Interfaces of Malignant and Immunologic Clonal Dynamics in Ovarian Cancer.

High-grade serous ovarian cancer (HGSC) exhibits extensive malignant clonal diversity with widespread but non-random patterns of disease dissemination. We investigated whether local immune microenvironment factors shape tumor progression properties at the interface of tumor-infiltrating lymphocytes (TILs) and cancer cells. Through multi-region study of 212 samples from 38 patients with whole-genome sequencing, immunohistochemistry, histologic image analysis, gene expression profiling, and T and B cell receptor sequencing, we identified three immunologic subtypes across samples and extensive within-patient diversity. Epithelial CD8+ TILs negatively associated with malignant diversity, reflecting immunological pruning of tumor clones inferred by neoantigen depletion, HLA I loss of heterozygosity, and spatial tracking between T cell and tumor clones. In addition, combinatorial prognostic effects of mutational processes and immune properties were observed, illuminating how specific genomic aberration types associate with immune response and impact survival. We conclude that within-patient spatial immune microenvironment variation shapes intraperitoneal malignant spread, provoking new evolutionary perspectives on HGSC clonal dispersion.

Single-cell genomic variation induced by mutational processes in cancer.

How cell-to-cell copy number alterations that underpin genomic instability1 in human cancers drive genomic and phenotypic variation, and consequently the evolution of cancer2, remains understudied. Here, by applying scaled single-cell whole-genome sequencing3 to wild-type, TP53-deficient and TP53-deficient;BRCA1-deficient or TP53-deficient;BRCA2-deficient mammary epithelial cells (13,818 genomes), and to primary triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSC) cells (22,057 genomes), we identify three distinct 'foreground' mutational patterns that are defined by cell-to-cell structural variation. Cell- and clone-specific high-level amplifications, parallel haplotype-specific copy number alterations and copy number segment length variation (serrate structural variations) had measurable phenotypic and evolutionary consequences. In TNBC and HGSC, clone-specific high-level amplifications in known oncogenes were highly prevalent in tumours bearing fold-back inversions, relative to tumours with homologous recombination deficiency, and were associated with increased clone-to-clone phenotypic variation. Parallel haplotype-specific alterations were also commonly observed, leading to phylogenetic evolutionary diversity and clone-specific mono-allelic expression. Serrate variants were increased in tumours with fold-back inversions and were highly correlated with increased genomic diversity of cellular populations. Together, our findings show that cell-to-cell structural variation contributes to the origins of phenotypic and evolutionary diversity in TNBC and HGSC, and provide insight into the genomic and mutational states of individual cancer cells.

The unique metabolome of clear cell ovarian carcinoma.

Clear cell ovarian carcinoma (CCOC) is an aggressive malignancy affecting younger women. Despite ovarian cancer subtypes having diverse molecular and clinical characteristics, the mainstay of treatment for advanced stage disease remains cytotoxic chemotherapy. Late stage CCOC is resistant to conventional chemotherapy, which means a suboptimal outcome for patients affected. Despite detailed genomic, epigenomic, transcriptomic, and proteomic characterisation, subtype-specific treatment for CCOC has shown little progress. The unique glycogen accumulation defining CCOC suggests altered metabolic pathway activity and dependency. This study presents the first metabolomic landscape of ovarian cancer subtypes, including 42 CCOC, 20 high-grade serous and 21 endometrioid ovarian carcinomas, together comprising the three most common ovarian carcinoma subtypes. We describe a distinct metabolomic landscape of CCOC compared with other ovarian cancer subtypes, including alterations in energy utilisation and cysteine metabolism. In addition, we identify CCOC-specific alterations in metabolic pathways including serine biosynthesis and ROS-associated pathways that could serve as potential therapeutic targets. Our study provides the first in-depth study into the metabolome of ovarian cancers and a rich resource to support ongoing research efforts to identify subtype-specific therapeutic targets that could improve the dismal outcome for patients with this devastating malignancy. © 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.

Changes in the tumour microenvironment mark the transition from serous borderline tumour to low-grade serous carcinoma.

