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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.

Digital Pathology (DP) is a platform which has the potential to develop a truly integrated and global pathology community. The generation of DP data at scale creates novel challenges for the histopathology community in managing, processing, and governing the use of these data. The current understanding of, and confidence in, the legal and ethical aspects of DP by pathologists is unknown. We developed an electronic survey (e‐survey) comprising of 22 questions, which was developed with input from the Royal College of Pathologists (RCPath) Digital Pathology Working Group. The e‐survey was circulated via e‐mail and social media (Twitter) through the RCPath Digital Pathology Working Group network, RCPath Trainee Committee network, the Pathology image data Lake for Analytics, Knowledge and Education (PathLAKE) digital pathology consortium, National Pathology Imaging Co‐operative (NPIC), local contacts, and to the membership of both The Pathological Society of Great Britain and Ireland and the British Division of the International Academy of Pathology (BDIAP). Between 14 July 2020 and 6 September 2020, we collected 198 responses representing a cross section of histopathologists, including individuals with experience of DP research. We ascertained that in the UK, DP is being used for diagnosis, research, and teaching, and that the platform is enabling data sharing. Our survey demonstrated that there is often a lack of confidence and understanding of the key issues of consent, legislation, and ethical guidelines. Of 198 respondents, 82 (41%) did not know when the use of digital scanned slide images would fall under the relevant legislation and 93 (47%) were ‘Not confident at all’ in their interpretation of consent for scanned slide images in research. With increasing uptake of DP, a working knowledge of these areas is essential but histopathologists often express a lack of confidence in these topics. The need for specific training in these areas is highlighted by the findings of this study.

Supplementary methods and figures

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.

Funding: This work is supported by the Industrial Centre for AI Research in digital Diagnostics (iCAIRD) which is funded by Innovate UK on behalf of UK Research and Innovation (UKRI) [project number: 104690], and in part by Chief Scientist Office, Scotland. Fully supervised learning for whole slide image based diagnostic tasks in histopathology is problematic due to the requirement for costly and time-consuming manual annotation by experts. Weakly supervised learning which utilises only slide-level labels during training is becoming more widespread as it relieves this burden, but has not yet been applied to endometrial whole slide images, in iSyntax format. In this work we apply a weakly supervised learning algorithm to a real-world dataset of this type for the first time, with over 85% validation accuracy and over 87% test accuracy. We then employ interpretability methods including attention heatmapping, feature visualisation, and a novel end-to-end saliency-mapping approach to identify distinct morphologies learned by the model and build an understanding of its behaviour. These interpretability methods, alongside consultation with expert pathologists, allow us to make comparisons between machine-learned knowledge and consensus in the field. This work contributes to the state of the art by demonstrating a robust practical application of weakly supervised learning on a real-world digital pathology dataset and shows the importance of fine-grained interpretability to support understanding and evaluation of model performance in this high-stakes use case. Publisher PDF

