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Hematogenous metastases are rarely present at diagnosis of ovarian clear cell carcinoma (OCC). Instead dissemination of these tumors is characteristically via direct extension of the primary tumor into nearby organs and the spread of exfoliated tumor cells throughout the peritoneum, initially via the peritoneal fluid, and later via ascites that accumulates as a result of disruption of the lymphatic system. The molecular mechanisms orchestrating these processes are uncertain. In particular, the signaling pathways used by malignant cells to survive the stresses of anchorage-free growth in peritoneal fluid and ascites, and to colonize remote sites, are poorly defined. We demonstrate that the transmembrane glycoprotein CUB-domain-containing protein 1 (CDCP1) has important and inhibitable roles in these processes. In vitro assays indicate that CDCP1 mediates formation and survival of OCC spheroids, as well as cell migration and chemoresistance. Disruption of CDCP1 via silencing and antibody-mediated inhibition markedly reduce the ability of TOV21G OCC cells to form intraperitoneal tumors and induce accumulation of ascites in mice. Mechanistically our data suggest that CDCP1 effects are mediated via a novel mechanism of protein kinase B (Akt) activation. Immunohistochemical analysis also suggested that CDCP1 is functionally important in OCC, with its expression elevated in 90% of 198 OCC tumors and increased CDCP1 expression correlating with poor patient disease-free and overall survival. This analysis also showed that CDCP1 is largely restricted to the surface of malignant cells where it is accessible to therapeutic antibodies. Importantly, antibody-mediated blockade of CDCP1 in vivo significantly increased the anti-tumor efficacy of carboplatin, the chemotherapy most commonly used to treat OCC. In summary, our data indicate that CDCP1 is important in the progression of OCC and that targeting pathways mediated by this protein may be useful for the management of OCC, potentially in combination with chemotherapies and agents targeting the Akt pathway.

BackgroundClassification of endometrial carcinomas (ECs) by morphologic features is inconsistent, and yields limited prognostic and predictive information. A new system for classification based on the molecular categories identified in The Cancer Genome Atlas is proposed.MethodsGenomic data from the Cancer Genome Atlas (TCGA) support classification of endometrial carcinomas into four prognostically significant subgroups; we used the TCGA data set to develop surrogate assays that could replicate the TCGA classification, but without the need for the labor-intensive and cost-prohibitive genomic methodology. Combinations of the most relevant assays were carried forward and tested on a new independent cohort of 152 endometrial carcinoma cases, and molecular vs clinical risk group stratification was compared.ResultsReplication of TCGA survival curves was achieved with statistical significance using multiple different molecular classification models (16 total tested). Internal validation supported carrying forward a classifier based on the following components: mismatch repair protein immunohistochemistry, POLE mutational analysis and p53 immunohistochemistry as a surrogate for 'copy-number' status. The proposed molecular classifier was associated with clinical outcomes, as was stage, grade, lymph-vascular space invasion, nodal involvement and adjuvant treatment. In multivariable analysis both molecular classification and clinical risk groups were associated with outcomes, but differed greatly in composition of cases within each category, with half of POLE and mismatch repair loss subgroups residing within the clinically defined 'high-risk' group. Combining the molecular classifier with clinicopathologic features or risk groups provided the highest C-index for discrimination of outcome survival curves.ConclusionsMolecular classification of ECs can be achieved using clinically applicable methods on formalin-fixed paraffin-embedded samples, and provides independent prognostic information beyond established risk factors. This pragmatic molecular classification tool has potential to be used routinely in guiding treatment for individuals with endometrial carcinoma and in stratifying cases in future clinical trials.

Aims: Decreased oestrogen receptor (ER) expression is a marker of poor prognosis in endometrial carcinomas (EC) of no specific molecular profile (NSMP), but the optimal cut-off to separate high-risk 'low ER' versus low-risk 'high ER' expression has not been defined. Here we characterised the distribution of ER staining in a cohort of ECs., Methods and Results: Biopsy specimens from 120 cases of NSMP EC were stained for ER and assigned an Allred score. In 66 additional cases ER staining of matched biopsy and hysterectomy were compared. Twelve of 120 tumours had an Allred score of 0-3, including three endometrioid carcinomas (EEA) (one G1, two G3), four clear cell carcinomas (CCC), two mesonephric-like adenocarcinoma (MLA) and one each of: gastric-type adenocarcinoma, carcinosarcoma and endometrial carcinoma NOS. Three had Allred scores of 4-5: two MLA and one high-grade carcinoma with yolk sac differentiation. Five had Allred scores of 6: four EEA (one G1, one G2, two G3) and one mixed clear cell and endometrioid carcinoma. The remaining 100 tumours with Allred scores ≥ 7 were all EEA (66 G1, 28 G2, five G3 and one grade unknown). Comparing the biopsy versus hysterectomy ER staining (n = 66), the results were within a single Allred score point, except two cases with strong diffuse expression in the biopsy (Allred 8) and moderate expression in the hysterectomy (Allred 5)., Conclusions: Most NSMP ECs (> 80%) show high ER expression (Allred score ≥ 7). All non-endometrioid carcinomas and a few endometrioid carcinomas had lower ER expression (Allred score ≤ 6) or were completely negative., (© 2024 The Author(s). Histopathology published by John Wiley & Sons Ltd.)

