Considerable efforts have been made to understand the leading causal mechanisms behind the development of platinum resistance in relapsed High Grade Serous ovarian Cancer (HGSC) patients. Accumulating evidence shows that epigenetic alterations play an important role in the emergence of platinum-resistant disease. To assess their subsequent effect on the transcriptome, we extracted and sequenced RNA from formalin-fixed, paraffin-embedded (FFPE) paired tumor samples in order to perform Differentially Gene Expression Analysis (DGEA) between diagnosis and platinum-resistant relapse.

The prognosis for patients with platinum-resistant ovarian High Grade Serous Carcinoma (HGSC) remains poor. BriTROC-1 data indicate that genomic alterations alone cannot explain acquired platinum resistance. We investigate epigenetic changes that may drive platinum resistance in HGSC. Using in an in vitro two-dimensional model, we recreated multiple cycles of chemotherapy. We utilised both established cell lines and primary cultures derived from HGSC ascites. Following cell characterisation (p53, PAX8 IHC), carboplatin sensitivity was assessed (sulforhodamine B assay). Cells were then pulsed with four cycles of carboplatin (50μM for 6 hours) with a week of recovery between each cycle. Methylation (Illumina 850k array), transcriptomic (RNAseq) and chromatin accessibility (ATACseq) assays were performed. Cells were also imaged using Stochastic Optical Reconstruction Microscopy. Firstly, we focused on RNAseq analysis to evaluate genes (and related biological processes) that are consistently enriched following carboplatin cycles and the unique genes that are conversely enriched at cycle level. We attempted to identify uniformly up-/down-regulated pathways across the different cultures. These data were compared with the same results obtained from the OvCar4, an established HGSC cell line and IVR01, an in-vivo resistant OvCar4 derivative. The ATACseq and methylation analyses are still ongoing, aiming to identify possible concordant changes in the chromatin accessibility and the methylation patterns that will allow us to clarify the changes observed in transcriptomic. The preliminary data obtained from the RNAseq analysis show that the highest number of enriched genes seems to occur after the first Carboplatin cycle, with a lower number of progressively enriched genes across all other ones. No constant biological processes were identified for the moment, across the different primary cell cultures. Further comparisons will be certainly needed. Understanding the epigenetic landscape of HGSC using physiologically relevant models will allow us to identify possible therapeutic targets that could eventually prevent platinum resistance.

AbstractPurpose: Ovarian high-grade serous carcinoma (HGSC) is usually diagnosed at late stage. We investigated whether late-stage HGSC has unique genomic characteristics consistent with acquisition of evolutionary advantage compared with early-stage tumors. Experimental Design: We performed targeted next-generation sequencing and shallow whole-genome sequencing (sWGS) on pretreatment samples from 43 patients with FIGO stage I–IIA HGSC to investigate somatic mutations and copy-number (CN) alterations (SCNA). We compared results to pretreatment samples from 52 patients with stage IIIC/IV HGSC from the BriTROC-1 study. Results: Age of diagnosis did not differ between early-stage and late-stage patients (median 61.3 years vs. 62.3 years, respectively). TP53 mutations were near-universal in both cohorts (89% early-stage, 100% late-stage), and there were no significant differences in the rates of other somatic mutations, including BRCA1 and BRCA2. We also did not observe cohort-specific focal SCNA that could explain biological behavior. However, ploidy was higher in late-stage (median, 3.0) than early-stage (median, 1.9) samples. CN signature exposures were significantly different between cohorts, with greater relative signature 3 exposure in early-stage and greater signature 4 in late-stage. Unsupervised clustering based on CN signatures identified three clusters that were prognostic. Conclusions: Early-stage and late-stage HGSCs have highly similar patterns of mutation and focal SCNA. However, CN signature analysis showed that late-stage disease has distinct signature exposures consistent with whole-genome duplication. Further analyses will be required to ascertain whether these differences reflect genuine biological differences between early-stage and late-stage or simply time-related markers of evolutionary fitness. See related commentary by Yang et al., p. 2730

Dataset containing pre-processed data files required to replicate analysis performed in the publication "The genomic landscape of recurrent ovarian high grade serous carcinoma: the BriTROC-1 study" (Smith & Bradley et al. 2023).

