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The molecular subtype classification of endometrial carcinomas has conceptually changed our approach to this disease. However, open questions remain about how to integrate certain histotype diagnoses with the molecular subtype. We report 2 cases with morphologic suspicion for endometrial carcinosarcoma, that still fell short of the essential criteria for diagnosing carcinosarcoma. On subsequent molecular testing pathogenic POLE mutations were detected and a descriptive diagnosis of endometrial endometrioid carcinomas, low-grade with a homologous sarcoma component was rendered. This challenges the existence of POLE-mutated "carcinosarcoma.", Competing Interests: M.K. is a consultant for HelixBiopharma, outside the scope of this study. The remaining authors declare no conflict of interest., (Copyright © 2024 by the International Society of Gynecological Pathologists.)

In this study, we aimed to test whether prognostic biomarkers can achieve a clinically relevant stratification of patients with stage I ovarian clear cell carcinoma (OCCC) and to survey the expression of 10 selected actionable targets (theranostic biomarkers) in stage II to IV cases. From the population-based Alberta Ovarian Tumor Type study, 160 samples of OCCC were evaluated by immunohistochemistry and/or silver-enhanced in situ hybridization for the status of 5 prognostic (p53, p16, IGF2BP3, CCNE1, FOLR1) and 10 theranostic biomarkers (ALK, BRAF V600E, ERBB2, ER, MET, MMR, PR, ROS1, NTRK1-3, VEGFR2). Kaplan-Meier survival analyses were performed. Cases with abnormal p53 or combined p16/IFG2BP3 abnormal expression identified a small subset of patients (6/54 cases) with stage I OCCC with an aggressive course (5-yr ovarian cancer-specific survival of 33.3%, compared with 91.5% in the other stage I cases). Among theranostic targets, ERBB2 amplification was present in 11/158 (7%) of OCCC, while MET was ubiquitously expressed in OCCC similar to a variety of normal control tissues. ER/PR showed a low prevalence of expression. No abnormal expression was detected for any of the other targets. We propose a combination of 3 biomarkers (p53, p16, IGF2BP3) to predict prognosis and the potential need for adjuvant therapy for patients with stage I OCCC. This finding requires replication in larger cohorts. In addition, OCCC could be tested for ERBB2 amplification for inclusion in gynecological basket trials targeting this alteration., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 by the International Society of Gynecological Pathologists.)

The International Endocervical adenocarcinoma Criteria and Classification (IECC) categorizes tumors into human papilloma virus (HPV) associated (HPVA), not associated (NHPV), and invasive adenocarcinoma not otherwise specified (IA NOS). HPVA and NHPV encompass 11 histotypes and an algorithm of mucin content, HPV ribonucleic acid (RNA), estrogen receptor and GATA3 is proposed for the diagnosis of most. In this study, the IECC algorithm's diagnoses were compared with hematoxylin and eosin (H&E) based IECC histotyping. Kappa statistics measured performance agreement. With additional markers, hierarchical clustering by random forest (RF) classification identified the most discriminating between tumor types, and investigated other algorithms. Three pathologists independently reviewed digitized H&E images of n=152 primary cervical adenocarcinomas for IECC histotype and mucin content, and tissue microarrays for expression of HPV RNA by in situ hybridization and 16 antibodies by immunohistochemistry. Results were finalized by consensus. There were n=113 HPVA, n=22 NHPV, and n=17 IA NOS. Mucin was obvious in n=36 and limited in n=116. Among n=124 with satisfactory test results, HPV RNA was positive in n=96, estrogen receptor in n=72, and GATA3 in n=15. The IECC algorithm diagnosed n=99 which agreed with H&E histotyping in n=64 for a fair κ of 0.36 (95% confidence interval, 0.21-0.50): n=12 were undiagnosed and n=13 were IA NOS. Small sample sizes restricted RF to HPVA versus NHPV which were discriminated by p16, HPV RNA, and MUC6 with an area under the curve of 0.74 (95% confidence interval, 0.58-0.90). The IECC algorithm for histotyping under-performed. The RF algorithmin for categorization was favorable, but validation in larger studies and investigation of additional algorithms to discriminate between all IECC histotypes are needed., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Most breast tumors are primary to this site; breast metastasis of endometrial origin is extremely rare. Low-grade endometrioid endometrial carcinomas can undergo dedifferentiation to undifferentiated carcinoma but such transformation at a metastatic site has been reported previously in only 2 cases. We report a case of dedifferentiation occurring in an isolated solitary breast metastasis of a low-grade endometrioid endometrial carcinoma. A 64-yr-old woman presented with a breast mass 2 yr after initial diagnosis of a grade 1 FIGO stage IIIA endometrioid endometrial carcinoma. Ultrasound guided biopsy of the breast mass showed a grade 1 endometrioid carcinoma which was diffusely estrogen receptor and PAX8-positive, consistent with metastasis from the previous endometrial carcinoma. The tumor initially responded to Letrozole therapy but then abruptly increased in size. Mastectomy revealed a poorly differentiated malignant tumor with morphology and immunophenotype (including loss of ARID1A and ARID1B immunoreactivity) consistent with undifferentiated endometrial carcinoma with no residual low-grade component. Awareness of the phenomenon of dedifferentiation of endometrial carcinoma in a metastatic site is important to avoid misdiagnosis as a primary breast cancer or metastasis from another primary site., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 by the International Society of Gynecological Pathologists.)