The classification of vulvar squamous cell carcinoma (VSCC), as in endometrial cancer, has shifted from the histology-based descriptors toward molecular-based identifiers. Recently, it has been reported that there are 3 genetically distinct and clinically significant subtypes of VSCC: HPV-associated VSCC, HPV-independent/p53 wild-type VSCC, and HPV-independent/p53-mutated VSCC. Each group has different prognostic implications as well as response to treatment, thus reinforcing the need for this 3-tier molecular classification. This molecular subtyping can easily be done on vulvar biopsies using p16 and p53 immunohistochemistry stains to further improve risk prediction and individualized treatment decisions, leading to better patient outcomes., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Summary: Our aim was to assess the molecular subtype(s) and perform a detailed morphologic review of tumors diagnosed as carcinosarcoma in a population-based cohort. Forty-one carcinosarcomas were identified from a cohort of 973 endometrial carcinomas diagnosed in 2016. We assessed immunostaining and sequencing data and undertook expert pathology reviews of these cases as well as all subsequently diagnosed (post-2016) carcinosarcomas of no specific molecular profile (NSMP) molecular subtype (n=3) from our institutions. In the 2016 cohort, 37 of the 41 carcinosarcomas (91.2%) were p53abn, 2 (4.9%) were NSMP, and 1 each (2.4%) were POLE mut and mismatch repair deficiency molecular subtypes, respectively. Of the 4 non-p53abn tumors on review, both NSMP tumors were corded and hyalinized (CHEC) pattern endometrioid carcinoma, the mismatch repair deficiency tumor was a grade 1 endometrioid carcinoma with reactive stromal proliferation, and the POLE mut tumor was grade 3 endometrioid carcinoma with spindle cell growth, that is, none were confirmed to be carcinosarcoma on review. We found 11 additional cases among the 37 p53abn tumors that were not confirmed to be carcinosarcoma on the review (3 undifferentiated or dedifferentiated carcinomas, 5 carcinomas with CHEC features, 2 carcinomas showing prominent reactive spindle cell stroma, and 1 adenosarcoma). In the review of institutional cases reported as NSMP carcinosarcoma after 2016, 3 were identified (1 adenosarcoma and 2 mesonephric-like adenocarcinoma on review). In this series, all confirmed endometrial carcinosarcomas were p53abn. The finding of any other molecular subtype in a carcinosarcoma warrants pathology review to exclude mimics., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 by the International Society of Gynecological Pathologists.)

Endocervical adenocarcinomas (EACs) are a group of malignant neoplasms associated with diverse pathogenesis, morphology, and clinical behavior. As a component of the International Society of Gynecological Pathologists International Endocervical Adenocarcinoma Project, a large international retrospective cohort of EACs was generated in an effort to study potential clinicopathological features with prognostic significance that may guide treatment in these patients. In this study, we endeavored to develop a robust human papillomavirus (HPV)-associated EAC prognostic model for surgically treated International Federation of Gynecology and Obstetrics (FIGO) stage IA2 to IB3 adenocarcinomas incorporating patient age, lymphovascular space invasion (LVSI) status, FIGO stage, and pattern of invasion according to the Silva system (traditionally a 3-tier system). Recently, a 2-tier/binary Silva pattern of invasion system has been proposed whereby adenocarcinomas are classified into low-risk (pattern A/pattern B without LVSI) and high-risk (pattern B with LVSI/pattern C) categories. Our cohort comprised 792 patients with HPV-associated EAC. Multivariate analysis showed that a binary Silva pattern of invasion classification was associated with recurrence-free and disease-specific survival (P < 0.05) whereas FIGO 2018 stage I substages were not. Evaluation of the current 3-tiered system showed that disease-specific survival for those patients with pattern B tumors did not significantly differ from that for those patients with pattern C tumors, in contrast to that for those patients with pattern A tumors. These findings underscore the need for prospective studies to further investigate the prognostic significance of stage I HPV-associated EAC substaging and the inclusion of the binary Silva pattern of invasion classification (which includes LVSI status) as a component of treatment recommendations., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 by the International Society of Gynecological Pathologists.)

Purpose: The purpose of this study was to evaluate RB1 expression and survival across ovarian carcinoma histotypes and how co-occurrence of BRCA1 or BRCA2 (BRCA) alterations and RB1 loss influences survival in tubo-ovarian high-grade serous carcinoma (HGSC)., Experimental Design: RB1 protein expression was classified by immunohistochemistry in ovarian carcinomas of 7,436 patients from the Ovarian Tumor Tissue Analysis consortium. We examined RB1 expression and germline BRCA status in a subset of 1,134 HGSC, and related genotype to overall survival (OS), tumor-infiltrating CD8+ lymphocytes, and transcriptomic subtypes. Using CRISPR-Cas9, we deleted RB1 in HGSC cells with and without BRCA1 alterations to model co-loss with treatment response. We performed whole-genome and transcriptome data analyses on 126 patients with primary HGSC to characterize tumors with concurrent BRCA deficiency and RB1 loss., Results: RB1 loss was associated with longer OS in HGSC but with poorer prognosis in endometrioid ovarian carcinoma. Patients with HGSC harboring both RB1 loss and pathogenic germline BRCA variants had superior OS compared with patients with either alteration alone, and their median OS was three times longer than those without pathogenic BRCA variants and retained RB1 expression (9.3 vs. 3.1 years). Enhanced sensitivity to cisplatin and paclitaxel was seen in BRCA1-altered cells with RB1 knockout. Combined RB1 loss and BRCA deficiency correlated with transcriptional markers of enhanced IFN response, cell-cycle deregulation, and reduced epithelial-mesenchymal transition. CD8+ lymphocytes were most prevalent in BRCA-deficient HGSC with co-loss of RB1., Conclusions: Co-occurrence of RB1 loss and BRCA deficiency was associated with exceptionally long survival in patients with HGSC, potentially due to better treatment response and immune stimulation., (©2024 The Authors; Published by the American Association for Cancer Research.)

