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BackgroundMedian overall survival (OS) for women with high-grade serous ovarian cancer (HGSOC) is ∼4 years, yet survival varies widely between patients. There are no well-established, gene expression signatures associated with prognosis. The aim of this study was to develop a robust prognostic signature for OS in patients with HGSOC.Patients and methodsExpression of 513 genes, selected from a meta-analysis of 1455 tumours and other candidates, was measured using NanoString technology from formalin-fixed paraffin-embedded tumour tissue collected from 3769 women with HGSOC from multiple studies. Elastic net regularization for survival analysis was applied to develop a prognostic model for 5-year OS, trained on 2702 tumours from 15 studies and evaluated on an independent set of 1067 tumours from six studies.ResultsExpression levels of 276 genes were associated with OS (false discovery rate < 0.05) in covariate-adjusted single-gene analyses. The top five genes were TAP1, ZFHX4, CXCL9, FBN1 and PTGER3 (P < 0.001). The best performing prognostic signature included 101 genes enriched in pathways with treatment implications. Each gain of one standard deviation in the gene expression score conferred a greater than twofold increase in risk of death [hazard ratio (HR) 2.35, 95% confidence interval (CI) 2.02-2.71; P < 0.001]. Median survival [HR (95% CI)] by gene expression score quintile was 9.5 (8.3 to -), 5.4 (4.6-7.0), 3.8 (3.3-4.6), 3.2 (2.9-3.7) and 2.3 (2.1-2.6) years.ConclusionThe OTTA-SPOT (Ovarian Tumor Tissue Analysis consortium - Stratified Prognosis of Ovarian Tumours) gene expression signature may improve risk stratification in clinical trials by identifying patients who are least likely to achieve 5-year survival. The identified novel genes associated with the outcome may also yield opportunities for the development of targeted therapeutic approaches.

Background: Despite an overall poor prognosis, about 15% of patients with advanced-stage tubo-ovarian high-grade serous carcinoma (HGSC) survive ten or more years after standard treatment., Methods: We evaluated the tumor microenvironment of this exceptional, understudied group using a large international cohort enriched for long-term survivors (LTS; 10+ years; n = 374) compared to medium-term (MTS; 5-7.99 years; n = 433) and short-term survivors (STS; 2-4.99 years; n = 416). Primary tumor samples were immunostained and scored for intra-epithelial and intra-stromal densities of 10 immune-cell subsets (including T cells, B cells, plasma cells, myeloid cells, PD-1+ cells, and PD-L1+ cells) and epithelial content., Results: Positive associations with LTS compared to STS were seen for 9/10 immune-cell subsets. In particular, the combination of intra-epithelial CD8+ T cells and intra-stromal B cells showed near five-fold increased odds of LTS compared to STS. All of these associations were stronger in tumors with high epithelial content and/or the C4/Differentiated molecular subtype, despite immune-cell densities generally being higher in tumors with low epithelial content and/or the C2/Immunoreactive molecular subtype., Conclusions: The tumor microenvironment of HGSC long-term survivors is distinguished by the intersection of T and B cell co-infiltration, high epithelial content and C4/Differentiated molecular subtype, features which may inspire new approaches to immunotherapy., Funding: Ovarian Cancer Research Program (OCRP) of the Congressionally Directed Medical Research Program (CDMRP), U.S. Department of Defense (DOD); American Cancer Society; BC Cancer Foundation; Canada's Networks of Centres of Excellence; Canadian Cancer Society; Canadian Institutes of Health Research; Cancer Councils of New South Wales, Victoria, Queensland, South Australia and Tasmania, Cancer Foundation of Western Australia; Cancer Institute NSW; Cancer Research UK; Deutsche Forschungsgesellschaft; ELAN Funds of the University of Erlangen-Nuremberg; Fred C. and Katherine B. Andersen Foundation; Genome BC; German Cancer Research Center; German Federal Ministry of Education and Research, Programme of Clinical Biomedical Research; Instituto de Salud Carlos III; Mayo Foundation; Minnesota Ovarian Cancer Alliance; Ministerio de Economía y Competitividad; MRC; National Center for Advancing Translational Sciences; National Health and Medical Research Council of Australia (NHMRC); Ovarian Cancer Australia; Peter MacCallum Foundation; Sydney West Translational Cancer Research Centre; Terry Fox Research Institute; The Eve Appeal (The Oak Foundation); UK National Institute for Health Research Biomedical Research Centres at the University of Cambridge; University of Pittsburgh School of Medicine; U.S. National Cancer Institute of the National Institutes of Health; VGH & UBC Hospital Foundation; Victorian Cancer Agency.

Cancer cells often exhibit DNA copy number aberrations and can vary widely in their ploidy. Correct estimation of the ploidy of single-cell genomes is paramount for downstream analysis. Based only on single-cell DNA sequencing information, scAbsolute achieves accurate and unbiased measurement of single-cell ploidy and replication status, including whole-genome duplications. We demonstrate scAbsolute's capabilities using experimental cell multiplets, a FUCCI cell cycle expression system, and a benchmark against state-of-the-art methods. scAbsolute provides a robust foundation for single-cell DNA sequencing analysis across different technologies and has the potential to enable improvements in a number of downstream analyses., (© 2024. The Author(s).)