BackgroundRisk-reducing salpingo-oophorectomy is the 'gold standard' for preventing tubo-ovarian cancer in women at increased risk. However, when performed in pre-menopausal women, it results in premature menopause and associated detrimental health consequences. This, together with acceptance of the central role of the fallopian tube in etiopathogenesis of high-grade serous carcinoma, by far the most common type of tubo-ovarian cancer, has led to risk-reducing early salpingectomy with delayed oophorectomy being proposed as a two-step surgical alternative for pre-menopausal women declining/delaying oophorectomy.Primary ObjectiveTo evaluate the impact on sexual function of risk-reducing early salpingectomy, within a two-step, risk-reducing, early salpingectomy with delayed oophorectomy tubo-ovarian cancer prevention strategy in pre-menopausal women at increased risk of tubo-ovarian cancer.Study HypothesisRisk-reducing early salpingectomy is non-inferior for sexual and endocrine function compared with controls; risk-reducing early salpingectomy is superior for sexual/endocrine function, non-inferior for quality-of-life, and equivalent in satisfaction to the standard risk-reducing salpingo-oophorectomy.Trial DesignMulti-center, observational cohort trial with three arms: risk-reducing early salpingectomy with delayed oophorectomy; risk-reducing salpingo-oophorectomy; controls (no surgery). Consenting individuals undergo an ultrasound, serum CA125, and follicle-stimulating hormone measurements and provide information on medical history, family history, quality-of-life, sexual function, cancer worry, psychological well-being, and satisfaction/regret. Follow-up by questionnaire takes place annually for 3 years. Women receiving risk-reducing early salpingectomy can undergo delayed oophorectomy at a later date of their choosing, or definitely by the menopause.Major Inclusion/Exclusion CriteriaInclusion criteria: pre-menopausal; aged >30 years; at increased risk of tubo-ovarian cancer (mutation carriers or on the basis of a strong family history); completed their family (for surgical arms). Exclusion criteria: post-menopausal; previous bilateral salpingectomy or bilateral oophorectomy; pregnancy; previous tubal/ovarian/peritoneal malignancy; Primary EndpointSexual function measured by validated questionnaires.Sample Size1000 (333 per arm).Estimated Dates for Completing Accrual and Presenting ResultsIt is estimated recruitment will be completed by 2023 and results published by 2027.Trial Registration NumberISRCTN registry: 25 173 360 (https://doi.org/10.1186/ISRCTN25173360).

Ovarian carcinosarcoma (OCS) is an aggressive and rare tumour type with limited treatment options. OCS is hypothesised to develop via the combination theory from 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). We show OCS from 18 women to be monoclonal through analysis of DNA variants from isolated carcinoma and sarcoma components. RNA sequencing indicated the carcinoma components were more mesenchymal when compared with pure ovarian carcinomas, supporting the conversion theory. We used pre-clinical OCS models to test the efficacy of microtubule-targeting drugs, including eribulin, which has been shown to reverse EMT characteristics. We demonstrated that microtubule inhibitors, vinorelbine and eribulin, were more effective than standard-of-care platinum-based chemotherapy. Eribulin reduced mesenchymal characteristics, N-MYC expression and cholesterol biosynthesis. Finally, eribulin induced a strong immune response, supporting immunotherapy combinations in the clinic.

Pathology departments must rise to new staffing challenges caused by the coronavirus disease-19 pandemic and may need to work more flexibly for the foreseeable future. In light of this, many pathologists and departments are considering the merits of remote or home reporting of digital cases. While some individuals have experience of this, little work has been done to determine optimum conditions for home reporting, including technical and training considerations. In this publication produced in response to the pandemic, we provide information regarding risk assessment of home reporting of digital slides, summarize available information on specifications for home reporting computing equipment, and share access to a novel point-of-use quality assurance tool for assessing the suitability of home reporting screens for digital slide diagnosis. We hope this study provides a useful starting point and some practical guidance in a difficult time. This study forms the basis of the guidance issued by the Royal College of Pathologists, available at: https://www.rcpath.org/uploads/assets/626ead77-d7dd-42e1-949988e43dc84c97/RCPath-guidance-for-remote-digital-pathology.pdf.