Investigation of histopathology slides by pathologists is an indispensable component of the routine diagnosis of cancer. Artificial intelligence (AI) has the potential to enhance diagnostic accuracy, improve efficiency, and patient outcomes in clinical pathology. However, variations in tissue preparation, staining protocols, and histopathology slide digitization could result in over-fitting of deep learning models when trained on the data from only one center, thereby underscoring the necessity to generalize deep learning networks for multi-center use. Several techniques, including the use of grayscale images, color normalization techniques, and Adversarial Domain Adaptation (ADA) have been suggested to generalize deep learning algorithms, but there are limitations to their effectiveness and discriminability. Convolutional Neural Networks (CNNs) exhibit higher sensitivity to variations in the amplitude spectrum, whereas humans predominantly rely on phase-related components for object recognition. As such, we propose Adversarial fourIer-based Domain Adaptation (AIDA) which applies the advantages of a Fourier transform in adversarial domain adaptation. We conducted a comprehensive examination of subtype classification tasks in four cancers, incorporating cases from multiple medical centers. Specifically, the datasets included multi-center data for 1113 ovarian cancer cases, 247 pleural cancer cases, 422 bladder cancer cases, and 482 breast cancer cases. Our proposed approach significantly improved performance, achieving superior classification results in the target domain, surpassing the baseline, color augmentation and normalization techniques, and ADA. Furthermore, extensive pathologist reviews suggested that our proposed approach, AIDA, successfully identifies known histotype-specific features. This superior performance highlights AIDA's potential in addressing generalization challenges in deep learning models for multi-center histopathology datasets., (© 2024. The Author(s).)

Vulvovaginal melanoma (VVM) is a rare but deadly disease, accounting for 5% of all vulvar malignancies, with a 5-yr survival rate of only 47% for all stages of the disease. VVM is a distinct subset of melanoma, with a unique genomic profile and underlying pathogenesis unassociated with sun exposure. Distinguishing these rare malignancies from very common pigmented lesions of the vulva and vagina is challenging as histologic features often overlap between entities. PReferentially expressed Antigen in MElanoma (PRAME) is a melanoma-associated protein, and immunohistochemistry (IHC) for PRAME distinguishes cutaneous, oral mucosal, and retinal melanoma from atypical nevi. Given the biological differences between VVM and cutaneous melanoma, the utility of PRAME IHC for the diagnosis of VVM is unknown. We accrued a cohort of 20 VVM and 21 benign vulvar melanocytic nevi. We found that nuclear PRAME IHC staining with 4+ intensity was present in 85% of the VVM and 0% of the nevi. With the assistance of PRAME IHC, we found evidence of close or positive margin involvement in 3 of 10 cases where margins were originally diagnosed as negative for melanoma in situ. Our study is the first to assess PRAME IHC in a cohort of VVM cases and provides confidence for using PRAME IHC to assist with diagnosis and margin assessment in this rare disease., Competing Interests: The authors declare no conflict of interest., (Copyright © 2023 by the International Society of Gynecological Pathologists.)

Lymphovascular space invasion (LVSI) is an important prognostic parameter in endometrial carcinoma (EC) and has gained increasing interest in recent years due to an expanding body of evidence of its independent prognostic value, especially when the presence of LVSI is quantified. A key strength of LVSI as a prognostic factor is that it can be detected on routine microscopic examination, without ancillary tests, and thus can be used in low-resource settings. A weakness, however, is the lack of uniformly applied criteria for assessment and quantification of LVSI, resulting in interobserver variation in diagnosis. This is confounded by artefacts and other morphological features that may mimic LVSI (commonly referred to as pseudo-LVSI). Despite these issues, multiple studies have shown that LVSI is strongly associated with lymph node (LN) metastasis and is an independent risk factor for LN recurrence and distant metastasis. Consequently, the presence of substantial/extensive LVSI has become an important consideration in formulating adjuvant treatment recommendations in patients with EC, and this has been incorporated in the recent International Federation of Gynecology and Obstetrics (FIGO) 2023 staging system. Herein, we review the current literature on LVSI in EC and discuss its role as a prognostic marker, the reproducibility of LVSI assessment and distinction between LVSI and its mimics. We provide illustrations of key diagnostic features and discuss the two-tiered (none/focal versus substantial) system of LVSI classification. This work is intended to provide guidance to practising pathologists and unify the approach towards LVSI assessment in EC., (© 2024 The Author(s). Histopathology published by John Wiley & Sons Ltd.)