Low-grade serous ovarian carcinoma (LGSC) is a rare and lethal subtype of ovarian cancer. LGSC is pathologically, biologically, and clinically distinct from the more common high-grade serous ovarian carcinoma (HGSC). LGSC arises from serous borderline ovarian tumours (SBTs). The mechanism of transformation for SBTs to LGSC remains poorly understood. To better understand the biology of LGSC, we performed whole proteome profiling of formalin-fixed, paraffin-embedded tissue blocks of LGSC (n = 11), HGSC (n = 19), and SBTs (n = 26). We identified that the whole proteome is able to distinguish between histotypes of the ovarian epithelial tumours. Proteins associated with the tumour microenvironment were differentially expressed between LGSC and SBTs. Fibroblast activation protein (FAP), a protein expressed in cancer-associated fibroblasts, is the most differentially abundant protein in LGSC compared with SBT. Multiplex immunohistochemistry (IHC) for immune markers (CD20, CD79a, CD3, CD8, and CD68) was performed to determine the presence of B cells, T cells, and macrophages. The LGSC FAP+ stroma was associated with greater abundance of Tregs and M2 macrophages, features not present in SBTs. Our proteomics cohort reveals that there are changes in the tumour microenvironment in LGSC compared with its putative precursor lesion, SBT. These changes suggest that the tumour microenvironment provides a supportive environment for LGSC tumourigenesis and progression. Thus, targeting the tumour microenvironment of LGSC may be a viable therapeutic strategy. © 2024 The Pathological Society of Great Britain and Ireland.

ARID1A regulates R-loop associated DNA replication stress.

ARID1A is a core DNA-binding subunit of the BAF chromatin remodeling complex, and is lost in up to 7% of all cancers. The frequency of ARID1A loss increases in certain cancer types, such as clear cell ovarian carcinoma where ARID1A protein is lost in about 50% of cases. While the impact of ARID1A loss on the function of the BAF chromatin remodeling complexes is likely to drive oncogenic gene expression programs in specific contexts, ARID1A also binds genome stability regulators such as ATR and TOP2. Here we show that ARID1A loss leads to DNA replication stress associated with R-loops and transcription-replication conflicts in human cells. These effects correlate with altered transcription and replication dynamics in ARID1A knockout cells and to reduced TOP2A binding at R-loop sites. Together this work extends mechanisms of replication stress in ARID1A deficient cells with implications for targeting ARID1A deficient cancers.

p53-Abnormal "Fields of Dysplasia" in Human Papillomavirus-Independent Vulvar Squamous Cell Carcinoma Impacts Margins and Recurrence Risk.

Abnormal p53 (p53abn) immunohistochemical (IHC) staining patterns can be found in vulvar squamous cell carcinoma (VSCC) and differentiated vulvar intraepithelial neoplasia (dVIN). They can also be found in the adjacent skin that shows morphology that falls short of the traditional diagnostic threshold for dVIN. Vulvectomy specimens containing human papillomavirus-independent p53abn VSCC with margins originally reported as negative for invasive and in situ disease were identified. Sections showing the closest approach by invasive or in situ neoplasia to margins were stained with p53 IHC stains. We evaluated the following: (1) detection of morphologically occult p53abn in situ neoplasia, (2) rates of margin status change after p53 IHC staining, and (3) effect of p53abn IHC staining at margins on the 2-year local recurrence rates. Seventy-three human papillomavirus-independent p53abn VSCCs were included. Half (35/73, 48%) had documented an in situ lesion in the original report. The use of p53 IHC staining identified 21 additional cases (29%) with the p53abn in situ lesions that were originally unrecognized. The histology of in situ lesions in the p53abn "field" varied and became more subtle (morphologically occult) farther away from the VSCC. Fifteen (21%) cases had a morphologically occult and previously unrecognized p53abn in situ lesion present at a resection margin, which conferred an increased risk of local recurrence (5/7 [71.4%] vs 6/22 [27.3%], P = .036). The p53abn in situ lesions at a margin were confirmed to have TP53 mutations by sequencing. p53 IHC staining identified morphologically occult p53abn in situ lesions surrounding human papillomavirus-independent VSCC. p53abn IHC staining at a margin was associated with a 3-fold increased risk of local recurrence.

Grade and Estrogen Receptor Expression Identify a Subset of No Specific Molecular Profile Endometrial Carcinomas at a Very Low Risk of Disease-Specific Death.