High grade serous ovarian carcinoma (HGSOC) is a highly heterogeneous disease that typically presents at an advanced, metastatic state. The multi-scale complexity of HGSOC is a major obstacle to predicting response to neoadjuvant chemotherapy (NACT) and understanding critical determinants of response. Here we present a framework to predict the response of HGSOC patients to NACT integrating baseline clinical, blood-based, and radiomic biomarkers extracted from all primary and metastatic lesions. We use an ensemble machine learning model trained to predict the change in total disease volume using data obtained at diagnosis (n = 72). The model is validated in an internal hold-out cohort (n = 20) and an independent external patient cohort (n = 42). In the external cohort the integrated radiomics model reduces the prediction error by 8% with respect to the clinical model, achieving an AUC of 0.78 for RECIST 1.1 classification compared to 0.47 for the clinical model. Our results emphasize the value of including radiomics data in integrative models of treatment response and provide methods for developing new biomarker-based clinical trials of NACT in HGSOC., (© 2023. Springer Nature Limited.)

High-grade serous ovarian carcinoma (HGSOC) is characterised by poor outcome and extreme chromosome instability (CIN). Therapies targeting centrosome amplification (CA), a key mediator of chromosome missegregation, may have significant clinical utility in HGSOC. However, the prevalence of CA in HGSOC, its relationship to genomic biomarkers of CIN and its potential impact on therapeutic response have not been defined. Using high-throughput multi-regional microscopy on 287 clinical HGSOC tissues and 73 cell lines models, here we show that CA through centriole overduplication is a highly recurrent and heterogeneous feature of HGSOC and strongly associated with CIN and genome subclonality. Cell-based studies showed that high-prevalence CA is phenocopied in ovarian cancer cell lines, and that high CA is associated with increased multi-treatment resistance; most notably to paclitaxel, the commonest treatment used in HGSOC. CA in HGSOC may therefore present a potential driver of tumour evolution and a powerful biomarker for response to standard-of-care treatment., (© 2023. Springer Nature Limited.)

The drivers of recurrence and resistance in ovarian high grade serous carcinoma remain unclear. We investigate the acquisition of resistance by collecting tumour biopsies from a cohort of 276 women with relapsed ovarian high grade serous carcinoma in the BriTROC-1 study. Panel sequencing shows close concordance between diagnosis and relapse, with only four discordant cases. There is also very strong concordance in copy number between diagnosis and relapse, with no significant difference in purity, ploidy or focal somatic copy number alterations, even when stratified by platinum sensitivity or prior chemotherapy lines. Copy number signatures are strongly correlated with immune cell infiltration, whilst diagnosis samples from patients with primary platinum resistance have increased rates of CCNE1 and KRAS amplification and copy number signature 1 exposure. Our data show that the ovarian high grade serous carcinoma genome is remarkably stable between diagnosis and relapse and acquired chemotherapy resistance does not select for common copy number drivers., (© 2023. The Author(s).)

Women with ovarian cancer have limited therapy options, with immunotherapy being unsatisfactory for a large group of patients. Tumor cells spread from the ovary or the fallopian tube into the abdominal cavity, which is commonly accompanied with massive ascites production. The ascites represents a unique peritoneal liquid tumor microenvironment with the presence of both tumor and immune cells, including cytotoxic lymphocytes. We characterized lymphocytes in ascites from patients with high-grade serous ovarian cancer. Our data reveal the presence of NK and CD8 + T lymphocytes expressing CD103 and CD49a, which are markers of tissue residency. Moreover, these cells express high levels of the inhibitory NKG2A receptor, with the highest expression level detected on tissue-resident NK cells. Lymphocytes with these features were also present at the primary tumor site. Functional assays showed that tissue-resident NK cells in ascites are highly responsive towards ovarian tumor cells. Similar results were observed in an in vivo mouse model, in which tissue-resident NK and CD8 + T cells were detected in the peritoneal fluid upon tumor growth. Together, our data reveal the presence of highly functional lymphocyte populations that may be targeted to improve immunotherapy for patients with ovarian cancer.

High-grade serous ovarian carcinoma (HGSOC) is the most genomically complex cancer, characterized by ubiquitous TP53 mutation, profound chromosomal instability, and heterogeneity. The mutational processes driving chromosomal instability in HGSOC can be distinguished by specific copy number signatures. To develop clinically relevant models of these mutational processes we derived 15 continuous HGSOC patient-derived organoids (PDOs) and characterized them using bulk transcriptomic, bulk genomic, single-cell genomic, and drug sensitivity assays. We show that HGSOC PDOs comprise communities of different clonal populations and represent models of different causes of chromosomal instability including homologous recombination deficiency, chromothripsis, tandem-duplicator phenotype, and whole genome duplication. We also show that these PDOs can be used as exploratory tools to study transcriptional effects of copy number alterations as well as compound-sensitivity tests. In summary, HGSOC PDO cultures provide validated genomic models for studies of specific mutational processes and precision therapeutics., Competing Interests: MV, LM, CS, DS, DC, SB, BH, MS, JH, FC No competing interests declared, AP, FM, GM, JB GM, FM, AMP and JDB are founders and shareholders of Tailor Bio Ltd, (© 2023, Vias, Morrill Gavarró et al.)

