Iain A. McNeish

Clinical validation of a DNA methylation biomarker associated with overall survival of relapsed ovarian cancer patients

Abstract Approximately 70% of ovarian cancer (OC) patients relapse after chemotherapy, underscoring the need to assess survival before second‐line treatment. We previously identified PLAT‐M8, an 8‐CpG blood‐based methylation signature linked to chemoresistance. This study validates its correlation with clinicopathological features and treatment profiles in additional cohorts. Extracted DNA from whole blood was provided from the BriTROC‐1 ( n  = 47) and OV04 cohorts ( n  = 57) upon the first relapse. Additional samples from Hammersmith Hospital ( n  = 100) were collected during first‐line chemotherapy (Cycles 3–4 and 6). Bisulphite pyrosequencing was used to quantify DNA methylation at the previously identified 8 CpG sites. The methylation data obtained were combined with previous data from ScoTROC‐1D and 1V ( n  = 141) and OCTIPS ( n  = 46). Cox regression was used to assess OS after relapse concerning clinicopathological characteristics. The DNA methylation Class (Class 1 vs. 2) was determined by consensus clustering. As for results, blood DNA methylation at relapse correlates with clinical outcomes, but it has no impact during first‐line treatment. Class 1 is linked to shorter survival (summary OS: HR 2.50, 1.64–3.79) and poorer prognosis on carboplatin monotherapy (OS: aHR 9.69, 95% CI: 2.38–39.47). It is associated with older (>75 years), advanced‐stage, platinum‐resistant patients, residual disease, and shorter PFS. In contrast, Class 2 is linked to platinum sensitivity, higher complete response rates (RECIST), and better prognosis but shows no correlation with CA‐125. These findings highlight PLAT‐M8's potential in guiding second‐line chemotherapy decisions. The PLAT‐M8 methylation biomarker is associated with survival in relapsed OC patients and may potentially predict their response to second‐line platinum treatment.

Identification of actionable targets using DEPArray ‐based sorting of pure carcinoma and stromal populations from formalin‐fixed paraffin‐embedded tissues followed by shallow whole‐genome sequencing

Abstract Formalin‐fixed paraffin‐embedded (FFPE) tissue specimens represent precious resources for clinical genomic profiling studies, especially when coupled with comprehensive medical records. Even though next‐generation sequencing (NGS) is an effective tool to detect somatic mutations and somatic copy number alterations (sCNA), the biggest challenges in unlocking clinically translatable genomic information from FFPE tissue are low DNA yields and degraded DNA, affected by variable formalin fixation. Another issue is that the proportion of carcinoma and other noncarcinoma cells is variable and can be confounded by intratumoral heterogeneity. To explore these challenges, we isolated pure carcinoma and stromal cells using the DEPArray™ NxT system, a microchip‐based digital sorter that allows isolation of pure, homogeneous subpopulations of cells from FFPE samples. We isolated pure carcinoma and stromal cell populations from 12 FFPE tissues, including tissues from nine primary and metastatic breast cancer and three primary ovarian high‐grade serous carcinomas. This was followed by downstream shallow whole‐genome sequencing (WGS) for copy number landscape profiling (10 samples) and/or a targeted panel for somatic mutation and sCNA analysis (seven samples), subject to cell availability. Seven out of 10 samples (even some with low tumour content or of old age) produced good‐quality genomic data, detecting sCNA in all carcinoma population samples but not in the stromal populations. Mutation analysis was performed successfully in 6/7 samples and somatic mutations were detected in all of them. Our workflow enabled the identification of clinically actionable targets, including PIK3CA, ERBB2, FGFR1/2, CDK6, CCNE1, KRAS amplifications and RB, BRCA1/2 losses in patients that would direct therapy. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Understanding and Addressing Challenges With Electronic Health Record Use in Gynecological Oncology: Cross-Sectional Survey of Multidisciplinary Professionals in the United Kingdom and Co-Design of an Integrated Informatics Platform to Support Clinical Decision-Making

Abstract Background Electronic health records (EHRs) are a cornerstone of modern health care delivery, but their current configuration often fragments information across systems, impeding timely and effective clinical decision-making. In gynecological oncology, where care involves complex, multidisciplinary coordination, these limitations can significantly impact the quality and efficiency of patient management. Few studies have examined how EHR systems support clinical decision-making from the perspective of end users. This study aimed to explore multiprofessional experiences of EHR use in gynecological oncology and to develop a co-designed informatics platform to improve decision-making for ovarian cancer care. Objective This study aims to evaluate the perspectives of health care professionals on retrieving routine clinical data from EHRs in the management of ovarian cancer and to design an integrated informatics platform that supports clinical decision-making. Methods We conducted a national cross-sectional survey of 92 UK-based professionals working in gynecological oncology, including oncologists, nurses, radiologists, and other specialists in ovarian cancer. The web-based questionnaire, combining quantitative and free-text responses, assessed their experiences with EHR use, focusing on information retrieval, usability challenges, perceived risks, and benefits. In parallel, a human-centered design approach involving health care professionals, data engineers, and informatics experts codeveloped a digital informatics platform that integrates structured and unstructured data from multiple clinical systems into a unified patient summary view for clinical decision-making. Natural language processing was applied to extract genomic and surgical information from free-text records, with data pipelines validated by clinicians against original clinical system sources. Results Among 92 respondents, 84 out of 91 (92%) routinely accessed multiple EHR systems, with 26 out of 91 (29%) using 5 or more. Notably, 16 out of 92 respondents (17%) reported spending more than 50% of their clinical time searching for patient information. Key challenges included lack of interoperability (35/141 reported challenges, 24.8%), difficulty locating critical data such as genetic results (57/85 respondents, 67%), and poor organization of information. Only 10 out of 92 professionals (11%) strongly agreed that their systems provided well-organized data for clinical use. While ease of access to patient data was a key benefit, 54 out of 90 respondents (60%) reported lacking access to comprehensive patient summaries. To address these issues, our co-designed informatics platform consolidates disparate patients’ data from different EHR systems into a single visual display to support clinical decision-making and audit. Conclusions Current EHR systems are suboptimal for supporting complex gynecological oncology care. Our findings highlight the urgent need for integrated, user-centered clinical decision tools. Fragmentation and lack of interoperability hinder information retrieval and may compromise patient care. Our co-designed ovarian cancer informatics platform is a potential real-world solution to improve data visibility, clinical efficiency, and ultimately the quality of ovarian cancer care.