Endometrial carcinoma (EC) can be divided into 4 prognostic molecular subtypes, and no specific molecular profile (NSMP) type is the most commonly occurring type (∼50%). Although described as having an intermediate to favorable prognosis, this subtype encompasses pathologically and molecularly diverse tumors. We aimed to identify factors associated with outcomes within the NSMP ECs that might be used to stratify prognosis and direct treatment. Clinicopathologic, immunohistochemical, and genetic features of a large series of NSMP EC were used to identify parameters that could identify the subset associated with a very favorable outcome (disease-specific death rate <5% at 5 years, termed low-risk NSMP). A total of 1110 NSMP ECs were profiled. In a univariate analysis, stage, grade, lymphovascular invasion, estrogen receptor (ER) and progesterone receptor (PR) expression, L1CAM overexpression, and mutations in PIK3CA were associated with disease-specific survival. Two critical features, grade and ER expression, identified a low-risk NSMP subset (grade 1-2, ER-positive [>1%], 84% of cases), which showed a 5-year disease-specific death rate of 1.6% across all stages and 1.4% within stage I. The remaining cases (high-risk NSMPs, grade 3, and/or ER-negative status) were responsible for most of the disease-specific deaths (disease-specific death rate at 5 years, 22.9%; hazard ratio compared with that of low-risk NSMPs: 16.3; 95% CI, 8.4-31.7). Within NSMP EC, the low-risk and high-risk categories were of prognostic significance independent of the stage on a multivariate analysis. Low-grade and ER-positive NSMP ECs are a homogeneous low-risk group associated with an exceptionally favorable prognosis in which de-escalation and/or endocrine therapy strategies can be applied. Grade 3 and/or ER-negative status identifies a high-risk NSMP subset, including rare high-grade histotypes (eg, clear cell, dedifferentiated, and mesonephric-like), responsible for most NSMP-related deaths. Subclassification of NSMPs allows for the category of low-risk EC molecular subtypes to be dramatically expanded because it now includes both POLEmut and the much more common low-risk NSMP EC.

Harmonized molecular classification; assessment of a single-test ProMisE NGS tool.

OBJECTIVES: Despite recommendations for integrating molecular classification of endometrial cancers (EC) into pathology reporting and clinical management, uptake is inconsistent. To assign ProMisE subtype, all molecular components must be available (POLE mutation status, mismatch repair (MMR) and p53 immunohistochemistry (IHC)) and often these are assessed at different stages of care and/or at different centres resulting in delays in treatment. We assessed a single-test DNA-based targeted next generation sequencing (NGS) molecular classifier (ProMisE NGS), comparing concordance and prognostic value to the original ProMisE classifier. METHODS: DNA was extracted from formalin-fixed paraffin embedded (FFPE) ECs that had previously undergone ProMisE molecular classification (POLE sequencing, IHC for p53 and MMR). DNA was sequenced using the clinically validated Imagia Canexia Health Find It™ amplicon-based NGS gene panel assay to assess for pathogenic POLE mutations (unchanged from original ProMisE), TP53 mutations (in lieu of p53 IHC), and microsatellite instability (MSI) (in lieu of MMR IHC),with the same order of segregation as original ProMisE used for subtype assignment. Molecular subtype assignment of both classifiers was compared by concordance metrics and Kaplan-Meier survival statistics. RESULTS: The new DNA-based NGS molecular classifier (ProMisE NGS) was used to determine the molecular subtype in 164 ECs previously classified with ProMisE. 159/164 cases were concordant with a kappa statistic of 0.96 and an overall accuracy of 0.97. Prognostic differences in progression-free, disease-specific and overall survival between the four molecular subtypes were observed for the new NGS classifier, recapitulating the survival curves of the original ProMisE classifier. ProMisE NGS was 100% concordant between matched biopsy and hysterectomy samples. CONCLUSION: ProMisE NGS is feasible on standard FFPE material, demonstrates high concordance with the original ProMisE classifier and maintains prognostic value in EC. This test has the potential to facilitate implementation of molecular classification of EC at the time of first diagnosis.

Molecular subtype stratified outcomes according to adjuvant therapy in endometrial cancer.