Objective: Mucinous ovarian carcinoma (MOC) is a rare histotype of ovarian cancer, with low response rates to standard chemotherapy, and very poor survival for patients diagnosed at advanced stage. There is a limited understanding of the MOC immune landscape, and consequently whether immune checkpoint inhibitors could be considered for a subset of patients., Methods: We performed multicolor immunohistochemistry (IHC) and immunofluorescence (IF) on tissue microarrays in a cohort of 126 MOC patients. Cell densities were calculated in the epithelial and stromal components for tumor-associated macrophages (CD68+/PD-L1+, CD68+/PD-L1-), T cells (CD3+/CD8-, CD3+/CD8+), putative T-regulatory cells (Tregs, FOXP3+), B cells (CD20+/CD79A+), plasma cells (CD20-/CD79a+), and PD-L1+ and PD-1+ cells, and compared these values with clinical factors. Univariate and multivariable Cox Proportional Hazards assessed overall survival. Unsupervised k-means clustering identified patient subsets with common patterns of immune cell infiltration., Results: Mean densities of PD1+ cells, PD-L1- macrophages, CD4+ and CD8+ T cells, and FOXP3+ Tregs were higher in the stroma compared to the epithelium. Tumors from advanced (Stage III/IV) MOC had greater epithelial infiltration of PD-L1- macrophages, and fewer PD-L1+ macrophages compared with Stage I/II cancers (p = 0.004 and p = 0.014 respectively). Patients with high epithelial density of FOXP3+ cells, CD8+/FOXP3+ cells, or PD-L1- macrophages, had poorer survival, and high epithelial CD79a + plasma cells conferred better survival, all upon univariate analysis only. Clustering showed that most MOC (86%) had an immune depleted (cold) phenotype, with only a small proportion (11/76,14%) considered immune inflamed (hot) based on T cell and PD-L1 infiltrates., Conclusion: In summary, MOCs are mostly immunogenically 'cold', suggesting they may have limited response to current immunotherapies., Competing Interests: Declaration of Competing Interest NSM has received a travel grant from NanoString technologies, unrelated to this work. DDLB has received research support grants from Roche-Genentech, AstraZeneca, and BeiGene (paid to institution); and personal consulting fees from Exo Therapeutics, that are outside the submitted work. MF has participated in Advisory Boards/Consulting from Astra Zeneca, Novartis, GSK, Takeda, Lilly, MSD Eisei and received honoraria/speakers' fees from AstraZeneca, GSK, ACT-Genomics; Research funding to institution: from AstraZeneca, Novartis, Beigene, all unrelated to this work. ADeF has received funding from AstraZeneca, unrelated this work. NS has received honoraria for invited participation in advisory boards hosted by Astra-Zeneca-MSD and Glaxo SmithKline, unrelated to this work. UM had stock ownership in Abcodia until October 2021, and UCL had a license agreement (2011–2021) with Abcodia not related to this work. BHN is a co-founder of Innovakine Therapeutics Inc., unrelated to this work., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Fewer than half of all patients with advanced-stage high-grade serous ovarian cancers (HGSCs) survive more than five years after diagnosis, but those who have an exceptionally long survival could provide insights into tumor biology and therapeutic approaches. We analyzed 60 patients with advanced-stage HGSC who survived more than 10 years after diagnosis using whole-genome sequencing, transcriptome and methylome profiling of their primary tumor samples, comparing this data to 66 short- or moderate-term survivors. Tumors of long-term survivors were more likely to have multiple alterations in genes associated with DNA repair and more frequent somatic variants resulting in an increased predicted neoantigen load. Patients clustered into survival groups based on genomic and immune cell signatures, including three subsets of patients with BRCA1 alterations with distinctly different outcomes. Specific combinations of germline and somatic gene alterations, tumor cell phenotypes and differential immune responses appear to contribute to long-term survival in HGSC., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Purpose: Chromosomal aberration and DNA copy number change are robust hallmarks of cancer. The gold standard for detecting copy number changes in tumor cells is fluorescence in situ hybridization (FISH) using locus-specific probes that are imaged as fluorescent spots. However, spot counting often does not perform well on solid tumor tissue sections due to partially represented or overlapping nuclei., Materials and Methods: To overcome these challenges, we have developed a computational approach called FrenchFISH, which comprises a nuclear volume correction method coupled with two types of Poisson models: either a Poisson model for improved manual spot counting without the need for control probes or a homogeneous Poisson point process model for automated spot counting., Results: We benchmarked the performance of FrenchFISH against previous approaches using a controlled simulation scenario and tested it experimentally in 12 ovarian carcinoma FFPE-tissue sections for copy number alterations at three loci (c-Myc, hTERC, and SE7). FrenchFISH outperformed standard spot counting with 74% of the automated counts having < 1 copy number difference from the manual counts and 17% having < 2 copy number differences, while taking less than one third of the time of manual counting., Conclusion: FrenchFISH is a general approach that can be used to enhance clinical diagnosis on sections of any tissue by both speeding up and improving the accuracy of spot count estimates.