Assessing PARP trapping dynamics in ovarian cancer using a CRISPR-engineered FRET biosensor

Poly(ADP-ribose) polymerase inhibitors (PARPi) have revolutionized the treatment of ovarian high-grade serous carcinoma (HGSC), particularly in homologous recombination-deficient tumors. However, the emergence of resistance poses a critical challenge, as over 50% of patients relapse within 3 years. The mechanisms underlying changes in PARP trapping, a central aspect of PARPi efficacy, are not well understood, as current experimental methodologies lack resolution and throughput. To address this, we develop an intramolecular fluorescence resonance energy transfer (FRET)-based biosensor by CRISPR-Cas9 dual labeling of endogenous PARP1 with EGFP and mCherryFP in OVCAR4 cells. This biosensor enables real-time, single-cell analysis of PARP trapping dynamics. Using fluorescence lifetime imaging microscopy (FLIM), we reveal dose-dependent PARP trapping, differentiate the trapping efficiencies of four clinically approved PARPi, and observe reduced trapping in PARPi-resistant models in vitro and in vivo. This biosensor provides critical insights into PARPi resistance mechanisms, with implications for developing more effective therapies and advancing personalized treatment for ovarian cancer patients.

The genomic trajectory of ovarian high‐grade serous carcinoma can be observed in STIC lesions

AbstractOvarian high‐grade serous carcinoma (HGSC) originates in the fallopian tube, with secretory cells carrying a TP53 mutation, known as p53 signatures, identified as potential precursors. p53 signatures evolve into serous tubal intraepithelial carcinoma (STIC) lesions, which in turn progress into invasive HGSC, which readily spreads to the ovary and disseminates around the peritoneal cavity. We recently investigated the genomic landscape of early‐ and late‐stage HGSC and found higher ploidy in late‐stage (median 3.1) than early‐stage (median 2.0) samples. Here, to explore whether the high ploidy and possible whole‐genome duplication (WGD) observed in late‐stage disease were determined early in the evolution of HGSC, we analysed archival formalin‐fixed paraffin‐embedded (FFPE) samples from five HGSC patients. p53 signatures and STIC lesions were laser‐capture microdissected and sequenced using shallow whole‐genome sequencing (sWGS), while invasive ovarian/fallopian tube and metastatic carcinoma samples underwent macrodissection and were profiled using both sWGS and targeted next‐generation sequencing. Results showed highly similar patterns of global copy number change between STIC lesions and invasive carcinoma samples within each patient. Ploidy changes were evident in STIC lesions, but not p53 signatures, and there was a strong correlation between ploidy in STIC lesions and invasive ovarian/fallopian tube and metastatic samples in each patient. The reconstruction of sample phylogeny for each patient from relative copy number indicated that high ploidy, when present, occurred early in the evolution of HGSC, which was further validated by copy number signatures in ovarian and metastatic tumours. These findings suggest that aberrant ploidy, suggestive of WGD, arises early in HGSC and is detected in STIC lesions, implying that the trajectory of HGSC may be determined at the earliest stages of tumour development. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Results of a randomised Phase II trial of olaparib, chemotherapy or olaparib and cediranib in patients with platinum-resistant ovarian cancer

Abstract Background OCTOVA compared the efficacy of olaparib (O) versus weekly paclitaxel (wP) or olaparib + cediranib (O + C) in recurrent ovarian cancer (OC). Aims The main aim of the OCTOVA trial was to determine the progression-free survival (PFS) of olaparib (O) versus the oral combination of olaparib plus cediranib (O + C) and weekly paclitaxel (wP) in recurrent ovarian cancer (OC). Methods In total, 139 participants who had relapsed within 12 months of platinum therapy were randomised to O (300 mg twice daily), wP (80 mg/m2 d1,8,15, q28) or O + C (300 mg twice daily/20 mg daily, respectively). The primary endpoint was progression-free survival (PFS) of olaparib (O) versus olaparib plus cediranib (O + C) or weekly paclitaxel (wP). The sample size was calculated to observe a PFS hazard ratio (HR) 0.64 in favour of O + C compared to O (20% one-sided type I error, 80% power). Results The majority had platinum-resistant disease (90%), 22% prior PARPi, 34% prior anti-angiogenic therapy, 30% germline BRCA1/2 mutations. The PFS was increased for O + C vs O (O + C 5.4 mo (2.3, 9.6): O 3.7 mo (1.8, 7.6) HR = 0.73; 60% CI: 0.59, 0.89; P = 0.1) and no different between wP and O (wP 3.9 m (1.9, 9.1); O 3.7 mo (1.8, 7.6) HR = 0.89, 60% CI: 0.72, 1.09; P = 0.69). The main treatment-related adverse events included manageable diarrhoea (4% Grade 3) and hypertension (4% Grade 3) in the O + C arm. Discussion OCTOVA demonstrated the activity of O + C in women with recurrent disease, offering a potential non-chemotherapy option. Trial registration ISRCTN14784018, registered on 19th January 2018 http://www.isrctn.com/ISRCTN14784018.