Introduction/Background Aims - To evaluate impact on sexual function, endocrine function, quality-of-life (QoL), health and well-being and determine cost-effectiveness of risk-reducing early-salpingectomy and delayed oophorectomy (RRESDO), as a two-step ovarian cancer (OC) prevention strategy in pre-menopausal women at increased risk of OC. Methodology Design - Multi-centre, observational cohort controlled trial with three arms: RRESDO; risk-reducing salpingo-oophorectomy (RRSO); controls (no surgery). Inclusion-criteria: Pre-menopausal women; >30-years; at increased risk of OC (mutation carriers or on basis of a strong family-history; completed their family (for surgical arms). Exclusion-criteria: Post-menopausal women; previous bilateral salpingectomy or bilateral oophorectomy; pregnancy; previous tubal/ovarian/peritoneal malignancy; Recruitment: Through NHS cancer genetics/high-risk familial cancer clinics/general gynaecology clinics/gynaecological oncology clinics/GP surgeries/clinical referrals/supporting charities/self-referral. Primary-outcome: Sexual function. Secondary-outcomes: Endocrine function/menopause; regret/satisfaction; surgical morbidity; QoL/psychological health; number of intraepithelial carcinomas/invasive cancers; utility scores for early-salpingectomy; cost-effectiveness; health and well-being. Consenting individuals for the surgical arms undergo an ultrasound, CA125, FSH and complete questionnaires collecting information on medical history, family-history, QoL, sexual function, cancer worry, psychological well-being and satisfaction/regret. All women undergoing surgery have cytological and histological assessment using a SEE-FIMM protocol, with centralised pathology review. Post-surgery FSH levels are tested and follow-up questionnaires sent at 3 months and annually. Controls have FSH levels checked and complete questionnaires at baseline and annually for three years. Participants from all three arms are invited to take part in semi-structured in-depth interviews. Results PROTECTOR is open to recruitment across several centres throughout the UK. Conclusion With growing evidence implicating the role of the fallopian tubes in ovarian carcinogenesis and the detrimental sequelae of surgical menopause in pre-menopausal women following RRSO, PROTECTOR is essential to investigate RRESDO as an alternative risk-reducing strategy in women who do not wish to undergo oophorectomy. Disclosure RM declares research funding from Barts and The London Charity and Roseetrees Trust for the PROTECTOR Study and is Chief Investigator. RM declares research funding from The Eve Appeal and Cancer Research UK outside this work, as well as an honorarium for grant review from Israel National Institute for Health Policy Research. RM is supported by an NHS Innovation Accelerator (NIA) Fellowship for population testing. FG is an investigator and the study coordinator for the PROTECTOR study. UM has a financial interest in Abcodia Ltd., a company formed to develop academic and commercial development of biomarkers for screening and risk prediction. NS, GM, NW, RG, GR, GB are members of the PROTECTOR central pathology review committee. GE, ES, HH, UM are members of the PROTECTOR trial management committee. MB, RL have nothing to declare.

Tumours with profound copy-number aberration elude molecular stratification due to their genomic complexity. By representing this complexity as a mixture of copy-number signatures, we provide molecular explanations for differing clinical outcomes. Here we present a method for copy-number signature identification, deriving eight signatures in 117 shallow whole-genome sequenced high-grade serous ovarian cancers (HGSOC), which validated on independent cohorts of 95 deep whole-genome sequenced, and 402 SNP array-profiled cases. Three copy-number signatures predicted longer overall survival, while the others predicted poorer outcome. We found evidence for the mutational processes giving rise to copy-number change for six of the eight signatures via correlations with other genomic features. Our results provide insights into the pathogenesis of HGSOC by uncovering multiple mutational processes that shape genomes following TP53 mutation. Importantly, our work shows that most HGSOC have a mixture of mutational processes suggesting that targeting a single mutator phenotype may be therapeutically suboptimal.

There have been occasional reports of primary cervical adenocarcinoma with areas of dedifferentiation resulting in morphologic mimicry of breast lobular carcinoma. We describe 4 cases of primary cervical squamous carcinoma with prominent acantholysis (3 cases), areas resembling breast lobular carcinoma (3 cases) or both (2 cases). All 4 tumors showed positivity with p63 and CK5/6 and 3 of 4 exhibited block-type immunoreactivity with p16. Two of the 4 cases contained high-risk human papillomavirus (types 16 and 18) on molecular testing; of the 2 cases which were human papillomavirus negative, 1 exhibited patchy nonblock immunoreactivity with p16. All cases exhibited some degree of loss of E-cadherin membranous staining in the areas of acantholysis and foci resembling breast lobular carcinoma. Three of 4 patients had extracervical spread at diagnosis; the fourth patient developed extracervical recurrence on follow-up. The initial FIGO stages were IB1, IIB (2 cases) and IVB. The 2 patients whose neoplasms were human papillomavirus negative developed distant metastases (supraclavicular, meningeal, and lung) during the course of their disease; the same 2 patients died of disease at periods of 4 mo and 1 yr after diagnosis. Cervical squamous carcinomas with acantholytic features and areas resembling breast lobular carcinoma are an unusual morphologic variant of squamous carcinoma. We consider the acantholysis and mimicry of breast lobular carcinoma to be part of a spectrum of morphologic changes, possibly related to loss of E-cadherin. These features can be regarded as a form of dedifferentiation which indicates a potential for aggressive behavior.