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 >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 ., (©2020 American Association for Cancer Research.)

High-grade serous ovarian carcinoma (HGSOC) remains the deadliest form of epithelial ovarian cancer and despite major efforts little improvement in overall survival has been achieved. Identification of recurring "driver" genetic lesions has the potential to enable design of novel therapies for cancer. Here, we report on a study to find such new therapeutic targets for HGSOC using exome-capture sequencing approach targeting all kinase genes in 127 patient samples. Consistent with previous reports, the most frequently mutated gene was TP53 (97% mutation frequency) followed by BRCA1 (10% mutation frequency). The average mutation frequency of the kinase genes mutated from our panel was 1.5%. Intriguingly, after BRCA1, JAK3 was the most frequently mutated gene (4% mutation frequency). We tested the transforming properties of JAK3 mutants using the Ba/F3 cell-based in vitro functional assay and identified a novel gain-of-function mutation in the kinase domain of JAK3 (p.T1022I). Importantly, p.T1022I JAK3 mutants displayed higher sensitivity to the JAK3-selective inhibitor Tofacitinib compared to controls. For independent validation, we re-sequenced the entire JAK3 coding sequence using tagged amplicon sequencing (TAm-Seq) in 463 HGSOCs resulting in an overall somatic mutation frequency of 1%. TAm-Seq screening of CDK12 in the same population revealed a 7% mutation frequency. Our data confirms that the frequency of mutations in kinase genes in HGSOC is low and provides accurate estimates for the frequency of JAK3 and CDK12 mutations in a large well characterized cohort. Although p.T1022I JAK3 mutations are rare, our functional validation shows that if detected they should be considered as potentially actionable for therapy. The observation of CDK12 mutations in 7% of HGSOC cases provides a strong rationale for routine somatic testing, although more functional and clinical characterization is required to understand which nonsynonymous mutations alterations are associated with homologous recombination deficiency., Competing Interests: The authors have declared that no competing interests exist.

Background: In colorectal and breast cancer, the density and localisation of immune infiltrates provides strong prognostic information. We asked whether similar automated quantitation and combined analysis of immune infiltrates could refine prognostic information in high-grade serous ovarian carcinoma (HGSOC) and tested associations between patterns of immune response and genomic driver alterations., Methods: Epithelium and stroma were semi-automatically segmented and the infiltration of CD45RO + , CD8 + and CD68 + cells was automatically quantified from images of 332 HGSOC patient tissue microarray cores., Results: Epithelial CD8 [p = 0.027, hazard ratio (HR) = 0.83], stromal CD68 (p = 3 × 10 -4 , HR = 0.44) and stromal CD45RO (p = 7 × 10 -4 , HR = 0.76) were positively associated with survival and remained so when averaged across the tumour and stromal compartments. Using principal component analysis, we identified optimised multiparameter survival models combining information from all immune markers (p = 0.016, HR = 0.88). There was no significant association between PTEN expression, type of TP53 mutation or presence of BRCA1/BRCA2 mutations and immune infiltrate densities or principal components., Conclusions: Combining measures of immune infiltration provided improved survival modelling and evidence for the multiple effects of different immune factors on survival. The presence of stromal CD68 + and CD45RO + populations was associated with survival, underscoring the benefits evaluating stromal immune populations may bring for prognostic immunoscores in HGSOC.