PTEN plays a central role in the pathogenesis of endometrial carcinoma. Previous studies reported a high interobserver reproducibility for the interpretation of PTEN immunohistochemistry (IHC). However, PTEN IHC and its interpretation remain challenging during laboratory practice. The purpose of this study was to reevaluate PTEN IHC pattern in direct comparison to next-generation sequencing in identifying PTEN abnormality. IHC and tagged-amplicon next-generation sequencing PTEN sequencing was performed on 182 endometrial carcinoma biopsy/curetting samples from five centers (Barts, Calgary, Cambridge, Leiden, and Vancouver). Sensitivity, specificity and accuracy of PTEN IHC to predict loss of function PTEN mutations were calculated. Abnormalities of PTEN in association with histotype and molecular subtype were assessed. A total of 5 PTEN IHC patterns were recorded: absent, subclonal loss, equivocal, reduced (relative to internal control) and retained. The absence of PTEN IHC has a sensitivity of 75.4% (95% confidence interval: 62.7-85.5%), a specificity of 84.6% (95% confidence interval: 76.2%-90.9%), and accuracy of 81.2% (95% confidence interval: 74.4%-86.9%) in predicting loss of function PTEN mutation. PTEN abnormality by complementary interpretation of both assays was present in 91.9% of endometrial endometrioid carcinoma, grade 1, and significantly higher in endometrial endometrioid carcinomas of all grades compared with endometrial serous carcinoma (80.0% vs. 19.4%, P<0.0001). PTEN abnormalities are common across all molecular subtypes of endometrioid carcinomas. Our data support the use of ancillary PTEN IHC for diagnostic purposes in endometrial neoplasms. However, for clinical trial design complementary testing of both IHC and sequencing of PTEN should be considered to assess the PTEN status in endometrial carcinomas., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 by the International Society of Gynecological Pathologists.)

