and other clinical data. Our results demonstrate that CLOBNET allows predictions to be made on the basis of EHR data to address clinically relevant questions.

A Functional Homologous Recombination Assay Predicts Primary Chemotherapy Response and Long-Term Survival in Ovarian Cancer Patients.

Purpose: Homologous recombination deficiency (HRD) correlates with platinum sensitivity in patients with ovarian cancer, which clinically is the most useful predictor of sensitivity to PARPi. To date, there are no reliable diagnostic tools to anticipate response to platinum-based chemotherapy, thus we aimed to develop an ex vivo functional HRD detection test that could predict both platinum-sensitivity and patient eligibility to targeted drug treatments.Experimental Design: We obtained a functional HR score by quantifying homologous recombination (HR) repair after ionizing radiation-induced DNA damage in primary ovarian cancer samples (n = 32). Samples clustered in 3 categories: HR-deficient, HR-low, and HR-proficient. We analyzed the HR score association with platinum sensitivity and treatment response, platinum-free interval (PFI) and overall survival (OS), and compared it with other clinical parameters. In parallel, we performed DNA-sequencing of HR genes to assess if functional HRD can be predicted by currently offered genetic screening.Results: Low HR scores predicted primary platinum sensitivity with high statistical significance (P = 0.0103), associated with longer PFI (HR-deficient vs. HR-proficient: 531 vs. 53 days), and significantly correlated with improved OS (HR score <35 vs. ≥35, hazard ratio = 0.08, P = 0.0116). At the genomic level, we identified a few unclear mutations in HR genes and the mutational signature associated with HRD, but, overall, genetic screening failed to predict functional HRD.Conclusions: We developed an ex vivo assay that detects tumor functional HRD and an HR score able to predict platinum sensitivity, which holds the clinically relevant potential to become the routine companion diagnostic in the management of patients with ovarian cancer. Clin Cancer Res; 24(18); 4482-93. ©2018 AACR.

Postoperative human epididymis protein 4 predicts primary therapy outcome in advanced epithelial ovarian cancer.

Primary chemotherapy treatment response monitoring in advanced epithelial ovarian cancer (EOC) is currently based on CT-imaging and serum CA125 values. Serum HE4 profile during first line chemotherapy has not been previously studied. We evaluated the HE4 profile during first line chemotherapy after primary (PDS) and interval debulking surgery (IDS). In total, 49 FIGO stage III/IV EOC patients were included in the study. 22 patients underwent PDS and 27 patients neoadjuvant chemotherapy (NACT) followed by IDS. Serial HE4 and CA125 serum samples were taken during first line chemotherapy. The association of postoperative tumor markers to surgery outcome, primary therapy outcome and progression free survival (PFS) were determined. The lowest HE4 and CA125 values during chemotherapy were compared to primary therapy outcome and PFS. The postoperative HE4 was associated to residual tumor after surgery (p = 0.0001), primary therapy outcome (p = 0.004) and PFS (p = 0.03) in all patients (n = 40). The postoperative CA125 was associated to PFS after IDS (n = 26, p = 0.006), but not after PDS. In multivariate analysis with FIGO stage (III/IV), residual tumor (0/>0) and postoperative CA125, the postoperative HE4 was the only statistically significant prognostic variable predicting PFS. Both HE4 and CA125 nadir corresponded to primary therapy outcome (HE4 p < 0.0001, CA125 p < 0.0001) and PFS (HE4 p = 0.009, CA125 p < 0.0001). HE4 is a promising candidate for EOC response monitoring. In our study, the performance of HE4 in response monitoring of first line chemotherapy was comparable to that of CA125. Of the postoperative values, only HE4 was statistically significantly associated to primary therapy outcome.

REG4 Is Highly Expressed in Mucinous Ovarian Cancer: A Potential Novel Serum Biomarker.

Preoperative diagnostics of ovarian neoplasms rely on ultrasound imaging and the serum biomarkers CA125 and HE4. However, these markers may be elevated in non-neoplastic conditions and may fail to identify most non-serous epithelial cancer subtypes. The objective of this study was to identify histotype-specific serum biomarkers for mucinous ovarian cancer. The candidate genes with mucinous histotype specific expression profile were identified from publicly available gene-expression databases and further in silico data mining was performed utilizing the MediSapiens database. Candidate biomarker validation was done using qRT-PCR, western blotting and immunohistochemical staining of tumor tissue microarrays. The expression level of the candidate gene in serum was compared to the serum CA125 and HE4 levels in a patient cohort of prospectively collected advanced ovarian cancer. Database searches identified REG4 as a potential biomarker with specificity for the mucinous ovarian cancer subtype. The specific expression within epithelial ovarian tumors was further confirmed by mRNA analysis. Immunohistochemical staining of ovarian tumor tissue arrays showed distinctive cytoplasmic expression pattern only in mucinous carcinomas and suggested differential expression between benign and malignant mucinous neoplasms. Finally, an ELISA based serum biomarker assay demonstrated increased expression only in patients with mucinous ovarian cancer. This study identifies REG4 as a potential serum biomarker for histotype-specific detection of mucinous ovarian cancer and suggests serum REG4 measurement as a non-invasive diagnostic tool for postoperative follow-up of patients with mucinous ovarian cancer.

18F-FDG-PET/CT can identify histopathological non-responders to platinum-based neoadjuvant chemotherapy in advanced epithelial ovarian cancer.

OBJECTIVE: The aim of this study was to examine the relationship between the reduction of maximum standardized uptake values (SUVmax) in 18F-FDG-PET/CT to histopathological changes obtained with neoadjuvant chemotherapy (NACT) in advanced epithelial ovarian cancer (EOC). We wanted to evaluate whether 18F-FDG-PET/CT is useful for identifying patients who will not respond to NACT and would therefore benefit from second-line chemotherapy instead of interval debulking surgery (IDS). METHODS: Twenty-six primarily inoperable EOC patients treated with NACT were enrolled in this study. 18F-FDG-PET/CT imaging was performed before diagnostic laparoscopy and after three to four NACT cycles. The relationship between the decrease in omental SUVmax from before to after NACT with omental histopathological response was examined in samples taken from the corresponding anatomical sites during IDS. Patients were divided into three groups according to chemotherapy-induced histopathological changes. Serum CA125 and HE4 halftimes during NACT as well as Ki-67 antigen expression in IDS samples were determined. RESULTS: The median omental SUVmax change during NACT was -64% (range-16% to -84%), and it was associated with histopathological response (p=0.004, OR 0.9, CI 0.84-0.97). A SUVmax decrease of less than 57% identified histopathological non-responders. Progression-free survival (PFS) differed between the poor, moderate and good histopathological response groups (0.9 year vs. 1.2 years vs. 1.4 years, respectively, p=0.05). The SUVmax change was not associated with PFS. CONCLUSION: 18F-FDG-PET/CT was able to identify patients who would not respond to NACT. To obtain a histopathological response in EOC, a substantial metabolic response in 18F-FDG-PET/CT is necessary.