SMARCA4 regulates the NK-mediated killing of senescent cells

Induction of senescence by chemotherapeutic agents arrests cancer cells and activates immune surveillance responses to contribute to therapy outcomes. In this investigation, we searched for ways to enhance the NK-mediated elimination of senescent cells. We used a staggered screen approach, first identifying siRNAs potentiating the secretion of immunomodulatory cytokines to later test for their ability to enhance NK-mediated killing of senescent cells. We identified that genetic or pharmacological inhibition of SMARCA4 enhanced senescent cell elimination by NK cells. SMARCA4 expression is elevated during senescence and its inhibition derepresses repetitive elements, inducing the SASP via activation of cGAS/STING and MAVS/MDA5 pathways. Moreover, a PROTAC targeting SMARCA4 synergized with cisplatin to increase the infiltration of CD8 T cells and mature, activated NK cells in an immunocompetent model of ovarian cancer. Our results indicate that SMARCA4 inhibitors enhance NK-mediated surveillance of senescent cells and may represent senotherapeutic interventions for ovarian cancer.

PTEN deficiency exposes a requirement for an ARF GTPase module for integrin‐dependent invasion in ovarian cancer

Abstract Dysregulation of the PI3K/AKT pathway is a common occurrence in high‐grade serous ovarian carcinoma (HGSOC), with the loss of the tumour suppressor PTEN in HGSOC being associated with poor prognosis. The cellular mechanisms of how PTEN loss contributes to HGSOC are largely unknown. We here utilise time‐lapse imaging of HGSOC spheroids coupled to a machine learning approach to classify the phenotype of PTEN loss. PTEN deficiency induces PI(3,4,5)P 3 ‐rich and ‐dependent membrane protrusions into the extracellular matrix (ECM), resulting in a collective invasion phenotype. We identify the small GTPase ARF6 as a crucial vulnerability of HGSOC cells upon PTEN loss. Through a functional proteomic CRISPR screen of ARF6 interactors, we identify the ARF GTPase‐activating protein (GAP) AGAP1 and the ECM receptor β1‐integrin (ITGB1) as key ARF6 interactors in HGSOC regulating PTEN loss‐associated invasion. ARF6 functions to promote invasion by controlling the recycling of internalised, active β1‐integrin to maintain invasive activity into the ECM. The expression of the CYTH2‐ARF6‐AGAP1 complex in HGSOC patients is inversely associated with outcome, allowing the identification of patient groups with improved versus poor outcome. ARF6 may represent a therapeutic vulnerability in PTEN‐depleted HGSOC.

Survival and modelled cancer antigen-125 ELIMination rate constant K score in ovarian cancer patients in first-line before poly(ADP-ribose) polymerase inhibitor era: A Gynaecologic Cancer Intergroup meta-analysis

In patients with advanced ovarian cancer, the modelled CA-125 ELIMination rate constant K (KELIM) is an early indicator of the tumour intrinsic chemosensitivity. We assessed the prognostic and surrogate values of KELIM with respect to those of surgery outcome (based on post-operative residual lesions) in the Gynaecologic Cancer Intergroup (GCIG) individual patient data meta-analysis MAOV (Meta-Analysis in OVarian cancer) built before the emergence of poly(ADP-ribose) polymerase (PARP) inhibitors. The dataset was split into learning and validation cohorts (ratio 1:2). The individual modelled KELIM values were estimated, standardised by the median value, then scored as unfavourable (<1.0) or favourable (≥1.0). Overall survival (OS) and progression-free survival (PFS) analyses were performed with a two-step meta-analytic approach and surrogacy through a two-level meta-analytic model. KELIM was assessed in 5884 patients from eight first-line trials (learning, 1962; validation, 3922). A favourable KELIM score was significantly associated with longer OS (validation set, median, 78.8 versus 28.4 months, hazard-ratios [HR] 0.46, 95% confidence interval [CI], 0.41-0.50, C-index 0.68), and longer PFS (validation set, median 30.5 versus 9.8 months, HR 0.49, 95% CI, 0.45-0.54, C-index 0.68), as were International Federation of Gynaecology and Obstetrics (FIGO) stage and debulking surgery outcome. Three prognostic groups were identified based on the surgery outcome and KELIM score, with large differences in OS (105.1, ∼45.0, and 22.1 months) and PFS (58.1, ∼15.0, and 8.0 months). Surrogacy for OS and for PFS was not established. KELIM is an independent prognostic biomarker for survival, complementary to surgery outcome, representing a new determinant of first-line treatment success.