BACKGROUND: The genomic complexity of profound copy-number aberration has prevented effective molecular stratification of high grade serous ovarian carcinoma (HGSOC). Recent algorithmic advances have enabled interpretation of complex genomic changes by identifying mutational signatures—genomic patterns that are the imprint of mutagenic processes accumulated over the lifetime of a cancer cell. We hypothesized that specific features of copy-number (CN) abnormalities could represent the imprints of distinct mutational processes, and developed methods to identify signatures from copy-number features in HGSOC. METHODS: We derived copy-number signatures from absolute copy number profiles from 253 primary and relapsed HGSOC samples from 132 patients in the BriTROC-1 cohort using low-cost shallow whole-genome sequencing (sWGS; 0.1×). A subset of 56 of these cases had deep whole-genome sequencing (dWGS) performed for mutation analysis and comparison with sWGS data. Independent validation was performed using 112 dWGS HGSOC cases from PCAWG and 415 HGSOC cases with SNP array and whole exome sequence from TCGA. CN signature exposures were correlated with mutation data, SNV signatures, and other measures derived from deep WGS and exome sequencing to identify statistically significant genomic associations using a false discovery rate RESULTS: We identified 7 CN signatures that provided a molecular framework to rederive the major defining elements of HGSOC genomes, including defective homologous recombination (HRD), tandem duplication, amplification of CCNE1 and amplification-associated fold-back inversions. Almost all patients with HGSOC demonstrated a mixture of signatures indicative of combinations of mutational processes, including those with early driver events such as BRCA2 mutation (in addition to HRD signatures). High exposure to CN signature 3, characterised by BRCA1/2-related HRD, was associated with improved overall survival. Conversely, high exposure to signature 1, which was characterised by oncogenic RAS signaling (including NF1, KRAS and NRAS mutation), predicted platinum-resistant relapse and poor survival. CONCLUSIONS: HGSOC lacks clinically-relevant patient stratification, which is reflected in poor survival and is a significant barrier to precision medicine. Copy-number signature exposures at diagnosis predict both overall survival and the probability of platinum-resistant relapse. Our results suggest that early TP53 mutation, the ubiquitous initiating event in HGSOC, may permit multiple mutational processes to co-evolve, potentially simultaneously and that additional signature exposures may alter the risk of developing therapeutic resistance. Thus, our results suggest that HGSOC is a continuum of genomes. We derived signatures using inexpensive sWGS of DNA from core biopsies. These approaches are rapid and cost effective, thus providing a clear path to clinical implementation. By dissecting the mutational forces shaping HGSOC genomes, our study paves the way to understanding extreme genomic complexity, as well as revealing the evolution of tumors as they relapse and acquire resistance to therapy. Citation Format: Geoff Macintyre, Teodora E. Goranova, Dilrini De Silva, Darren Ennis, Anna M. Piskorz, Matthew Eldridge, Daoud Sie, Liz-Anne Lewsley, Aishah Hanif, Cheryl Wilson, Suzanne Dowson, Rosalind M. Glasspool, Michelle Lockley, Elly Brockbank, Ana Montes, Axel Walther, Sudha Sundar, Richard Edmondson, Geoff D. Hall, Andrew Clamp, Charlie Gourley, Marcia Hall, Christina Fotopoulou, Hani Gabra, James Paul, Anna Supernat, David Millan, Aoisha Hoyle, Gareth Bryson, Craig Nourse, Laura Mincarelli, Luis Navarro Sanchez, Bauke Ylstra, Mercedes Jimenez-Linan, Luiza Moore, Oliver Hofmann, Florian Markowetz, Iain A. McNeish, James D. Brenton. COPY-NUMBER SIGNATURES AND MUTATIONAL PROCESSES IN HIGH GRADE SEROUS OVARIAN CARCINOMA [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP09.