Endometrial cancer (EC) has four molecular subtypes with strong prognostic value and therapeutic implications. The most common subtype (NSMP; No Specific Molecular Profile) is assigned after exclusion of the defining features of the other three molecular subtypes and includes patients with heterogeneous clinical outcomes. In this study, we employ artificial intelligence (AI)-powered histopathology image analysis to differentiate between p53abn and NSMP EC subtypes and consequently identify a sub-group of NSMP EC patients that has markedly inferior progression-free and disease-specific survival (termed 'p53abn-like NSMP'), in a discovery cohort of 368 patients and two independent validation cohorts of 290 and 614 from other centers. Shallow whole genome sequencing reveals a higher burden of copy number abnormalities in the 'p53abn-like NSMP' group compared to NSMP, suggesting that this group is biologically distinct compared to other NSMP ECs. Our work demonstrates the power of AI to detect prognostically different and otherwise unrecognizable subsets of EC where conventional and standard molecular or pathologic criteria fall short, refining image-based tumor classification. This study's findings are applicable exclusively to females., (© 2024. The Author(s).)

Purpose: Shallow whole-genome sequencing (sWGS) can detect copy-number (CN) aberrations. In high-grade serous ovarian cancer (HGSOC) sWGS identified CN signatures such as homologous recombination deficiency (HRD) to direct therapy. We applied sWGS with targeted sequencing to p53abn endometrial cancers to identify additional prognostic stratification and therapeutic opportunities., Experimental Design: sWGS and targeted panel sequencing was performed on formalin-fixed, paraffin-embedded p53abn endometrial cancers. CN alterations, mutational data and CN signatures were derived, and associations to clinicopathologic and outcomes data were assessed., Results: In 187 p53abn endometrial cancers, 5 distinct CN signatures were identified. Signature 5 was associated with BRCA1/2 CN loss with features similar to HGSOC HRD signature. Twenty-two percent of potential HRD cases were identified, 35 patients with signature 5, and 8 patients with BRCA1/2 somatic mutations. Signatures 3 and 4 were associated with a high ploidy state, and CCNE1, ERBB2, and MYC amplifications, with mutations in PIK3CA enriched in signature 3. We observed improved overall survival (OS) for patients with signature 2 and worse OS for signatures 1 and 3. Twenty-eight percent of patients had CCNE1 amplification and this subset was enriched with carcinosarcoma histotype. Thirty-four percent of patients, across all histotypes, had ERBB2 amplification and/or HER2 overexpression on IHC, which was associated with worse outcomes. Mutations in PPP2R1A (29%) and FBXW7 (16%) were among the top 5 most common mutations., Conclusions: sWGS and targeted sequencing identified therapeutic opportunities in 75% of patients with p53abn endometrial cancer. Further research is needed to determine the efficacy of treatments targeting these identified pathways within p53abn endometrial cancers., (©2024 The Authors; Published by the American Association for Cancer Research.)

Objective: Vulvar squamous cell carcinoma (VSCC) can be stratified into three molecular subtypes based on the immunoexpression of p16 and p53: HPV-independent p53-abnormal (p53abn) (most common, biologically aggressive), HPV-associated, with p16-overexpression (second most common, prognostically more favourable) and more recently recognised HPV-independent p53-wildtype (p53wt) (rarest subtype, prognostically intermediate). Our aim was to determine whether molecular subtypes can be reliably identified in pre-operative biopsies and whether these correspond to the subsequent vulvectomy specimen., Methods: Matched-paired pre-surgical biopsies and subsequent resection specimen of 57 patients with VSCC were analysed for the immunohistochemical expression of p16 and p53 by performing a three-tiered molecular subtyping to test the accuracy rate., Results: Most cases 36/57 (63.2%) belonged to the HPV-independent (p53-abn) molecular subtype, followed by HPV-associated 17/57 (29.8%) and HPV-independent (p53wt) 4/57 (7.0%). The overall accuracy rate on biopsy was 91.2% (52/57): 97.3% for p53-abnormal, 94.1% for p16-overexpression and 50% for p16-neg/p53-wt VSCC. Incorrect interpretation of immunohistochemical p53 staining pattern was the reason for discordant results in molecular subtyping in all five cases. In one case there was an underestimation of p53 pattern (wildtype instead of abnormal/aberrant) and in one case an overestimation of the p53 staining pattern (abnormal/aberrant instead of wildtype). In 3/5 there was a "double positive" staining result (p16 overexpression and abnormal/aberrant p53 staining pattern). In that cases additional molecular workup is required for correct molecular subtyping, resulting in an overall need for molecular examination of 3/57 (3.5%)., Conclusions: Compared to the final resections specimen, the three-tiered molecular classification of VSCC can be determined on pre-surgical biopsies with a high accuracy rate. This enables more precise surgical planning, prediction of the response to (chemo) radiation, selection of targeted therapies and planning of the optimal follow-up strategy for patients in the age of personalised medicine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

In clinical oncology, many diagnostic tasks rely on the identification of cells in histopathology images. While supervised machine learning techniques necessitate the need for labels, providing manual cell annotations is time-consuming. In this paper, we propose a self-supervised framework (enVironment-aware cOntrastive cell represenTation learning: VOLTA) for cell representation learning in histopathology images using a technique that accounts for the cell's mutual relationship with its environment. We subject our model to extensive experiments on data collected from multiple institutions comprising over 800,000 cells and six cancer types. To showcase the potential of our proposed framework, we apply VOLTA to ovarian and endometrial cancers and demonstrate that our cell representations can be utilized to identify the known histotypes of ovarian cancer and provide insights that link histopathology and molecular subtypes of endometrial cancer. Unlike supervised models, we provide a framework that can empower discoveries without any annotation data, even in situations where sample sizes are limited., (© 2024. The Author(s).)

Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article.

An updated International Federation of Gynecology and Obstetrics (FIGO) staging system for endometrial carcinoma was introduced in June 2023. The new system represents a significant departure from traditional endometrial and other gynecological carcinoma staging systems which are agnostic of parameters such as tumor type, tumor grade, lymphovascular space invasion, and molecular alterations. The updated system, which incorporates all of these 'non-anatomical' parameters, is an attempt to make staging more personalized and relevant to patient prognostication and management, and to align with the European Society of Gynaecological Oncology/European Society for Radiotherapy and Oncology/European Society of Pathology (ESGO/ESTRO/ESP) risk stratification. Herein, we present a critical review of the new staging system and discuss its advantages and disadvantages. The authors propose that the new FIGO staging system should be first appraised at a multi-institutional and global level with the input of all relevant societies (gynecology, pathology, gynecologic oncology, medical oncology, radiation oncology) to understand the impact, scope, and supporting evidence of the proposed changes. Such a process is fundamental to produce a robust system that pathologists and treating clinicians can adopt., Competing Interests: Competing interests: None declared., (© IGCS and ESGO 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Mesonephric-like adenocarcinoma (MLA) of the female genital tract is an uncommon histotype that can arise in both the endometrium and the ovary. The exact cell of origin and histogenesis currently remain unknown. Here, we investigated whole genome DNA methylation patterns and copy number variations (CNVs) in a series of MLAs in the context of a large cohort of various gynaecological carcinoma types. CNV analysis of 19 MLAs uncovered gains of chromosomes 1q (18/19, 95%), 10 (15/19, 79%), 12 (14/19, 74%), and 2 (10/19, 53%), as well as loss of chromosome 1p (7/19, 37%). Gains of chromosomes 1q, 10, and 12 were also identified in the majority of mesonephric adenocarcinomas of the uterine cervix (MAs) as well as subsets of endometrioid carcinomas (ECs) and low-grade serous carcinomas of the ovary (LGSCs) but only in a minority of serous carcinomas of the uterine corpus (USCs), clear cell carcinomas (CCCs), and tubo-ovarian high-grade serous carcinomas (HGSCs). While losses of chromosome 1p together with gains of chromosome 1q were also identified in both MA and LGSC, gains of chromosome 2 were almost exclusively identified in MLA and MA. Unsupervised hierarchical clustering and t-SNE analysis of DNA methylation data (Illumina EPIC array) identified a co-clustering for MLAs and MAs, which was distinct from clusters of ECs, USCs, CCCs, LGSCs, and HGSCs. Group-wise comparisons confirmed a close epigenetic relationship between MLA and MA. These findings, in conjunction with the established histological and immunophenotypical overlap, suggest bona fide mesonephric differentiation, and support a more precise terminology of mesonephric-type adenocarcinoma instead of MLA in these tumours. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Introduction: Targeted-immunotherapies such as antibody-drug conjugates (ADC), chimeric antigen receptor (CAR) T cells or bispecific T-cell engagers (eg, BiTE ® ) all aim to improve cancer treatment by directly targeting cancer cells while sparing healthy tissues. Success of these therapies requires tumor antigens that are abundantly expressed and, ideally, tumor specific. The CD34-related stem cell sialomucin, podocalyxin (PODXL), is a promising target as it is overexpressed on a variety of tumor types and its expression is consistently linked to poor prognosis. However, PODXL is also expressed in healthy tissues including kidney podocytes and endothelia. To circumvent this potential pitfall, we developed an antibody, named PODO447, that selectively targets a tumor-associated glycoform of PODXL. This tumor glycoepitope is expressed by 65% of high-grade serous ovarian carcinoma (HGSOC) tumors., Methods: In this study we characterize these PODO447-expressing tumors as a distinct subset of HGSOC using four different patient cohorts that include pre-chemotherapy, post-neoadjuvant chemotherapy (NACT) and relapsing tumors as well as tumors from various peritoneal locations., Results: We find that the PODO447 epitope expression is similar across tumor locations and negligibly impacted by chemotherapy. Invariably, tumors with high levels of the PODO447 epitope lack infiltrating CD8 + T cells and CD20 + B cells/plasma cells, an immune phenotype consistently associated with poor outcome., Discussion: We conclude that the PODO447 glycoepitope is an excellent biomarker of immune "cold" tumors and a candidate for the development of targeted-therapies for these hard-to-treat cancers., Competing Interests: Authors MH, PD, KM and CR hold patents for the PODXL antibodies described in this manuscript including issued US Patents US11090383B2, US11390673B2. KM and CR hold a patent claiming methods for detecting and treating cancer related to podocalyxin US9309323B2. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Brassard, Hughes, Dean, Hernaez, Thornton, Banville, Smazynski, Warren, Zhang, Milne, Gilks, Mes-Masson, Huntsman, Nelson, Roskelley and McNagny.)