Benefit From Fractionated Dose-Dense Chemotherapy in Patients With Poor Prognostic Ovarian Cancer: ICON-8 Trial

PURPOSE An international meta-analysis identified a group of patients with advanced epithelial ovarian cancer (EOC) with a very poor survival because of two unfavorable features: (1) a poor chemosensitivity defined by an unfavorable modeled CA-125 ELIMination rate constant K (KELIM) score &lt;1.0 with the online calculator CA-125—Biomarker Kinetics, and (2) an incomplete debulking surgery. We assumed that patients belonging to this poor prognostic group would benefit from a fractionated densified chemotherapy regimen. METHODS The data set of ICON-8 phase III trial (ClinicalTrials.gov identifier: NCT01654146 ), where patients with EOC were treated with the standard three-weekly, or the weekly dose-dense, carboplatin-paclitaxel regimens and debulking primary surgery (immediate primary surgery [IPS] or delayed primary [or interval] surgery [DPS]), was investigated. The association between treatment arm efficacy, standardized KELIM (scored as favorable ≥1.0, or unfavorable &lt;1.0), and surgery completeness was assessed by univariate/multivariate analyses in IPS and DPS cohorts. RESULTS Of 1,566 enrolled patients, KELIM was calculated with the online model in 1,334 with ≥3 CA-125 available values (85%). As previously reported, both KELIM and surgery completeness were complementary prognostic covariates, and could be combined into three prognostic groups with large OS differences: (1) good if favorable KELIM and complete surgery; (2) intermediate if either unfavorable KELIM or incomplete surgery; and (3) poor if unfavorable KELIM and incomplete surgery. Weekly dose-dense chemotherapy was associated with PFS/OS improvement in the poor prognostic group in both the IPS cohort (PFS: hazard ratio [HR], 0.50; 95% CI, 0.31 to 0.79; OS: HR, 0.58; 95% CI, 0.35 to 0.95) and the DPS cohort (PFS: HR, 0.53; 95% CI, 0.37 to 0.76; OS: HR, 0.57; 95% CI, 0.39 to 0.82). CONCLUSION Fractionated dose-dense chemotherapy might be beneficial for patients belonging to the poor prognostic group characterized by lower tumor chemosensitivity assessed with the online calculator CA-125—Biomarker Kinetics and incomplete debulking surgery. Further investigation in the future SALVOVAR trial is warranted.

Development and Validation of the Gene Expression Predictor of High-grade Serous Ovarian Carcinoma Molecular SubTYPE (PrOTYPE)

Abstract Purpose: Gene expression–based molecular subtypes of high-grade serous tubo-ovarian cancer (HGSOC), demonstrated across multiple studies, may provide improved stratification for molecularly targeted trials. However, evaluation of clinical utility has been hindered by nonstandardized methods, which are not applicable in a clinical setting. We sought to generate a clinical grade minimal gene set assay for classification of individual tumor specimens into HGSOC subtypes and confirm previously reported subtype-associated features. Experimental Design: Adopting two independent approaches, we derived and internally validated algorithms for subtype prediction using published gene expression data from 1,650 tumors. We applied resulting models to NanoString data on 3,829 HGSOCs from the Ovarian Tumor Tissue Analysis consortium. We further developed, confirmed, and validated a reduced, minimal gene set predictor, with methods suitable for a single-patient setting. Results: Gene expression data were used to derive the predictor of high-grade serous ovarian carcinoma molecular subtype (PrOTYPE) assay. We established a de facto standard as a consensus of two parallel approaches. PrOTYPE subtypes are significantly associated with age, stage, residual disease, tumor-infiltrating lymphocytes, and outcome. The locked-down clinical grade PrOTYPE test includes a model with 55 genes that predicted gene expression subtype with &amp;gt;95% accuracy that was maintained in all analytic and biological validations. Conclusions: We validated the PrOTYPE assay following the Institute of Medicine guidelines for the development of omics-based tests. This fully defined and locked-down clinical grade assay will enable trial design with molecular subtype stratification and allow for objective assessment of the predictive value of HGSOC molecular subtypes in precision medicine applications. See related commentary by McMullen et al., p. 5271

Paclitaxel Induces Immunogenic Cell Death in Ovarian Cancer via TLR4/IKK2/SNARE-Dependent Exocytosis

Abstract Emerging evidence shows that the efficacy of chemotherapeutic drugs is reliant on their capability to induce immunogenic cell death (ICD), thus transforming dying tumor cells into antitumor vaccines. We wanted to uncover potential therapeutic strategies that target ovarian cancer by having a better understanding of the standard-of-care chemotherapy treatment. Here, we showed in ovarian cancer that paclitaxel induced ICD-associated damage-associated molecular patterns (DAMP, such as CALR exposure, ATP secretion, and HMGB1 release) in vitro and elicited significant antitumor responses in tumor vaccination assays in vivo. Paclitaxel-induced TLR4 signaling was essential to the release of DAMPs, which led to the activation of NF-κB–mediated CCL2 transcription and IkappaB kinase 2–mediated SNARE-dependent vesicle exocytosis, thus exposing CALR on the cell surface. Paclitaxel induced endoplasmic reticulum stress, which triggered protein kinase R–like ER kinase activation and eukaryotic translation initiation factor 2α phosphorylation independent of TLR4. Paclitaxel chemotherapy induced T-cell infiltration in ovarian tumors of the responsive patients; CALR expression in primary ovarian tumors also correlated with patients' survival and patient response to chemotherapy. These findings suggest that the effectiveness of paclitaxel relied upon the activation of antitumor immunity through ICD via TLR4 and highlighted the importance of CALR expression in cancer cells as an indicator of response to paclitaxel chemotherapy in ovarian cancer.