OBJECTIVES: Recent data support the predictive implications of molecular subtype assignment in endometrial cancer (EC). Our objective was to retrospectively assess clinical outcomes according to adjuvant treatment received within EC molecular subtypes. METHODS: Clinical outcomes (disease-specific and progression-free survival DSS/PFS) of EC patients from a single institution and population-based cohorts that had undergone molecular classification were assessed with respect to adjuvant therapy received and 2016 ESMO risk group. RESULTS: 2472 ECs were assessed; 184 (7.4%) POLEmut, 638 (25.8%) MMRd, 1223 (49.5%) NSMP and 427 (17.3%) p53abn. N = 774 (34.6%) of the cohort were ESMO 2016 high risk and 109 (4.8%) were advanced or metastatic. In patients with MMRd EC, assessed across and within stage, there was no observed benefit in DSS or PFS with the addition of chemotherapy +/- radiation compared to radiation alone in ESMO high risk (p = 0.694) or ESMO high, advanced, metastatic risk groups combined (p = 0.852). In patients with p53abn EC, adjuvant chemotherapy given with radiation was associated with significantly longer DSS compared to radiation alone in ESMO high risk (p = 0.007) and ESMO high, advanced and metastatic risk groups combined (p = 0.015), even when restricted to stage I disease (p < 0.001) and when compared in serous vs. non-serous histotypes (p = 0.009). CONCLUSIONS: Adjuvant chemotherapy is associated with more favorable outcomes for patients with p53abn EC, including stage I disease and non-serous histotypes, but does not appear to add benefit within MMRd ECs for any stage of disease, consistent with PORTEC-3 molecular subanalysis. Prospective trials, assessing treatment efficacy within molecular subtype are needed, however these 'real-world' data should be considered when discussing adjuvant treatment with patients.

Profiling the immune landscape in mucinous ovarian carcinoma.

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.

The utility of color normalization for AI-based diagnosis of hematoxylin and eosin-stained pathology images.

The color variation of hematoxylin and eosin (H&E)-stained tissues has presented a challenge for applications of artificial intelligence (AI) in digital pathology. Many color normalization algorithms have been developed in recent years in order to reduce the color variation between H&E images. However, previous efforts in benchmarking these algorithms have produced conflicting results and none have sufficiently assessed the efficacy of the various color normalization methods for improving diagnostic performance of AI systems. In this study, we systematically investigated eight color normalization algorithms for AI-based classification of H&E-stained histopathology slides, in the context of using images both from one center and from multiple centers. Our results show that color normalization does not consistently improve classification performance when both training and testing data are from a single center. However, using four multi-center datasets of two cancer types (ovarian and pleural) and objective functions, we show that color normalization can significantly improve the classification accuracy of images from external datasets (ovarian cancer: 0.25 AUC increase, p = 1.6 e-05; pleural cancer: 0.21 AUC increase, p = 1.4 e-10). Furthermore, we introduce a novel augmentation strategy by mixing color-normalized images using three easily accessible algorithms that consistently improves the diagnosis of test images from external centers, even when the individual normalization methods had varied results. We anticipate our study to be a starting point for reliable use of color normalization to improve AI-based, digital pathology-empowered diagnosis of cancers sourced from multiple centers. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The proteome of clear cell ovarian carcinoma.

Clear cell ovarian carcinoma (CCOC) is the second most common subtype of epithelial ovarian carcinoma. Late-stage CCOC is not responsive to gold-standard chemotherapy and results in suboptimal outcomes for patients. In-depth molecular insight is urgently needed to stratify the disease and drive therapeutic development. We conducted global proteomics for 192 cases of CCOC and compared these with other epithelial ovarian carcinoma subtypes. Our results showed distinct proteomic differences in CCOC compared with other epithelial ovarian cancer subtypes including alterations in lipid and purine metabolism pathways. Furthermore, we report potential clinically significant proteomic subgroups within CCOC, suggesting the biologic plausibility of stratified treatment for this cancer. Taken together, our results provide a comprehensive understanding of the CCOC proteomic landscape to facilitate future understanding and research of this disease. © 2022 The Pathological Society of Great Britain and Ireland.