Purpose: The clinical significance of the p53-abnormal (p53abn) molecular subtype in stage I low-grade endometrioid endometrial carcinoma (EEC) is debated. We aimed to review pathologic and molecular characteristics, and outcomes of stage I low-grade p53abn EEC in a large international cohort., Experimental Design: Previously diagnosed stage I p53abn EC (POLE-wild-type, mismatch repair-proficient) low-grade EEC from Canadian retrospective cohorts and PORTEC-1&2 trials were included. Pathology review was performed by six expert gynecologic pathologists blinded to p53 status. IHC profiling, next-generation sequencing, and shallow whole-genome sequencing was performed. Kaplan-Meier method was used for survival analysis., Results: We identified 55 stage I p53abn low-grade EEC among 3,387 cases (2.5%). On pathology review, 17 cases (31%) were not diagnosed as low-grade EEC by any pathologists, whereas 26 cases (47%) were diagnosed as low-grade EEC by at least three pathologists. The IHC and molecular profile of the latter cases were consistent with low-grade EEC morphology (ER/PR positivity, patchy p16 expression, PIK3CA and PTEN mutations) but they also showed features of p53abn EC (TP53 mutations, many copy-number alterations). These cases had a clinically relevant risk of disease recurrence (5-year recurrence-free survival 77%), with pelvic and/or distant recurrences observed in 12% of the patients., Conclusions: A subset of p53abn EC is morphologically low-grade EEC and exhibit genomic instability. Even for stage I disease, p53abn low-grade EEC are at substantial risk of disease recurrence. These findings highlight the clinical relevance of universal p53-testing, even in low-grade EEC, to identify women at increased risk of recurrence., (©2023 The Authors; Published by the American Association for Cancer Research.)

Aims: The significance of subclonal expression of p53 (abrupt transition from wild-type to mutant-pattern staining) is not well understood, and the arbitrary diagnostic cut-off of 10% between NSMP and p53abn molecular subtypes of endometrial carcinoma (EC) has not been critically assessed. Our aim was to characterise subclonal p53 and discrepant p53 expression/TP53 sequencing results in EC and assess their clinical significance., Methods and Results: Subclonal p53 immuostaining on whole sections from 957 ECs was recorded. Agreement between TP53 mutational assessment and p53 immunostaining was evaluated. Subclonal p53 IHC staining was seen in 4.0% (38 of 957) of cases, with 23 of 957 (2.4%) showing mutant-pattern p53 staining in ≥10% of tumour cells. It was most commonly seen in POLEmut (nine of 65, 14%) and MMRd (13 of 274, 4.7%) EC ('multiple classifier' ECs), where subclonal p53 staining does not impact the molecular subtype diagnosis. Excluding POLEmut and MMRd EC, 11 of 957 (1.1%) showed ≥10% subclonal p53 from which four patients died of disease, while there were no deaths due to disease in the five patients with <10% mutant-pattern p53 staining. Agreement between p53 immunostaining and TP53 sequencing was 92.6%; most of the discrepant results were in the ultramutated POLEmut or hypermutated MMRd ECs. In NSMP and p53abn EC the agreement between IHC and sequencing was 95.8%., Conclusions: Subclonal p53 staining ≥10% is present in only 1.1% of EC after excluding 'multiple classifier' ECs. The cut-off of ≥10% subclonal p53 staining identified patients at increased risk of dying from EC, supporting its use to diagnose p53abn molecular subtype., (© 2023 The Authors. Histopathology published by John Wiley & Sons Ltd.)

DICER1 is an RNase III enzyme essential for miRNA biogenesis through cleaving precursor-miRNA hairpins. Germline loss-of-function DICER1 mutations underline the development of DICER1 syndrome, a rare genetic disorder that predisposes children to cancer development in organs such as lung, gynecologic tract, kidney, and brain. Unlike classical tumor suppressors, the somatic "second hit" in DICER1 syndrome-associated cancers does not fully inactivate DICER1 but impairs its RNase IIIb activity only, suggesting a noncanonical two-hit hypothesis. Here, we developed a genetically engineered conditional compound heterozygous Dicer1 mutant mouse strain that fully recapitulates the biallelic DICER1 mutations in DICER1 syndrome-associated human cancers. Crossing this tool strain with tissue-specific Cre strains that activate Dicer1 mutations in gynecologic tract cells at two distinct developmental stages revealed that embryonic biallelic Dicer1 mutations caused infertility in females by disrupting oviduct and endometrium development and ultimately drove cancer development. These multicystic tubal and intrauterine tumors histologically resembled a subset of DICER1 syndrome-associated human cancers. Molecular analysis uncovered accumulation of additional oncogenic events (e.g., aberrant p53 expression, Kras mutation, and Myc activation) in murine Dicer1 mutant tumors and validated miRNA biogenesis defects in 5P miRNA strand production, of which, loss of let-7 family miRNAs was identified as a putative key player in transcriptomic rewiring and tumor development. Thus, this DICER1 syndrome-associated cancer model recapitulates the biology of human cancer and provides a unique tool for future investigation and therapeutic development., Significance: Generation of a Dicer1 mutant mouse model establishes the oncogenicity of missense mutations in the DICER1 RNase IIIb domain and provides a faithful model of DICER1 syndrome-associated cancer for further investigation., (©2023 American Association for Cancer Research.)