Oncologist-led BRCA ‘mainstreaming’ in the ovarian cancer clinic: A study of 255 patients and its impact on their management

AbstractAlthough guidelines recommend BRCA testing for all women with non-mucinous epithelial ovarian cancer, there is significant variability in access to testing across the UK. A germline BRCA mutation (BRCAm) in ovarian cancer patients provides prognostic and predictive information and influences clinical management, such as the use of PARP inhibitors, which have demonstrated a progression-free survival benefit in the BRCAm cohort. Additionally, the finding of a BRCAm has significant implications for patients and their families in terms of cancer risk and prevention. We studied the impact of a newly-formed, oncologist-led ‘mainstreaming’ germline BRCA testing pathway in 255 ovarian cancer patients at Imperial College NHS Trust. Prior to the establishment of ‘mainstreaming’, uptake of germline BRCA testing was 14% with a mean turnaround time of 148.2 calendar days. The ‘mainstreaming’ approach led to a 95% uptake of germline BRCA testing and a mean turnaround time of 20.6 days. Thirty-four (13.33%) BRCAm patients were identified. At the time of data collection nine BRCAm patients had received a PARP inhibitor off-trial, three had entered a PARP inhibitor trial and 5 were receiving platinum-based chemotherapy with a plan to receive PARP inhibitor maintenance. This study provides further evidence of the impact of oncologist-led ‘mainstreaming’ programs.

Prophylactic In Vivo Hematopoietic Stem Cell Gene Therapy with an Immune Checkpoint Inhibitor Reverses Tumor Growth in Syngeneic Mouse Tumor Models

Abstract Population-wide testing for cancer-associated mutations has established that more than one-fifth of ovarian and breast carcinomas are associated with inherited risk. Salpingo-oophorectomy and/or mastectomy are currently the only effective options offered to women with high-risk germline mutations. Our goal here is to develop a long-lasting approach that provides immunoprophylaxis for mutation carriers. Our approach leverages the fact that at early stages, tumors recruit hematopoietic stem/progenitor cells (HSPC) from the bone marrow and differentiate them into tumor-supporting cells. We developed a technically simple technology to genetically modify HSPCs in vivo. The technology involves HSPC mobilization and intravenous injection of an integrating HDAd5/35++ vector. In vivo HSPC transduction with a GFP-expressing vector and subsequent implantation of syngeneic tumor cells showed &amp;gt;80% GFP marking in tumor-infiltrating leukocytes. To control expression of transgenes, we developed a miRNA regulation system that is activated only when HSPCs are recruited to and differentiated by the tumor. We tested our approach using the immune checkpoint inhibitor anti-PD-L1-γ1 as an effector gene. In in vivo HSPC-transduced mice with implanted mouse mammary carcinoma (MMC) tumors, after initial tumor growth, tumors regressed and did not recur. Conventional treatment with an anti-PD-L1 mAb had no significant antitumor effect, indicating that early, self-activating expression of anti-PD-L1-γ1 can overcome the immunosuppressive environment in MMC tumors. The efficacy and safety of this approach was further validated in an ovarian cancer model with typical germline mutations (ID8 p53−/− brca2−/−), both in a prophylactic and therapeutic setting. This HSPC gene therapy approach has potential for clinical translation. Significance: Considering the limited prophylactic options that are currently offered to women with high-risk germ-line mutations, the in vivo HSPC gene therapy approach is a promising strategy that addresses a major medical problem.

Safety and efficacy of the tumor-selective adenovirus enadenotucirev with or without paclitaxel in platinum-resistant ovarian cancer: a phase 1 clinical trial

Background Treatment outcomes remain poor in recurrent platinum-resistant ovarian cancer. Enadenotucirev, a tumor-selective and blood stable adenoviral vector, has demonstrated a manageable safety profile in phase 1 studies in epithelial solid tumors. Methods We conducted a multicenter, open-label, phase 1 dose-escalation and dose-expansion study (OCTAVE) to assess enadenotucirev plus paclitaxel in patients with platinum-resistant epithelial ovarian cancer. During phase 1a, the maximum tolerated dose of intraperitoneally administered enadenotucirev monotherapy (three doses; days 1, 8 and 15) was assessed using a 3+3 dose-escalation model. Phase 1b included a dose-escalation and an intravenous dosing dose-expansion phase assessing enadenotucirev plus paclitaxel. For phase 1a/b, the primary objective was to determine the maximum tolerated dose of enadenotucirev (with paclitaxel in phase 1b). In the dose-expansion phase, the primary endpoint was progression-free survival (PFS). Additional endpoints included response rate and T-cell infiltration. Results Overall, 38 heavily pretreated patients were enrolled and treated. No dose-limiting toxicities were observed at any doses. However, frequent catheter complications led to the discontinuation of intraperitoneal dosing during phase 1b. Intravenous enadenotucirev (1×1012 viral particles; days 1, 3 and 5 every 28-days for two cycles) plus paclitaxel (80 mg/m2; days 9, 16 and 23 of each cycle) was thus selected for dose-expansion. Overall, 24/38 (63%) patients experienced at least 1 Grade ≥3 treatment-emergent adverse event (TEAE); most frequently neutropenia (21%). Six patients discontinued treatment due to TEAEs, including one patient due to a grade 2 treatment-emergent serious AE of catheter site infection (intraperitoneal enadenotucirev monotherapy). Among the 20 patients who received intravenous enadenotucirev plus paclitaxel, 4-month PFS rate was 64% (median 6.2 months), objective response rate was 10%, 35% of patients achieved stable disease and 65% of patients had a reduction in target lesion burden at ≥1 time point. Five out of six patients with matched pre-treatment and post-treatment biopsies treated with intravenous enadenotucirev plus paclitaxel had increased (mean 3.1-fold) infiltration of CD8 +T cells in post-treatment biopsies. Conclusions Intravenously dosed enadenotucirev plus paclitaxel demonstrated manageable tolerability, an encouraging median PFS and increased tumor immune-cell infiltration in platinum-resistant ovarian cancer. Trial registration number NCT02028117.