Opportunistic Salpingectomy Between 2017 and 2020: A Descriptive Analysis.

OBJECTIVES: Opportunistic salpingectomy (OS) is the removal of fallopian tubes during another pelvic surgery for the purpose of ovarian cancer prevention. Herein, we describe the rates of OS at the time of hysterectomy and tubal sterilization between 2017 and 2020. METHODS: This study uses the Canadian Institute of Health Information's Discharge Abstract Database and National Ambulatory Care Reporting System for all Canadian provinces and territories except for Quebec between the fiscal years 2017 and 2020. A descriptive analysis on all people aged 15 years and older who had hysterectomies or tubal sterilizations was conducted to determine the proportion of hysterectomies that included bilateral salpingectomy (OS) and the proportion of tubal sterilizations that were OS compared to tubal ligation. RESULTS: There were 174 006 people included in the study. The proportion of hysterectomies that included OS increased from 31.7% in 2017 to 39.9% by 2020. With respect to tubal sterilizations, rates of OS increased from 26.3% of all tubal sterilizations in 2017 to 42.5% in 2020. British Columbia remained the jurisdiction with the highest rates of OS, but rates increased significantly in many jurisdictions, particularly at the time of tubal sterilization. CONCLUSION: The rates of OS have continued to increase in all Canadian jurisdictions following the official Society of Obstetricians and Gynaecologists of Canada recommendation to consider OS in 2015. Assuming that all tubal ligations could have been OS and 75% of hysterectomies with ovarian conservation could have included OS, our data indicate 76 932 missed opportunities for ovarian cancer prevention.

BRCA mutations lead to XIAP overexpression and sensitise ovarian cancer to inhibitor of apoptosis (IAP) family inhibitors.

BACKGROUND: We tested the hypothesis that inhibitor of apoptosis family (IAP) proteins may be altered in BRCA1-mutated ovarian cancers and that could affect the sensitivity to IAP inhibitors. METHODS: The levels of IAP proteins were evaluated in human cancers and cell lines. Cell lines were used to determine the effects of IAP inhibitors. The in vivo effects of treatments were evaluated in PDX mouse models. RESULTS: Expression of X-linked inhibitor of apoptosis (XIAP) is increased in BRCA1-mutated cancers and high levels are associated with improved patient outcomes after platinum chemotherapy. XIAP overexpression is mediated by NF-kB activation and is associated with an optimisation of PARP. BRCA1-mutated cell lines are particularly sensitive to IAP inhibitors due to an inhibitory effect on PARP. Both a BRCA1-mutated cell line with acquired resistance to PARP inhibitors and one with restored BRCA1 remain sensitive to IAP inhibitors. Treatment with IAP inhibitors restores the efficacy of PARP inhibition in these cell lines. The IAP inhibitor LCL161 alone and in combination with a PARP inhibitor, exhibited antitumour effects in PDX mouse models of resistant BRCA2 and 1-mutated ovarian cancer, respectively. CONCLUSION: A clinical trial may be justified to further investigate the utility of IAP inhibitors.

Significance of p53 immunostaining in mesothelial proliferations and correlation with TP53 mutation status.

p53 immunohistochemistry has long been proposed for the separation of benign from malignant mesothelial proliferations, with the older literature suggesting that any degree of positivity supported a diagnosis of mesothelioma. However, using modern immunohistochemistry platforms in other organ systems, notably gynecologic tumors, it has become clear that p53 staining can represent wild-type protein, and only specific staining patterns (absent, overexpression, or cytoplasmic expression) are indicative of a TP53 mutation. We applied these principles to two tissue microarrays containing 94 mesotheliomas and 66 reactive mesothelial proliferations. Seven/65 (11%) epithelioid mesotheliomas showed aberrant staining (four absent and three overexpression patterns) as did 5/29 (17%) of sarcomatoid mesotheliomas (all overexpression patterns). We sequenced the TP53 gene (exons 2-11) in five of the epithelioid and three of the sarcomatoid cases with aberrant staining as well as 12 epithelioid and eight sarcomatoid mesotheliomas with wild-type staining. All three sarcomatoid cases with aberrant staining showed mutated TP53, as did three of the epithelioid cases; in two of the epithelioid cases no mutation was detected, most likely because of large deletions not detected by this assay. In contrast, none of the 20 mesotheliomas with wild-type staining contained mutated TP53. We conclude that absent or overexpression p53 staining patterns can be used as a marker of a malignant vs. a benign mesothelial proliferation. The sensitivity of p53 staining by itself is low, but here addition of p53 to BAP1/MTAP staining increased sensitivity from 72 to 81% for epithelioid and 38 to 50% for sarcomatoid mesotheliomas.