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., Competing Interests: Declaration of Competing Interest Dr. McConechy is a former employee and Dr. Huntsman is a founder and previous Chief Medical Officer of Imagia Canexia Health., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Incorporation of molecular classification into clinicopathologic assessment of endometrial carcinoma (EC) improves risk stratification. Four EC molecular subtypes, as identified by The Cancer Genome Atlas, can be diagnosed through a validated algorithm Pro active M olecular R is k Classifier for E ndometrial Cancer (ProMisE) using p53 and mismatch repair (MMR) protein immunohistochemistry (IHC), and DNA polymerase epsilon ( POLE) mutational testing. Cost and access are major barriers to universal testing, particularly POLE analysis. We assessed a selective ProMisE algorithm (ProMisE-S): p53 and MMR IHC on all EC's with POLE testing restricted to those with abnormal MMR or p53 IHC (to identify POLEmut EC with secondary abnormalities in MMR and/or p53) and those with high-grade or non-endometrioid morphology, stage >IA or presence of lymphovascular space invasion (so as to avoid testing on the lowest risk tumors). We retrospectively compared the known ProMisE molecular classification to ProMisE-S in 912 EC. We defined a group of "very low-risk" EC (G1/G2, endometrioid, MMR-proficient, p53 wild-type, stage IA, no lymphovascular space invasion) in whom POLE testing will not impact on patient care; using ProMisE-S, POLE testing would not be required in 55% of biopsies and 38% of all EC's, after evaluation of the hysterectomy specimen, in a population-based cohort. "Very low-risk" endometrioid EC with unknown POLE status showed excellent clinical outcomes. Fifteen of 166 (9%) of all p53abn EC showed G1/G2 endometrioid morphology, supporting the potential value of universal p53 IHC. The addition of molecular testing changed the risk category in 89/896 (10%) EC's. In routine practice, POLE testing could be further restricted to only those patients in whom this would alter adjuvant therapy recommendations., Competing Interests: N.S. has served on advisory boards for Astra-Zeneca-MSD and Glaxo SmithKline. A.T. has served on advisory board for MSD. The remaining authors declare no conflict of interest., (Copyright © 2022 by the International Society of Gynecological Pathologists.)

Purpose: Detection of 11 pathogenic variants in the POLE gene in endometrial cancer (EC) is critically important to identify women with a good prognosis and reduce overtreatment. Currently, POLE status is determined by DNA sequencing, which can be expensive, relatively time-consuming, and unavailable in hospitals without specialized equipment and personnel. This may hamper the implementation of POLE -testing in clinical practice. To overcome this, we developed and validated a rapid, low-cost POLE hotspot test by a quantitative polymerase chain reaction (qPCR) assay, QPOLE ., Materials and Methods: Primer and fluorescence-labeled 5'-nuclease probe sequences of the 11 established pathogenic POLE mutations were designed. Three assays, QPOLE -frequent for the most common mutations and QPOLE -rare-1 and QPOLE-rare-2 for the rare variants, were developed and optimized using DNA extracted from formalin-fixed paraffin-embedded tumor tissues. The simplicity of the design enables POLE status assessment within 4-6 hours after DNA isolation. An interlaboratory external validation study was performed to determine the practical feasibility of this assay., Results: Cutoffs for POLE wild-type, POLE -mutant, equivocal, and failed results were predefined on the basis of a subset of POLE mutants and POLE wild-types for the internal and external validation. For equivocal cases, additional DNA sequencing is recommended. Performance in 282 EC cases, of which 99 were POLE -mutated, demonstrated an overall accuracy of 98.6% (95% CI, 97.2 to 99.9), a sensitivity of 95.2% (95% CI, 90.7 to 99.8), and a specificity of 100%. After DNA sequencing of 8.8% equivocal cases, the final sensitivity and specificity were 96.0% (95% CI, 92.1 to 99.8) and 100%. External validation confirmed feasibility and accuracy., Conclusion: QPOLE is a qPCR assay that is a quick, simple, and reliable alternative for DNA sequencing. QPOLE detects all pathogenic variants in the exonuclease domain of the POLE gene. QPOLE will make low-cost POLE -testing available for all women with EC around the globe.