Cell-autonomous inflammation of BRCA1-deficient ovarian cancers drives both tumor-intrinsic immunoreactivity and immune resistance via STING

In this study, we investigate mechanisms leading to inflammation and immunoreactivity in ovarian tumors with homologous recombination deficiency (HRD). BRCA1 loss is found to lead to transcriptional reprogramming in tumor cells and cell-intrinsic inflammation involving type I interferon (IFN) and stimulator of IFN genes (STING). BRCA1-mutated (BRCA1

Targeting autocrine amphiregulin robustly and reproducibly inhibits ovarian cancer in a syngeneic model: roles for wildtype p53

AbstractOvarian cancer (OvCA) remains one of the most devastating malignancies, but treatment options are still limited. We report that amphiregulin (AREG) can serve as an effective and safe pharmacological target in a syngeneic murine model. AREG is highly abundant in abdominal fluids of patients with advanced OvCa. In immunocompetent animals, depletion or overexpression of AREG respectively prolonged or shortened animal survival. A new antibody we generated in AREG-knockout mice recognized murine AREG and reproducibly prolonged animal survival in the syngeneic model. The underlying mechanism likely involves binding of wildtype p53 to AREG’s promoter and autocrine activation of the epidermal growth factor receptor (EGFR), a step blocked by the antibody. Accordingly, depletion of p53 downregulated AREG secretion and conferred tolerance, whereas blocking an adaptive process involving CXCL1, which transactivates EGFR, might increase therapeutic efficacy. Consistent with these observations, analysis of OvCa patients revealed that high AREG correlates with poor prognosis of patients expressing wildtype TP53. In conclusion, clinical tests of the novel antibody are warranted; high AREG, normal TP53, and reduced CXCL1 activity might identify patients with OvCa who may derive therapeutic benefit.

Structural Variants at the BRCA1/2 Loci are a Common Source of Homologous Repair Deficiency in High-grade Serous Ovarian Carcinoma

Abstract Purpose: The abundance and effects of structural variation at BRCA1/2 in tumors are not well understood. In particular, the impact of these events on homologous recombination repair deficiency (HRD) has yet to be demonstrated. Experimental Design: Exploiting a large collection of whole-genome sequencing data from high-grade serous ovarian carcinoma (N = 205) together with matched RNA sequencing for the majority of tumors (N = 150), we have comprehensively characterized mutation and expression at BRCA1/2. Results: In addition to the known spectrum of short somatic mutations (SSM), we discovered that multi-megabase structural variants (SV) were a frequent, unappreciated source of BRCA1/2 disruption in these tumors, and we found a genome-wide enrichment for large deletions at the BRCA1/2 loci across the cohort. These SVs independently affected a substantial proportion of patients (16%) in addition to those affected by SSMs (24%), conferring HRD and impacting patient survival. We also detail compound deficiencies involving SSMs and SVs at both loci, demonstrating that the strongest risk of HRD emerges from combined SVs at both BRCA1 and BRCA2 in the absence of SSMs. Furthermore, these SVs are abundant and disruptive in other cancer types. Conclusions: These results extend our understanding of the mutational landscape underlying HRD, increase the number of patients predicted to benefit from therapies exploiting HRD, and suggest there is currently untapped potential in SV detection for patient stratification.

Epithelial-to-Mesenchymal Transition Supports Ovarian Carcinosarcoma Tumorigenesis and Confers Sensitivity to Microtubule Targeting with Eribulin

Abstract Ovarian carcinosarcoma (OCS) is an aggressive and rare tumor type with limited treatment options. OCS is hypothesized to develop via the combination theory, with a single progenitor resulting in carcinomatous and sarcomatous components, or alternatively via the conversion theory, with the sarcomatous component developing from the carcinomatous component through epithelial-to-mesenchymal transition (EMT). In this study, we analyzed DNA variants from isolated carcinoma and sarcoma components to show that OCS from 18 women is monoclonal. RNA sequencing indicated that the carcinoma components were more mesenchymal when compared with pure epithelial ovarian carcinomas, supporting the conversion theory and suggesting that EMT is important in the formation of these tumors. Preclinical OCS models were used to test the efficacy of microtubule-targeting drugs, including eribulin, which has previously been shown to reverse EMT characteristics in breast cancers and induce differentiation in sarcomas. Vinorelbine and eribulin more effectively inhibited OCS growth than standard-of-care platinum-based chemotherapy, and treatment with eribulin reduced mesenchymal characteristics and N-MYC expression in OCS patient-derived xenografts. Eribulin treatment resulted in an accumulation of intracellular cholesterol in OCS cells, which triggered a downregulation of the mevalonate pathway and prevented further cholesterol biosynthesis. Finally, eribulin increased expression of genes related to immune activation and increased the intratumoral accumulation of CD8+ T cells, supporting exploration of immunotherapy combinations in the clinic. Together, these data indicate that EMT plays a key role in OCS tumorigenesis and support the conversion theory for OCS histogenesis. Targeting EMT using eribulin could help improve OCS patient outcomes. Significance: Genomic analyses and preclinical models of ovarian carcinosarcoma support the conversion theory for disease development and indicate that microtubule inhibitors could be used to suppress EMT and stimulate antitumor immunity.