Multifocal squamous cervical carcinomas account for up to 25% of IA1 tumors identified on excisional biopsy, yet there are no uniformly accepted histopathologic criteria for defining and staging these lesions. Here, we use a strict case definition and meticulous specimen processing from colposcopist to pathologist to identify and follow-up 25 cases of multifocal IA1 cervical squamous carcinomas identified in excisional biopsies. We stage these tumors using the dimensions of the largest focus and a minimum of 2 mm between each foci to define multifocality. The cases are followed up for a median of 7 yr with no episodes of tumor recurrence or metastasis. We also show that the prevalence of residual preinvasive (20%) and invasive disease (5%) on repeat excision/surgery are comparable to data available for unifocal IA1 cases. Our study supports the hypothesis that multifocal lesions should be staged according to largest individual focus of invasion and we emphasize the importance of meticulous specimen handling to appropriately identify multifocal tumors. In addition, our analysis suggests that outcomes are comparable to unifocal lesions and supports the hypothesis that they may be managed in a similar manner.

A previous audit conducted in the West of Scotland (WoS) suggested that anatomical factors accounted for a substantial proportion of invaded surgical margins after resection of an oral or oropharyngeal squamous cell carcinoma (SCC). Since then a number of technical improvements have taken place, the most important of which has been advanced digital imaging that has enabled better surgical planning. In this study we compare the incidence of involved surgical margins in a recent group with those found in the earlier audit. The earlier (WoS) group comprised a consecutive series of patient operated on for a primary SCC of the oral cavity or oropharynx between November 1999 and November 2001 (n=296). The later series comprised 178 patients operated on for oral or oropharyngeal SCC at the Southern General Hospital (SGH), Glasgow, between 2006 and 2009. A total of 245 patients in the WoS cohort had information available on the invasion of the margins of whom 68 (28%) had an invaded margin. Of 177 patients in the SGH group, 9 (5%) had an invaded margin (p=0.001). An anatomical approach to the resection of oral and oropharyngeal SCC is appropriate, as it results in a rate of invaded margins of less than 10% irrespective of size and site of the primary lesion.

Erosive injury of the upper gastrointestinal tract resulting from therapeutic oral iron supplements is an uncommon phenomenon. We present a case of a large gastric ulcer with clinical, endoscopic and radiological features suggestive of malignancy, which resolved completely on cessation of iron therapy.

The p38 mitogen-activated protein kinase (MAPK) system is increasingly recognized as an important inflammatory pathway in systemic vascular disease but its role in pulmonary vascular disease is unclear. Previous in vitro studies suggest p38 MAPKα is critical in the proliferation of pulmonary artery fibroblasts, an important step in the pathogenesis of pulmonary vascular remodeling (PVremod). In this study the role of the p38 MAPK pathway was investigated in both in vitro and in vivo models of pulmonary hypertension and human disease. Pharmacological inhibition of p38 MAPKα in both chronic hypoxic and monocrotaline rodent models of pulmonary hypertension prevented and reversed the pulmonary hypertensive phenotype. Furthermore, with the use of a novel and clinically available p38 MAPKα antagonist, reversal of pulmonary hypertension was obtained in both experimental models. Increased expression of phosphorylated p38 MAPK and p38 MAPKα was observed in the pulmonary vasculature from patients with idiopathic pulmonary arterial hypertension, suggesting a role for activation of this pathway in the PVremod A reduction of IL-6 levels in serum and lung tissue was found in the drug-treated animals, suggesting a potential mechanism for this reversal in PVremod. This study suggests that the p38 MAPK and the α-isoform plays a pathogenic role in both human disease and rodent models of pulmonary hypertension potentially mediated through IL-6. Selective inhibition of this pathway may provide a novel therapeutic approach that targets both remodeling and inflammatory pathways in pulmonary vascular disease.