TP53 mutations are considered a surrogate biomarker of the serous-like 'copy number high' molecular subtype of endometrial carcinoma (EC). In ovarian carcinoma, p53 immunohistochemistry (IHC) accurately reflects mutational status with almost 100% specificity but its performance in EC has not been established. This study tested whether p53 IHC reliably predicts TP53 mutations identified by next-generation sequencing (NGS) in EC biopsy samples for all ECs and as part of a molecular classification algorithm after exclusion of cases harbouring mismatch repair defects (MMRd) or pathogenic DNA polymerase epsilon exonuclease domain mutations (POLEmut). A secondary aim assessed inter-laboratory variability in p53 IHC. From a total of 207 cases from five centres (37-49 cases per centre), p53 IHC carried out at a central reference laboratory was compared with local IHC (n = 164) and curated tagged-amplicon NGS TP53 sequencing results (n = 177). Following consensus review, local and central p53 IHC results were concordant in 156/164 (95.1%) tumours. Discordant results were attributable to both interpretive and technical differences in staining between the local and central laboratories. When results were considered as any mutant pattern versus wild-type pattern staining, however, there was disagreement between local and central review in only one case. The concordance between p53 IHC and TP53 mutation was 155/168 (92.3%) overall, and 117/123 (95.1%) after excluding MMRd and POLEmut EC. Three (3/6) discordant results were in serous carcinomas with complete absence of p53 staining but no detectable TP53 mutation. Subclonal mutant p53 IHC expression was observed in 9/177 (5.1%) cases, of which four were either MMRd or POLEmut. Mutant pattern p53 IHC was observed in 63/63 (100%) serous carcinomas that were MMR-proficient/POLE exonuclease domain wild-type. Optimised p53 IHC performs well as a surrogate test for TP53 mutation in EC biopsies, demonstrates excellent inter-laboratory reproducibility, and has high clinical utility for molecular classification algorithms in EC. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (© 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Existing methods to improve detection of circulating tumor DNA (ctDNA) have focused on genomic alterations but have rarely considered the biological properties of plasma cell-free DNA (cfDNA). We hypothesized that differences in fragment lengths of circulating DNA could be exploited to enhance sensitivity for detecting the presence of ctDNA and for noninvasive genomic analysis of cancer. We surveyed ctDNA fragment sizes in 344 plasma samples from 200 patients with cancer using low-pass whole-genome sequencing (0.4×). To establish the size distribution of mutant ctDNA, tumor-guided personalized deep sequencing was performed in 19 patients. We detected enrichment of ctDNA in fragment sizes between 90 and 150 bp and developed methods for in vitro and in silico size selection of these fragments. Selecting fragments between 90 and 150 bp improved detection of tumor DNA, with more than twofold median enrichment in >95% of cases and more than fourfold enrichment in >10% of cases. Analysis of size-selected cfDNA identified clinically actionable mutations and copy number alterations that were otherwise not detected. Identification of plasma samples from patients with advanced cancer was improved by predictive models integrating fragment length and copy number analysis of cfDNA, with area under the curve (AUC) >0.99 compared to AUC <0.80 without fragmentation features. Increased identification of cfDNA from patients with glioma, renal, and pancreatic cancer was achieved with AUC > 0.91 compared to AUC < 0.5 without fragmentation features. Fragment size analysis and selective sequencing of specific fragment sizes can boost ctDNA detection and could complement or provide an alternative to deeper sequencing of cfDNA., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The genomic complexity of profound copy number aberrations has prevented effective molecular stratification of ovarian cancers. Here, to decode this complexity, we derived copy number signatures from shallow whole-genome sequencing of 117 high-grade serous ovarian cancer (HGSOC) cases, which were validated on 527 independent cases. We show that HGSOC comprises a continuum of genomes shaped by multiple mutational processes that result in known patterns of genomic aberration. Copy number signature exposures at diagnosis predict both overall survival and the probability of platinum-resistant relapse. Measurement of signature exposures provides a rational framework to choose combination treatments that target multiple mutational processes.

Background: Investigating tumour evolution and acquired chemotherapy resistance requires analysis of sequential tumour material. We describe the feasibility of obtaining research biopsies in women with relapsed ovarian high-grade serous carcinoma (HGSC)., Methods: Women with relapsed ovarian HGSC underwent either image-guided biopsy or intra-operative biopsy during secondary debulking, and samples were fixed in methanol-based fixative. Tagged-amplicon sequencing was performed on biopsy DNA., Results: We screened 519 patients in order to enrol 220. Two hundred and two patients underwent successful biopsy, 118 of which were image-guided. There were 22 study-related adverse events (AE) in the image-guided biopsies, all grades 1 and 2; pain was the commonest AE. There were pre-specified significant AE in 3/118 biopsies (2.5%). 87% biopsies were fit-for-purpose for genomic analyses. Median DNA yield was 2.87 μg, and was higher in biopsies utilising 14 G or 16 G needles compared to 18 G. TP53 mutations were identified in 94.4% patients., Conclusions: Obtaining tumour biopsies for research in relapsed HGSC is safe and feasible. Adverse events are rare. The large majority of biopsies yield sufficient DNA for genomic analyses-we recommend use of larger gauge needles and methanol fixation for such biopsies, as DNA yields are higher but with no increase in AEs.

To identify common alleles associated with different histotypes of epithelial ovarian cancer (EOC), we pooled data from multiple genome-wide genotyping projects totaling 25,509 EOC cases and 40,941 controls. We identified nine new susceptibility loci for different EOC histotypes: six for serous EOC histotypes (3q28, 4q32.3, 8q21.11, 10q24.33, 18q11.2 and 22q12.1), two for mucinous EOC (3q22.3 and 9q31.1) and one for endometrioid EOC (5q12.3). We then performed meta-analysis on the results for high-grade serous ovarian cancer with the results from analysis of 31,448 BRCA1 and BRCA2 mutation carriers, including 3,887 mutation carriers with EOC. This identified three additional susceptibility loci at 2q13, 8q24.1 and 12q24.31. Integrated analyses of genes and regulatory biofeatures at each locus predicted candidate susceptibility genes, including OBFC1, a new candidate susceptibility gene for low-grade and borderline serous EOC.