Endometrial carcinoma molecular subtype correlates with the presence of lymph node metastases.

BACKGROUND: The role of lymph node assessment/dissection (LND) in endometrial cancer (EC) has been debated for decades, with significant practice variation between centers. Molecular classification of EC provides prognostic information and can be accurately performed on preoperative endometrial biopsies. We assessed the association between molecular subtype and lymph node metastases (LNM) in order to determine if this tool could be used to stratify surgical decision making. METHODS: All EC patients undergoing primary staging surgery with planned complete pelvic +/- para-aortic LND from a single institution in the 2015 calendar year were identified, with clinicopathological and outcome data assessed in the context of retrospectively assigned molecular classification. RESULTS: 172 patients were included. Molecular classification of the total cohort showed 21 POLEmut (12.2%), 47 MMRd (27.3%), 74 NSMP (43.1%), and 30 p53abn (17.4%) ECs. Complete pelvic +/- para-aortic LND was performed in 171 of 172 patients, and LNM were found in 31/171 (18.1%). This included macrometastases (19/31), micrometastases (5/31), and isolated tumour cells (ITCs) (7/31). LNM were pelvic only in 83.9%, and pelvic plus para-aortic in 16.1%. There were no isolated para-aortic LNM. Molecular subtype was significantly associated with LNM (p = 0.004). There was a strong association between the presence of LNM and p53abn EC (nodal involvement in 44.8% of cases), with LNM detected in 14.2% of POLEmut, 14.9% of MMRd, and 10.8% of NSMP EC. On multivariate analysis, molecular subtype and preoperative CA 125 > 25 were significantly associated with LNM (p = 0.021 and p = 0.022 respectively) but preoperative grade and histotype were not (p = 0.24). CONCLUSION: EC molecular subtype is significantly associated with the presence of LNM. As molecular classification can be obtained on preoperative diagnostic specimens, this information can be used to guide surgical treatment planning and may reduce the cost and morbidity of unnecessary lymph node staging in EC care.

Variation in practice in endometrial cancer and potential for improved care and equity through molecular classification.

OBJECTIVES: We measured the variation in practice across all aspects of endometrial cancer (EC) management and assessed the potential impact of implementation of molecular classification. METHODS: Centers from across Canada provided representative tumor samples and clinical data, including preoperative workup, operative management, hereditary cancer program (HCP) referrals, adjuvant therapy, surveillance and outcomes, for all EC patients diagnosed in 2016. Tumors were classified into the four ProMisE molecular subtypes. RESULTS: A total of 1336 fully evaluable EC patients were identified from 10 tertiary cancer centers (TC; n = 1022) and 19 community centers (CC; n = 314). Variation of surgical practice across TCs was profound (14-100%) for lymphadenectomy (LND) (mean 57% Gr1/2, 82% Gr3) and omental sampling (20% Gr1/2, 79% Gr3). Preoperative CT scans were inconsistently obtained (mean 32% Gr1/2, 51% Gr3) and use of adjuvant chemo or chemoRT in high risk EC ranged from 0-55% and 64-100%, respectively. Molecular subtyping was performed retrospectively and identified 6% POLEmut, 28% MMRd, 48% NSMP and 18% p53abn ECs, and was significantly associated with survival. Within patients retrospectively diagnosed with MMRd EC only 22% had been referred to HCP. Of patients with p53abn EC, LND and omental sampling was not performed in 21% and 23% respectively, and 41% received no chemotherapy. Comparison of management in 2016 with current 2020 ESGO/ESTRO/ESP guidelines identified at least 26 and 95 patients that would have been directed to less or more adjuvant therapy, respectively (10% of cohort). CONCLUSION: Molecular classification has the potential to mitigate the profound variation in practice demonstrated in current EC care, enabling reproducible risk assessment, guiding treatment and reducing health care disparities.