Immunohistochemistry (IHC) improves the diagnosis of cervical adenocarcinoma but is not adequately studied. The performance of 16 antibodies previously reported as potentially discriminating between some histotypes was investigated in 184 tumors comprised of 12 histotype groups collapsed into 3 categories [47 adenocarcinomas in situ (AIS), 121 probable human papillomavirus-dependent adenocarcinomas (HPVD), and 16 of probable independence (HPVI)]. IHC sections from 5 tissue microarrays were scanned, and 3 pathologists independently reviewed images to assess staining percentages and intensities. Biomarker expression was based on published positive and negative cutoffs and agreement between any 2 pathologists. Differences between the 3 categories in the hierarchical ranking of biomarker positivity were analyzed by Random Forest classification, and between select groups by Unsupervised Hierarchical Clustering. Important category discriminants were combined in logistic regression models and the area under the curve (AUC) computed. Potential group discriminants were terminal cluster biomarkers with a 50% or more difference in positivity. Strong associations occurred between the lower expression of carcinoembryonic antigen and stromal actin in AIS compared with HPVD [AUC=0.70, 95% confidence interval (CI), 0.59-0.80] and in the higher expression of p16 and estrogen receptor in comparison to HPVI (AUC=0.86, 95% CI, 0.73-0.98), and between the higher expression of p16, carcinoembryonic antigen and estrogen receptor in HPVD compared with HPVI (AUC=0.88, 95% CI, 0.77-0.99). Between select groups, 9 biomarkers emerged as potential discriminants. Select IHC biomarkers can discriminate AIS from invasive adenocarcinomas, and invasive adenocarcinomas stratified by human papillomavirus dependency from each other. Independent replication in larger studies is needed, and to confirm discriminants of histotype groups., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Endometrial serous carcinoma (ESC) is an aggressive neoplasm mainly seen in older women. The objective of this study was to refine immunohistochemical (IHC) panels for the differential diagnoses against endometrial endometrioid grade 3 (EC3), endometrial clear cell, and ovarian high-grade serous carcinoma as well as exploring the prognostic role of selected IHC markers. Fifty-two ESC from a single institution were assessed for 20 IHC markers, including ARID1A, CCNE1, CDKN2A, ERBB2, ESR1, HNF1B, FBXW7, IGF2BP3, MLH1, MSH2, MSH6, NAPSA, PAX8, PGR, PMS2, PTEN, TFF3, TP53, VIM, and WT1. ERBB2 chromogenic in situ hybridization was evaluated on tissue microarrays. Statistical analysis was performed. All ESC showed aberrant TP53, normal mismatch repair protein, and retained ARID1A and PTEN expression. ESR1 expression was present in 80% of ESC. A combination of TP53, PTEN, and CDKN2A had a sensitivity of 93.6% [95% confidence interval (CI), 84%-98%] and specificity of 87.8% (95% CI, 75%-95%) for ESC versus EC3. A combination of NAPSA and ESR1 had a sensitivity of 97.9% (95% CI, 89%-99%) and specificity of 72.2% (95% CI, 46%-90%) for ESC versus clear cell carcinoma. Absence of WT1 alone had a sensitivity of 66.0% (95% CI, 51%-79%) and specificity of 98.0% (95% CI, 94%-99%) for ESC versus ovarian high-grade serous carcinoma. Among all 52 ESCs, ERBB2 amplification was present in 23%, FBXW7 expression was absent in 10%, and CCNE1 was overexpressed in 59%, however, none were associated with prognosis. Our data support the value of IHC marker panels for histotyping of high-grade endometrial carcinomas.

Growing insights into the biological features and molecular underpinnings of ovarian cancer has prompted a shift toward histotype-specific treatments and clinical trials. As a result, the preoperative diagnosis of ovarian carcinomas based on small tissue sampling is rapidly gaining importance. The data on the accuracy of ovarian carcinoma histotype-specific diagnosis based on small tissue samples, however, remains very limited in the literature. Herein, we describe a prospective series of 30 ovarian tumors diagnosed using cytology, frozen section, core needle biopsy, and immunohistochemistry (p53, p16, WT1, HNF-1β, ARID1A, TFF3, vimentin, and PR). The accuracy of histotype diagnosis using each of these modalities was 52%, 81%, 85%, and 84% respectively, using the final pathology report as the reference standard. The accuracy of histotype diagnosis using the Calculator for Ovarian Subtype Prediction (COSP), which evaluates immunohistochemical stains independent of histopathologic features, was 85%. Diagnostic accuracy varied across histotype and was lowest for endometrioid carcinoma across all diagnostic modalities (54%). High-grade serous carcinomas were the most overdiagnosed on core needle biopsy (accounting for 45% of misdiagnoses) and clear cell carcinomas the most overdiagnosed on frozen section (accounting for 36% of misdiagnoses). On core needle biopsy, 2/30 (7%) cases had a higher grade lesion missed due to sampling limitations. In this study, we identify several challenges in the diagnosis of ovarian tumors based on limited tissue sampling. Recognition of these scenarios can help improve diagnostic accuracy as we move forward with histotype-specific therapeutic strategies.