Background: Circulating tumour DNA (ctDNA) carrying tumour-specific sequence alterations may provide a minimally invasive means to dynamically assess tumour burden and response to treatment in cancer patients. Somatic TP53 mutations are a defining feature of high-grade serous ovarian carcinoma (HGSOC). We tested whether these mutations could be used as personalised markers to monitor tumour burden and early changes as a predictor of response and time to progression (TTP)., Methods and Findings: We performed a retrospective analysis of serial plasma samples collected during routine clinical visits from 40 patients with HGSOC undergoing heterogeneous standard of care treatment. Patient-specific TP53 assays were developed for 31 unique mutations identified in formalin-fixed paraffin-embedded tumour DNA from these patients. These assays were used to quantify ctDNA in 318 plasma samples using microfluidic digital PCR. The TP53 mutant allele fraction (TP53MAF) was compared to serum CA-125, the current gold-standard response marker for HGSOC in blood, as well as to disease volume on computed tomography scans by volumetric analysis. Changes after one cycle of treatment were compared with TTP. The median TP53MAF prior to treatment in 51 relapsed treatment courses was 8% (interquartile range [IQR] 1.2%-22%) compared to 0.7% (IQR 0.3%-2.0%) for seven untreated newly diagnosed stage IIIC/IV patients. TP53MAF correlated with volumetric measurements (Pearson r = 0.59, p < 0.001), and this correlation improved when patients with ascites were excluded (r = 0.82). The ratio of TP53MAF to volume of disease was higher in relapsed patients (0.04% per cm3) than in untreated patients (0.0008% per cm3, p = 0.004). In nearly all relapsed patients with disease volume > 32 cm3, ctDNA was detected at ≥20 amplifiable copies per millilitre of plasma. In 49 treatment courses for relapsed disease, pre-treatment TP53MAF concentration, but not CA-125, was associated with TTP. Response to chemotherapy was seen earlier with ctDNA, with a median time to nadir of 37 d (IQR 28-54) compared with a median time to nadir of 84 d (IQR 42-116) for CA-125. In 32 relapsed treatment courses evaluable for response after one cycle of chemotherapy, a decrease in TP53MAF of >60% was an independent predictor of TTP in multivariable analysis (hazard ratio 0.22, 95% CI 0.07-0.67, p = 0.008). Conversely, a decrease in TP53MAF of ≤60% was associated with poor response and identified cases with TTP < 6 mo with 71% sensitivity (95% CI 42%-92%) and 88% specificity (95% CI 64%-99%). Specificity was improved when patients with recent drainage of ascites were excluded. Ascites drainage led to a reduction of TP53MAF concentration. The limitations of this study include retrospective design, small sample size, and heterogeneity of treatment within the cohort., Conclusions: In this retrospective study, we demonstrated that ctDNA is correlated with volume of disease at the start of treatment in women with HGSOC and that a decrease of ≤60% in TP53MAF after one cycle of chemotherapy was associated with shorter TTP. These results provide evidence that ctDNA has the potential to be a highly specific early molecular response marker in HGSOC and warrants further investigation in larger cohorts receiving uniform treatment., Competing Interests: We have read the journal's policy and the authors of this manuscript have the following competing interests: DG, NR and JDB are co-founders, shareholders and officers/consultants of Inivata Ltd, a cancer genomics company that commercialises ctDNA analysis.

TP53 mutations are ubiquitous in high-grade serous ovarian carcinomas (HGSOC), and the presence of TP53 mutation discriminates between high and low-grade serous carcinomas and is now an important biomarker for clinical trials targeting mutant p53. p53 immunohistochemistry (IHC) is widely used as a surrogate for TP53 mutation but its accuracy has not been established. The objective of this study was to test whether improved methods for p53 IHC could reliably predict TP53 mutations independently identified by next generation sequencing (NGS). Four clinical p53 IHC assays and tagged-amplicon NGS for TP53 were performed on 171 HGSOC and 80 endometrioid carcinomas (EC). p53 expression was scored as overexpression (OE), complete absence (CA), cytoplasmic (CY) or wild type (WT). p53 IHC was evaluated as a binary classifier where any abnormal staining predicted deleterious TP53 mutation and as a ternary classifier where OE, CA or WT staining predicted gain-of-function (GOF or nonsynonymous), loss-of-function (LOF including stopgain, indel, splicing) or no detectable TP53 mutations (NDM), respectively. Deleterious TP53 mutations were detected in 169/171 (99%) HGSOC and 7/80 (8.8%) EC. The overall accuracy for the best performing IHC assay for binary and ternary prediction was 0.94 and 0.91 respectively, which improved to 0.97 (sensitivity 0.96, specificity 1.00) and 0.95 after secondary analysis of discordant cases. The sensitivity for predicting LOF mutations was lower at 0.76 because p53 IHC detected mutant p53 protein in 13 HGSOC with LOF mutations. CY staining associated with LOF was seen in 4 (2.3%) of HGSOC. Optimized p53 IHC can approach 100% specificity for the presence of TP53 mutation and its high negative predictive value is clinically useful as it can exclude the possibility of a low-grade serous tumour. 4.1% of HGSOC cases have detectable WT staining while harboring a TP53 LOF mutation, which limits sensitivity for binary prediction of mutation to 96%.