The Genomic Landscape of Early-Stage Ovarian High-Grade Serous Carcinoma

Abstract Purpose: 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

Dual G9A/EZH2 Inhibition Stimulates Antitumor Immune Response in Ovarian High-Grade Serous Carcinoma

Abstract Ovarian high-grade serous carcinoma (HGSC) prognosis correlates directly with presence of intratumoral lymphocytes. However, cancer immunotherapy has yet to achieve meaningful survival benefit in patients with HGSC. Epigenetic silencing of immunostimulatory genes is implicated in immune evasion in HGSC and re-expression of these genes could promote tumor immune clearance. We discovered that simultaneous inhibition of the histone methyltransferases G9A and EZH2 activates the CXCL10–CXCR3 axis and increases homing of intratumoral effector lymphocytes and natural killer cells while suppressing tumor-promoting FoxP3+ CD4 T cells. The dual G9A/EZH2 inhibitor HKMTI-1–005 induced chromatin changes that resulted in the transcriptional activation of immunostimulatory gene networks, including the re-expression of elements of the ERV-K endogenous retroviral family. Importantly, treatment with HKMTI-1–005 improved the survival of mice bearing Trp53−/− null ID8 ovarian tumors and resulted in tumor burden reduction. These results indicate that inhibiting G9A and EZH2 in ovarian cancer alters the immune microenvironment and reduces tumor growth and therefore positions dual inhibition of G9A/EZH2 as a strategy for clinical development.

Homologous recombination deficiency in newly diagnosed FIGO stage III/IV high-grade epithelial ovarian cancer: a multi-national observational study

Olaparib plus bevacizumab maintenance therapy improves survival outcomes in women with newly diagnosed, advanced, high-grade ovarian cancer with a deficiency in homologous recombination. We report data from the first year of routine homologous recombination deficiency testing in the National Health Service (NHS) in England, Wales, and Northern Ireland between April 2021 and April 2022. The Myriad myChoice companion diagnostic was used to test DNA extracted from formalin-fixed, paraffin-embedded tumor tissue in women with newly diagnosed International Federation of Gynecology and Obstetrics (FIGO) stage III/IV high-grade epithelial ovarian, fallopian tube, or primary peritoneal cancer. Tumors with homologous recombination deficiency were those with a The myChoice assay was performed on 2829 tumors. Of these, 2474 (87%) and 2178 (77%) successfully underwent This is the largest real-world evaluation of homologous recombination deficiency testing in newly diagnosed FIGO stage III/IV high-grade epithelial ovarian, fallopian tube, or primary peritoneal cancer. It is important to select tumor tissue with adequate tumor content and quality to reduce the risk of assay failure. The rapid uptake of testing across England, Wales, and Northern Ireland demonstrates the power of centralized NHS funding, center specialization, and the NHS Genomic Laboratory Hub network.

Rucaparib for maintenance treatment of platinum-sensitive, recurrent ovarian carcinoma: Final results of the phase 3, randomized, placebo-controlled ARIEL3 trial.

In ARIEL3, rucaparib maintenance significantly improved progression-free survival (PFS; primary endpoint) and long-term follow-up (LTFU) outcomes (including PFS2: time to disease progression on subsequent therapy or death) versus placebo in patients with recurrent, platinum-sensitive ovarian cancer. Here we report the final analysis of overall survival (OS; key secondary endpoint), LTFU outcomes, and safety. OS and updated LTFU efficacy outcomes were analyzed (data cutoff date: April 4, 2022) across three nested populations (BRCA-mutated, homologous recombination deficient [HRD], and intention to treat [ITT]). Patients were randomized 2:1 to rucaparib (600 mg BID; n = 375) or placebo (n = 189). Median follow-up was 77.0 months. 168 patients in the placebo arm received subsequent treatment; of these, 77 (46 %) received a poly(ADP-ribose) polymerase inhibitor-containing treatment. Median OS from randomization post chemotherapy for rucaparib vs placebo was 45.9 vs 47.8 months (HR 0.83, 95 % CI 0.58-1.19) for the BRCA-mutated population; no OS benefit was found with rucaparib in the HRD and ITT populations. Median PFS2 for rucaparib vs placebo was 26.1 vs 18.4 months (HR 0.67, 95 % CI 0.48-0.94) for the BRCA-mutated population. Rucaparib numerically improved PFS2 and other LTFU outcomes versus placebo in the HRD and ITT populations. Safety was consistent with prior reports; myelodysplastic syndrome and/or acute myeloid leukemia occurred in 4 % and 3 % of patients in the rucaparib and placebo arms, respectively. OS was similar between treatment arms. PFS benefit with rucaparib was maintained through the subsequent therapy line. These data support rucaparib as maintenance treatment for recurrent ovarian carcinoma.

RAS/PI3K pathway mutations sensitise epithelial ovarian cancer cells to a PARP/NAMPT inhibitor combination

Abstract The combination of PARP and NAMPT inhibitors (PARPi/NAMPTi) has been explored for the treatment of triple-negative breast cancer, Ewing sarcoma and high-grade serous carcinoma (HGSC). However, dose limiting toxicity has hampered NAMPTi in clinical trials. To maximise the therapeutic window, we set out to identify predictive genomic biomarkers. Bioinformatic analysis and screening of a panel of epithelial ovarian cancer (EOC) cell lines revealed that cells with RAS/PI3K pathway mutations are sensitive to the NAMPTi FK866. Combined exposure to olaparib and FK866 is associated with a reduction in nicotinamide mononucleotide (NMN) and the PARP substrate nicotinamide adenine dinucleotide (NAD + ), with coincident increases in ROS production, DNA damage and apoptosis induction. Caspase 3/7 activity is upregulated to a greater extent in RAS/PI3K mutant cell lines. Finally, the combination significantly reduces omental tumour weight and increases overall survival in mice injected with ID8 Trp53 -/- ;Pten -/- cells. This study highlights the potential of the PARPi/NAMPTi combination in RAS/PI3K pathway mutant EOC.