FOXL2 in adult-type granulosa cell tumour of the ovary: oncogene or tumour suppressor gene?

A recurrent mutation in FOXL2 (c.402C>G; p.C134W) is present in over 95% of adult-type granulosa cell tumours (AGCTs). In contrast, various loss-of-function mutations in FOXL2 lead to the development of blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES). BPES is characterised by an eyelid malformation often accompanied with primary ovarian insufficiency. Two recent studies suggest that FOXL2 C402G is a gain- or change-of-function mutation with altered DNA-binding specificity. Another study proposes that FOXL2 C402G is selectively targeted for degradation, inducing somatic haploinsufficiency, suggesting its role as a tumour suppressor. The latter study relies on data indicative of an FOXL2 allelic imbalance in AGCTs. Here we present RNA-seq data as genetic evidence that no real allelic imbalance is observed at the transcriptomic level in AGCTs. Additionally, there is no loss of protein expression in tumours harbouring the mutated allele. These data and other features of this mutation compared to other oncogenes and tumour suppressor genes argue strongly against FOXL2 being a tumour suppressor in this context. Given the likelihood that FOXL2 C402G is oncogenic, targeting the variant protein or its downstream consequences is the most viable path forward to identifying an effective treatment for this cancer. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Genomic analysis of low-grade serous ovarian carcinoma to identify key drivers and therapeutic vulnerabilities.

Low-grade serous ovarian carcinoma (LGSOC) is associated with a poor response to existing chemotherapy, highlighting the need to perform comprehensive genomic analysis and identify new therapeutic vulnerabilities. The data presented here represent the largest genetic study of LGSOCs to date (n = 71), analysing 127 candidate genes derived from whole exome sequencing cohorts to generate mutation and copy-number variation data. Additionally, immunohistochemistry was performed on our LGSOC cohort assessing oestrogen receptor, progesterone receptor, TP53, and CDKN2A status. Targeted sequencing identified 47% of cases with mutations in key RAS/RAF pathway genes (KRAS, BRAF, and NRAS), as well as mutations in putative novel driver genes including USP9X (27%), MACF1 (11%), ARID1A (9%), NF2 (4%), DOT1L (6%), and ASH1L (4%). Immunohistochemistry evaluation revealed frequent oestrogen/progesterone receptor positivity (85%), along with CDKN2A protein loss (10%) and CDKN2A protein overexpression (6%), which were linked to shorter disease outcomes. Indeed, 90% of LGSOC samples harboured at least one potentially actionable alteration, which in 19/71 (27%) cases were predictive of clinical benefit from a standard treatment, either in another cancer's indication or in LGSOC specifically. In addition, we validated ubiquitin-specific protease 9X (USP9X), which is a chromosome X-linked substrate-specific deubiquitinase and tumour suppressor, as a relevant therapeutic target for LGSOC. Our comprehensive genomic study highlighted that there is an addiction to a limited number of unique 'driver' aberrations that could be translated into improved therapeutic paths. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Modelling hereditary diffuse gastric cancer initiation using transgenic mouse-derived gastric organoids and single-cell sequencing.

Hereditary diffuse gastric cancer (HDGC) is a cancer syndrome caused by germline variants in CDH1, the gene encoding the cell-cell adhesion molecule E-cadherin. Loss of E-cadherin in cancer is associated with cellular dedifferentiation and poor prognosis, but the mechanisms through which CDH1 loss initiates HDGC are not known. Using single-cell RNA sequencing, we explored the transcriptional landscape of a murine organoid model of HDGC to characterize the impact of CDH1 loss in early tumourigenesis. Progenitor populations of stratified squamous and simple columnar epithelium, characteristic of the mouse stomach, showed lineage-specific transcriptional programs. Cdh1 inactivation resulted in shifts along the squamous differentiation trajectory associated with aberrant expression of genes central to gastrointestinal epithelial differentiation. Cytokeratin 7 (CK7), encoded by the differentiation-dependent gene Krt7, was a specific marker for early neoplastic lesions in CDH1 carriers. Our findings suggest that deregulation of developmental transcriptional programs may precede malignancy in HDGC. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.