The Canadian Immunohistochemistry Quality Control provides proficiency testing for immunohistochemistry in Canadian laboratories. Canadian Immunohistochemistry Quality Control Run 42 assessed WT1, Napsin A, and p53; commonly used markers for histotyping ovarian carcinomas. A 42-core tissue microarray, which included the 5 major histotypes of ovarian carcinomas with a subset having known TP53 mutational status, was used for this Canadian Immunohistochemistry Quality Control challenge. Participants included 43 laboratories for p53, 29 for WT1, and 26 for Napsin A. p53 was scored as aberrant if the staining was strong and diffuse or absent. Napsin A and WT1 were scored positive if any tumor cells stained. The reference p53 expression pattern was inferred by TP53 mutation type when available. For WT1, Napsin A, and cases lacking mutational data, the reference staining pattern was based on the majority staining result. The error rate for p53 was 8.8%. Most errors (84%) were due to weak staining. The sensitivity and specificity of aberrant p53 expression for an underlying TP53 mutation was 91.6% and 87.9%, respectively. The error rate for WT1 was 0.76% with all errors occurring in laboratories using the 6F-h2 clone. The average errors for laboratories using 6F-h2 were 2.4 compared with 0 for WT-49. The error rate for Napsin A was 4%. The average errors for laboratories using polyclonal Napsin A were 3 compared with 1.1 for monoclonal Napsin A. Weak p53 staining increases interpretative errors, primarily due to absence of staining in tumors with wild-type TP53. p53 immunohistochemistry correlates strongly with TP53 mutational status. Polyclonal Napsin A and 6F-h2 may lack specificity in comparison to monoclonal Napsin A and WT-49.

Background: Next-generation sequencing (NGS) of tumour samples is a critical component of personalised cancer treatment, but it requires high-quality DNA samples. Routine neutral-buffered formalin (NBF) fixation has detrimental effects on nucleic acids, causing low yields, as well as fragmentation and DNA base changes, leading to significant artefacts., Patients and Methods: We have carried out a detailed comparison of DNA quality from matched samples isolated from high-grade serous ovarian cancers from 16 patients fixed in methanol and NBF. These experiments use tumour fragments and mock biopsies to simulate routine practice, ensuring that results are applicable to standard clinical biopsies., Results: Using matched snap-frozen tissue as gold standard comparator, we show that methanol-based fixation has significant benefits over NBF, with greater DNA yield, longer fragment size and more accurate copy-number calling using shallow whole-genome sequencing (WGS). These data also provide a new approach to understand and quantify artefactual effects of fixation using non-negative matrix factorisation to analyse mutational spectra from targeted and WGS data., Conclusion: We strongly recommend the adoption of methanol fixation for sample collection strategies in new clinical trials. This approach is immediately available, is logistically simple and can offer cheaper and more reliable mutation calling than traditional NBF fixation., (© The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology.)

Background: The major clinical challenge in the treatment of high-grade serous ovarian cancer (HGSOC) is the development of progressive resistance to platinum-based chemotherapy. The objective of this study was to determine whether intra-tumour genetic heterogeneity resulting from clonal evolution and the emergence of subclonal tumour populations in HGSOC was associated with the development of resistant disease., Methods and Findings: Evolutionary inference and phylogenetic quantification of heterogeneity was performed using the MEDICC algorithm on high-resolution whole genome copy number profiles and selected genome-wide sequencing of 135 spatially and temporally separated samples from 14 patients with HGSOC who received platinum-based chemotherapy. Samples were obtained from the clinical CTCR-OV03/04 studies, and patients were enrolled between 20 July 2007 and 22 October 2009. Median follow-up of the cohort was 31 mo (interquartile range 22-46 mo), censored after 26 October 2013. Outcome measures were overall survival (OS) and progression-free survival (PFS). There were marked differences in the degree of clonal expansion (CE) between patients (median 0.74, interquartile range 0.66-1.15), and dichotimization by median CE showed worse survival in CE-high cases (PFS 12.7 versus 10.1 mo, p = 0.009; OS 42.6 versus 23.5 mo, p = 0.003). Bootstrap analysis with resampling showed that the 95% confidence intervals for the hazard ratios for PFS and OS in the CE-high group were greater than 1.0. These data support a relationship between heterogeneity and survival but do not precisely determine its effect size. Relapsed tissue was available for two patients in the CE-high group, and phylogenetic analysis showed that the prevalent clonal population at clinical recurrence arose from early divergence events. A subclonal population marked by a NF1 deletion showed a progressive increase in tumour allele fraction during chemotherapy., Conclusions: This study demonstrates that quantitative measures of intra-tumour heterogeneity may have predictive value for survival after chemotherapy treatment in HGSOC. Subclonal tumour populations are present in pre-treatment biopsies in HGSOC and can undergo expansion during chemotherapy, causing clinical relapse.

Epithelial ovarian cancer is a highly heterogeneous disease and remains the most lethal gynaecological malignancy in the Western world. Therapeutic approaches need to account for inter-patient and intra-tumoural heterogeneity and detailed characterization of in vitro models representing the different histological and molecular ovarian cancer subtypes is critical to enable reliable preclinical testing. There are approximately 100 publicly available ovarian cancer cell lines but their cellular and molecular characteristics are largely undescribed. We have characterized 39 ovarian cancer cell lines under uniform conditions for growth characteristics, mRNA/microRNA expression, exon sequencing, drug response for clinically-relevant therapeutics and collated all available information on the original clinical features and site of origin. We tested for statistical associations between the cellular and molecular features of the lines and clinical features. Of the 39 ovarian cancer cell lines, 14 were assigned as high-grade serous, four serous-type, one low-grade serous and 20 non-serous type. Three morphological subtypes: Epithelial (n = 21), Round (n = 7) and Spindle (n = 12) were identified that showed distinct biological and molecular characteristics, including overexpression of cell movement and migration-associated genes in the Spindle subtype. Comparison with the original clinical data showed association of the spindle-like tumours with metastasis, advanced stage, suboptimal debulking and poor prognosis. In addition, the expression profiles of Spindle, Round and Epithelial morphologies clustered with the previously described C1-stromal, C5-mesenchymal and C4 ovarian subtype expression profiles respectively. Comprehensive profiling of 39 ovarian cancer cell lines under controlled, uniform conditions demonstrates clinically relevant cellular and genomic characteristics. This data provides a rational basis for selecting models to develop specific treatment approaches for histological and molecular subtypes of ovarian cancer.