Our objective was to test whether p53 expression status is associated with survival for women diagnosed with the most common ovarian carcinoma histotypes (high-grade serous carcinoma [HGSC], endometrioid carcinoma [EC], and clear cell carcinoma [CCC]) using a large multi-institutional cohort from the Ovarian Tumor Tissue Analysis (OTTA) consortium. p53 expression was assessed on 6,678 cases represented on tissue microarrays from 25 participating OTTA study sites using a previously validated immunohistochemical (IHC) assay as a surrogate for the presence and functional effect of TP53 mutations. Three abnormal expression patterns (overexpression, complete absence, and cytoplasmic) and the normal (wild type) pattern were recorded. Survival analyses were performed by histotype. The frequency of abnormal p53 expression was 93.4% (4,630/4,957) in HGSC compared to 11.9% (116/973) in EC and 11.5% (86/748) in CCC. In HGSC, there were no differences in overall survival across the abnormal p53 expression patterns. However, in EC and CCC, abnormal p53 expression was associated with an increased risk of death for women diagnosed with EC in multivariate analysis compared to normal p53 as the reference (hazard ratio [HR] = 2.18, 95% confidence interval [CI] 1.36-3.47, p = 0.0011) and with CCC (HR = 1.57, 95% CI 1.11-2.22, p = 0.012). Abnormal p53 was also associated with shorter overall survival in The International Federation of Gynecology and Obstetrics stage I/II EC and CCC. Our study provides further evidence that functional groups of TP53 mutations assessed by abnormal surrogate p53 IHC patterns are not associated with survival in HGSC. In contrast, we validate that abnormal p53 IHC is a strong independent prognostic marker for EC and demonstrate for the first time an independent prognostic association of abnormal p53 IHC with overall survival in patients with CCC., (© 2023 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.)

Sertoli-Leydig cell tumors (SLCTs) are uncommon ovarian sex cord-stromal neoplasms which are currently classified into well, moderately, and poorly differentiated and retiform types. Well-differentiated SLCT is the least common and typically occurs in pure form, whereas moderately and poorly differentiated and retiform types often comprise a morphologic spectrum with an admixture of all 3. DICER1 pathogenic variants are very common in SLCTs but, as far as we are aware, have not been reported in well-differentiated neoplasms, although the number of cases studied is small due to the rarity of this neoplasm. We undertook DICER1 molecular testing in a cohort of 18 well-differentiated SLCTs and show all these to be DICER1 wild-type. None of the cases harbored the p. FOXL2 C134W hotspot mutation. Based upon the DICER1 molecular results, together with morphologic observations, we propose that well-differentiated SLCT is an unrelated neoplasm to the more common moderately/poorly differentiated and retiform SLCTs and is a fundamentally distinct and unrelated tumor type within the ovarian sex cord-stromal tumor family. The implications for tumor nomenclature and recommendations for future tumor classification are discussed within the context of tumors collectively known as SLCTs., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

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., (Copyright © 2022 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

DICER1 syndrome is a tumor predisposition syndrome that is associated with up to 30 different neoplastic lesions, usually affecting children and adolescents. Here we identify a group of mesenchymal tumors which is highly associated with DICER1 syndrome, and molecularly distinct from other DICER1-associated tumors. This group of DICER1-associated mesenchymal tumors encompasses multiple well-established clinicopathological tumor entities and can be further divided into three clinically meaningful classes designated "low-grade mesenchymal tumor with DICER1 alteration" (LGMT DICER1), "sarcoma with DICER1 alteration" (SARC DICER1), and primary intracranial sarcoma with DICER1 alteration (PIS DICER1). Our study not only provides a combined approach to classify DICER1-associated neoplasms for improved clinical management but also suggests a role for global hypomethylation and other recurrent molecular events in sarcomatous differentiation in mesenchymal tumors with DICER1 alteration. Our results will facilitate future investigations into prognostication and therapeutic approaches for affected patients., (© 2023. The Author(s).)

Background: Cyclin E1 (CCNE1) is a potential predictive marker and therapeutic target in tubo-ovarian high-grade serous carcinoma (HGSC). Smaller studies have revealed unfavorable associations for CCNE1 amplification and CCNE1 overexpression with survival, but to date no large-scale, histotype-specific validation has been performed. The hypothesis was that high-level amplification of CCNE1 and CCNE1 overexpression, as well as a combination of the two, are linked to shorter overall survival in HGSC., Methods: Within the Ovarian Tumor Tissue Analysis consortium, amplification status and protein level in 3029 HGSC cases and mRNA expression in 2419 samples were investigated., Results: High-level amplification (>8 copies by chromogenic in situ hybridization) was found in 8.6% of HGSC and overexpression (>60% with at least 5% demonstrating strong intensity by immunohistochemistry) was found in 22.4%. CCNE1 high-level amplification and overexpression both were linked to shorter overall survival in multivariate survival analysis adjusted for age and stage, with hazard stratification by study (hazard ratio [HR], 1.26; 95% CI, 1.08-1.47, p = .034, and HR, 1.18; 95% CI, 1.05-1.32, p = .015, respectively). This was also true for cases with combined high-level amplification/overexpression (HR, 1.26; 95% CI, 1.09-1.47, p = .033). CCNE1 mRNA expression was not associated with overall survival (HR, 1.00 per 1-SD increase; 95% CI, 0.94-1.06; p = .58). CCNE1 high-level amplification is mutually exclusive with the presence of germline BRCA1/2 pathogenic variants and shows an inverse association to RB1 loss., Conclusion: This study provides large-scale validation that CCNE1 high-level amplification is associated with shorter survival, supporting its utility as a prognostic biomarker in HGSC., (© 2022 The Authors. Cancer published by Wiley Periodicals LLC on behalf of American Cancer Society.)