Background Preclinical studies support targeting PI3K/AKT/mTOR signalling in platinum-resistant ovarian cancer (PROC). A phase I study of the dual mTORC1/mTORC2 inhibitor vistusertib with weekly paclitaxel (wP) showed activity. We report the results of Arm 1 of OCTOPUS, the first randomised trial of weekly paclitaxel and dual mTORC1/2 inhibition in ovarian cancer.Methods Patients with platinum-resistant or refractory high grade serous carcinoma were randomised (1:1) to wP (80mg/m2 D1,8,15 of 28 day cycle) plus oral vistusertib (50mg BD) or placebo (P). The primary endpoint was progression-free survival (PFS). Secondary endpoints included response rate (RR) and overall survival (OS).Results 140 patients (median age 63, range: 36-86; 18% platinum-refractory; 54% ≥3 prior therapies) were randomised. There was no difference in PFS (median 4.5 vs 4.1m (HR 0.84; 80% CI (0.67, 1.07); 1-sided p=0.18), OS (median 9.7 vs 11.1m (HR 1.21; 80% CI (0.91, 1.60); 1-sided p=0.80) or RR (odds ratio 0.86; 80% CI (0.55, 1.36); 1-sided p=0.66). Grade 3/4 adverse events were 41.2% (wP+V) vs 36.7% (wP+P). Low tumour PTEN expression was associated with longer PFS in the wP+V arm (9.4 vs 4.1m p=0.003) but not in the wP arm (4.8 vs 4.2m p=0.60). Tumour genome-wide copy number (CN) analysis suggested that high CN signature 4 was associated with worse outcome in the wP+P arm (2.3 vs 4.6m p=0.018) but not the wP+V arm (5.4 vs 3.3m).Conclusions Vistusertib did not improve clinical activity of wP in PROC. However, low tumour cell PTEN expression may be a predictive biomarker for vistusertib activity.Translational Relevance Preclinical studies suggest that activation of the PI3K/AKT/mTOR signalling pathway contributes to platinum-resistance in ovarian high grade serous carcinoma (HGSC). Based on activity in a phase I study, we evaluated the clinical efficacy of the dual mTORC1/mTORC2 inhibitor vistusertib in combination with weekly paclitaxel in the OCTOPUS study - a multi-centre, randomised, placebo-controlled, phase II trial in platinum-resistant ovarian (HGSC). In the first randomised trial of weekly paclitaxel and dual mTORC1/2 inhibition in ovarian cancer, vistusertib did not improve clinical activity of weekly paclitaxel. However, translational analyses indicated that low tumour cell PTEN expression may be a predictive biomarker for vistusertib activity. We also showed genome-wide copy number (CN) analysis, in particular high exposure to CN signature 4, may also allow identification of patients with greater chance of benefit from dual mTORC inhibition. Potential predictive biomarkers identified in our study should be evaluated in ongoing/future studies.### Competing Interest StatementS Banerjee Research Grants: Astrazeneca, GlaxoSmithKline; Advisory Boards: Amgen, Astrazeneca, Genmabs, GlaxoSmithKline Immunogen, Merck Sharpe Dohme, Merck Sereno, Mersana, Oncxerna, Seagen, Shattuck Labs; Honoraria for lectures: Amgen, Astrazeneca, Clovis, GlaxoSmithKine, Immunogen, Merck Sharpe Dohme, Mersana, Pfizer, Roche, Takeda. Clinical Advisory Board (uncompensated) Epsilogen G Giannone Payment for educational events from Mylan, outside the submitted manuscript. A Clamp Research grant AstraZeneca, Advisory Board- GSK, MSD, Honoraria- Clovis Oncology, AstraZeneca R Glasspool Rosalind Glasspool is Principal Investigator for trials sponsored by AstraZeneca, Clovis, GlaxoSmithKline/Tesaro, Immunogen, AbbVie, Pfizer, Lilly, and Novartis, and reports research funding from Clovis, Boehringer Ingelheim, and Lilly/Ignyta; institution research funding from GlaxoSmithKline/Tesaro; personal speaker and/or consultancy fees from AstraZeneca, MSD, Clovis, Tesaro, GlaxoSmithKline, Immunogen, and Sotio; and institutional consultancy fees from Novartis R Kristeleit Grants: Clovis, MSD Advisory Boards/Consulting: GSK, Eisai, Astra Zeneca, Clovis, MSD, Basilea, Shattuck Labs, Zydus Cadila, iTEOS, InCyte, Regeneron. IA McNeish COI Advisory boards and speaker fees for AZ, Clovis Oncology, GSK/Tesaro, Roche, OncoC4, Epsila Bio, Theolytics and Duke St Bio, all outside the scope of OCTOPUS. Institutional grant income from AZ also outside the scope of OCTOPUS. ### Clinical TrialISRCTN16426935### Funding StatementFunding was provided from AstraZeneca via the NIHR Alliance and Endorsed by Cancer Research UK (CRUKE/14/053); Funding for translational research was provided by AstraZeneca, the Lady Garden Foundation and NIHR Imperial Biomedical Research Centre.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:The study was conducted in accordance with the Research Governance Framework for Health and Community Care (Second edition; 2006) and the Medicines for Human Use (Clinical Trials) Regulations, 2004 SI 2004:1031 (as amended) and World Medical Association Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects 1964 (as amended) and was co-sponsored by University of Glasgow and NHS Greater Glasgow and Clyde. Ethical approval was obtained from London, Brighton and Sussex Research Ethics Committee (reference 15/LO/1302) and all patients provided written informed consent.I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

BackgroundIn arm 1 of the phase II randomised OCTOPUS trial (ISRCTN16426935), no significant differences in Progression-Free Survival (PFS) or Overall Survival were observed with the addition of vistusertib (V), a dual mTORC1/2 inhibitor, to weekly paclitaxel (wP) in platinum-resistant/refractory ovarian high-grade serous carcinoma. However, preliminary immunohistochemistry (IHC) data suggested that PTEN status may be predictive of benefit of addition of V to wP. Aim: We evaluated if PTEN expression (scored using quantitative digital IHC) or specific genomic features might be predictive of V benefit. We also compared genomic profiles in archival and study entry specimens.MethodsPTEN expression in archival samples (N=68) was scored using QuPath Histo-score (H-score; range 0-300), and compared to pathologist scoring. In archival (N=43) and study entry (N=35) samples, DNA copy number (CN) and CN signature exposure were assessed using shallow whole genome sequencing; target sequencing was performed using a custom panel (Illumina AmpliSeq).ResultsDigital quantification of PTEN status was feasible with a high correlation between QuPath and pathologist scores (r=0.94, p<0.0001 for tumour; r=0.70, p=0.009 for non-tumour). H-score variability was lower in non-tumour than in tumour cells. Patients with low PTEN tumours (defined as tumour