Background: Serum concentrations of the tumor-associated folate receptor 1 (FOLR1) protein may be a marker for early cancer detection, yet concentrations have also been detected in cancer-free women. We investigated the conditions associated with circulating FOLR1 protein in healthy individuals and sought to clarify the range of normal serum values., Methods: Sera of cancer-free men and women (N = 60) enrolled in a population-based cohort study in Alberta, Canada were analyzed for FOLR1 protein using an electrochemical luminescence immunoassay. Dietary, lifestyle, medical and reproductive history information was collected by questionnaires. Differences in serum FOLR1 concentrations between groups were assessed by non-parametric tests, and predictors of serum FOLR1 concentrations were estimated using multivariable linear regression., Results: Median serum FOLR1 concentration was higher in women (491 pg/ml, range = 327-693 pg/ml) than in men (404 pg/ml, range = 340-682 pg/ml), P = 0.001. FOLR1 concentration was also positively associated with vitamin A intake (P = 0.02), and showed positive trends with age and with oral contraceptive hormone use among women and an inverse trend with body mass index. All variables examined explained almost half of the variation in serum FOLR1 (model R2 = 0.44, P = 0.04); however, the retention of gender (P = 0.003) and vitamin A intake (P = 0.03) together explained 20% (P = 0.001) of serum FOLR1 variation. No other predictor was significant at P<0.05., Conclusions: The positive association between serum FOLR1 concentration and female gender independent of an age effect suggests caution against statements to exploit serum FOLR1 for early cancer detection without further understanding the biological underpinnings of these observations. Serum FOLR1 concentrations may be influenced by the steroid retinoic acid (vitamin A) but do not appear to be associated with folate nutritional status. These findings require confirmation in larger independent studies.

Cancers acquire resistance to systemic treatment as a result of clonal evolution and selection. Repeat biopsies to study genomic evolution as a result of therapy are difficult, invasive and may be confounded by intra-tumour heterogeneity. Recent studies have shown that genomic alterations in solid cancers can be characterized by massively parallel sequencing of circulating cell-free tumour DNA released from cancer cells into plasma, representing a non-invasive liquid biopsy. Here we report sequencing of cancer exomes in serial plasma samples to track genomic evolution of metastatic cancers in response to therapy. Six patients with advanced breast, ovarian and lung cancers were followed over 1-2 years. For each case, exome sequencing was performed on 2-5 plasma samples (19 in total) spanning multiple courses of treatment, at selected time points when the allele fraction of tumour mutations in plasma was high, allowing improved sensitivity. For two cases, synchronous biopsies were also analysed, confirming genome-wide representation of the tumour genome in plasma. Quantification of allele fractions in plasma identified increased representation of mutant alleles in association with emergence of therapy resistance. These included an activating mutation in PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha) following treatment with paclitaxel; a truncating mutation in RB1 (retinoblastoma 1) following treatment with cisplatin; a truncating mutation in MED1 (mediator complex subunit 1) following treatment with tamoxifen and trastuzumab, and following subsequent treatment with lapatinib, a splicing mutation in GAS6 (growth arrest-specific 6) in the same patient; and a resistance-conferring mutation in EGFR (epidermal growth factor receptor; T790M) following treatment with gefitinib. These results establish proof of principle that exome-wide analysis of circulating tumour DNA could complement current invasive biopsy approaches to identify mutations associated with acquired drug resistance in advanced cancers. Serial analysis of cancer genomes in plasma constitutes a new paradigm for the study of clonal evolution in human cancers.

Plasma of cancer patients contains cell-free tumor DNA that carries information on tumor mutations and tumor burden. Individual mutations have been probed using allele-specific assays, but sequencing of entire genes to detect cancer mutations in circulating DNA has not been demonstrated. We developed a method for tagged-amplicon deep sequencing (TAm-Seq) and screened 5995 genomic bases for low-frequency mutations. Using this method, we identified cancer mutations present in circulating DNA at allele frequencies as low as 2%, with sensitivity and specificity of >97%. We identified mutations throughout the tumor suppressor gene TP53 in circulating DNA from 46 plasma samples of advanced ovarian cancer patients. We demonstrated use of TAm-Seq to noninvasively identify the origin of metastatic relapse in a patient with multiple primary tumors. In another case, we identified in plasma an EGFR mutation not found in an initial ovarian biopsy. We further used TAm-Seq to monitor tumor dynamics, and tracked 10 concomitant mutations in plasma of a metastatic breast cancer patient over 16 months. This low-cost, high-throughput method could facilitate analysis of circulating DNA as a noninvasive "liquid biopsy" for personalized cancer genomics.