Divergent trajectories to structural diversity impact patient survival in high grade serous ovarian cancer

Abstract Deciphering the structural variation across tumour genomes is crucial to determine the events driving tumour progression and better understand tumour adaptation and evolution. High grade serous ovarian cancer (HGSOC) is an exemplar tumour type showing extreme, but poorly characterised structural diversity. Here, we comprehensively describe the mutational landscape driving HGSOC, exploiting a large (N = 324), deeply whole genome sequenced dataset. We reveal two divergent evolutionary trajectories, affecting patient survival and involving differing genomic environments. One involves homologous recombination repair deficiency (HRD) while the other is dominated by whole genome duplication (WGD) with frequent chromothripsis, breakage-fusion-bridges and extra-chromosomal DNA. These trajectories contribute to structural variation hotspots, containing candidate driver genes with significantly altered expression. While structural variation predominantly drives tumorigenesis, we find high mtDNA mutation loads associated with shorter patient survival. We show that a combination of mutations in the mitochondrial and nuclear genomes impact prognosis, suggesting strategies for patient stratification.

Study protocol for Adaptive ChemoTherapy for Ovarian cancer (ACTOv): a multicentre phase II randomised controlled trial to evaluate the efficacy of adaptive therapy (AT) with carboplatin, based on changes in CA125, in patients with relapsed platinum-sensitive high-grade serous or high-grade endometrioid ovarian cancer

Introduction Adaptive ChemoTherapy for Ovarian cancer (ACTOv) is a phase II, multicentre, randomised controlled trial, evaluating an adaptive therapy (AT) regimen with carboplatin in women with relapsed, platinum-sensitive high-grade serous or high-grade endometrioid cancer of the ovary, fallopian tube and peritoneum whose disease has progressed at least 6 months after day 1 of the last cycle of platinum-based chemotherapy. AT is a novel, evolutionarily informed approach to cancer treatment, which aims to exploit intratumoral competition between drug-sensitive and drug-resistant tumour subpopulations by modulating drug dose according to a patient’s own response to the last round of treatment. ACTOv is the first clinical trial of AT in this disease setting. Methods and analysis 80 patients will be randomised 1:1 to standard therapy (control) or AT (investigational) arms. The starting and maximum carboplatin dose in both arms is area under the curve (AUC) ×5 according to absolute nuclear medicine glomerular filtration rate. The AT regimen will modify the carboplatin dose according to changes in the serum biomarker CA125, a proxy measure of total tumour burden. Patients will receive treatment intravenously every 21 days for a maximum of 6 and 12 cycles in the control and investigational arms, respectively. The primary endpoint is modified progression-free survival (investigator-assessed using RECIST 1.1 (Response Evaluation Criteria in Solid Cancers) compared with the baseline prerandomisation scan rather than the radiological nadir), clinical progression or death from any cause. Secondary endpoints will include acceptability, deliverability, compliance, toxicity, CA125, quality of life and overall survival. ACTOv is open to National Health Service hospitals throughout the UK, recruitment is anticipated to take 36 months across 10 sites and will be managed by the Cancer Research UK and University College London Cancer Trials Centre. Ethics and dissemination The trial has been reviewed and received approval from the London—Dulwich Research Ethics Committee (REC). Results of the trial will be disseminated through publication in peer-reviewed journals. Trial registration number NCT05080556 .

Tertiary lymphoid structures and B cells determine clinically relevant T cell phenotypes in ovarian cancer

AbstractIntratumoral tertiary lymphoid structures (TLSs) have been associated with improved outcome in various cohorts of patients with cancer, reflecting their contribution to the development of tumor-targeting immunity. Here, we demonstrate that high-grade serous ovarian carcinoma (HGSOC) contains distinct immune aggregates with varying degrees of organization and maturation. Specifically, mature TLSs (mTLS) as forming only in 16% of HGSOCs with relatively elevated tumor mutational burden (TMB) are associated with an increased intratumoral density of CD8+ effector T (TEFF) cells and TIM3+PD1+, hence poorly immune checkpoint inhibitor (ICI)-sensitive, CD8+ T cells. Conversely, CD8+ T cells from immunologically hot tumors like non-small cell lung carcinoma (NSCLC) are enriched in ICI-responsive TCF1+ PD1+ T cells. Spatial B-cell profiling identifies patterns of in situ maturation and differentiation associated with mTLSs. Moreover, B-cell depletion promotes signs of a dysfunctional CD8+ T cell compartment among tumor-infiltrating lymphocytes from freshly isolated HGSOC and NSCLC biopsies. Taken together, our data demonstrate that – at odds with NSCLC – HGSOC is associated with a low density of follicular helper T cells and thus develops a limited number of mTLS that might be insufficient to preserve a ICI-sensitive TCF1+PD1+ CD8+ T cell phenotype. These findings point to key quantitative and qualitative differences between mTLSs in ICI-responsive vs ICI-irresponsive neoplasms that may guide the development of alternative immunotherapies for patients with HGSOC.