Endometrial clear cell carcinoma (CC) is an uncommon tumor and often carries a poor prognosis. It has histologic features that overlap with other endometrial carcinomas and is frequently misclassified. Accurate classification is crucial, however, to improve treatment options. The objectives of this study were (1) to assess diagnostic interobserver variability among 5 gynecologic pathologists for tumors originally diagnosed as CC or with a component of CC (n=44); (2) to determine the utility of immunohistochemical markers estrogen receptor and HNF-1β; and (3) to detect mutations in select genes. Clinical data and morphologic features were also recorded. Agreement among reviewers was only moderate: only 46% of the original CC remained classified as such. After reclassification, estrogen receptor was positive in 8% of CC, 67% of endometrioid carcinomas (EC), and 47% of serous carcinomas (SC). Sensitivities of HNF-1β in CC, SC, and EC were 62%, 27%, and 17%, respectively, whereas specificity for CC versus EC or SC was 78%. Mutations in PIK3CA, PIK3R1, PTEN, KRAS, and NRAS were detected in 41% of 37 cases that had adequate material for study. At least 1 mutation was identified in 33% of CC, 67% of EC, and 33% of SC. This group of patients had poor outcomes: 72% of the patients with follow-up information had died of disease. In summary, this study suggests that the current pool of CC is a heterogeneous group of tumors from the morphologic, immunophenotypic, and molecular point of views and that only a percentage of them represent true CC.

Immunohistochemistry results for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 are used to guide breast carcinoma patient management and it is essential to monitor these tests in external quality assurance (EQA) programs. Canadian Immunohistochemistry Quality Control is a web-based program with novel approach to EQA. Canadian Immunohistochemistry Quality Control RUN2 included tissue microarray slides with 38 samples tested by 18 immunohistochemical laboratories. Deidentified results were posted for viewing at www.ciqc.ca including all used protocols matched with scanned slides for virtual microscopy and garrattograms. Sensitivity, specificity, Kendall W test (concordance between laboratories), and kappa statistics (agreement with designated reference values) were calculated. Kappa values were within the target range (>0.8, or "near perfect" agreement) for 85% results. Kendall coefficient was 0.942 for estrogen receptor, 0.930 for progesterone receptor, and 0.958 for human epidermal growth factor receptor 2. The anonymous participation, quick feedback, and unrestricted full access in EQA results provides rapid insight into technical or interpretive deficiencies, allowing appropriate corrective action to be taken whereas the use of tissue microarrays enables meaningful statistical analysis.

The majority of patients diagnosed with ovarian carcinoma are classified as being in advanced stage of disease. In a situation of cancer spread throughout the abdominal cavity, a successful curative treatment is difficult to achieve. Therefore, preventing binding of tumor cells to the mesothelium is crucial for patients' outcome. One important mechanism is the interaction between hyaluronic acid and the CD44 receptor with its submembrane linking complex. This consists of ezrin, radixin, and moesin and connects the CD44 receptor with the cytoskeleton. To assess the role of ezrin and moesinfor ovarian carcinoma progression, we analyzed ovarian carcinoma samples from 105 patients for expression of ezrin and moesin by immunohistochemistry and correlated these data with several clinicopathological parameters. To elucidate the functional importance of ezrin and moesin, their expression was inhibited in SKOV-3 cells by RNA interference. Ezrin and moesin were strongly expressed in 49 and 48% of ovarian carcinoma samples, respectively, and their presence correlated with reduced overall survival in univariate analysis (ezrin, p=0.0189; moesin, p=0.0351). In multivariate analysis (including FIGO stage, residual tumor, histological type, and Silverberg grading), ezrin still remained significant as an independent risk factor (relative risk, 2.39; p=0.012). In SKOV-3 cells, siRNA against ezrin but not moesin inhibited in vitro invasion. These data imply that ezrin is necessary for tumor cell invasion, and the better prognosis of ovarian carcinomas lacking ezrin is probably related to their impaired invasion.