Uterine mesenchymal tumours harboring the KAT6B/A::KANSL1 gene fusion represent a distinct type of uterine sarcoma based on DNA methylation profiles.

Uterine mesenchymal tumours harboring KAT6B/A::KANSL1 gene fusions typically exhibit histological and immunophenotypic overlap with endometrial stromal and smooth muscle tumours. To date it remains uncertain whether such neoplasms should be regarded as variants of smooth muscle or endometrial stromal neoplasm, or if they constitute a distinct tumour type. In this study we investigated DNA methylation patterns and copy number variations (CNVs) in a series of uterine tumours harboring KAT6B/A::KANSL1 gene fusions in comparison to other mesenchymal neoplasms of the gynecological tract. Unsupervised hierarchical clustering and t-SNE analysis of DNA methylation data (Illumina EPIC array) identified a distinct cluster for 8/13 KAT6B/A::KANSL1 tumours (herein referred to as core cluster). The other 5 tumours (herein referred to as outliers) did not assign to the core cluster but clustered near various other tumour types. CNV analysis did not identify significant alterations in the core cluster. In contrast, various alterations, including deletions at the CDKN2A/B and NF1 loci were identified in the outlier group. Analysis of the DNA methylation clusters in relation to histological features revealed that while tumours in the core KAT6B/A::KANSL1 cluster were histologically bland, outlier tumours frequently exhibited "high-grade" histologic features in the form of significant nuclear atypia, increased mitotic activity and necrosis. Three of the five patients with outlier tumours died from their disease while clinical follow-up in the remaining two patients was limited (less than 12 months). In comparison, none of the 7 out of 8 patients with tumors in the core KAT6B/A::KANSL1 sarcoma cluster, where follow-up was available, died from disease. Furthermore, only 1 out of 7 patients recurred (mean follow-up of 30 months). In conclusion, KAT6B/A::KANSL1 uterine sarcoma is a molecularly unique type of uterine tumour that should be recognized as a distinct entity. These tumors typically exhibit low-grade histologic features but are occasionally morphologically high-grade; the latter have a DNA methylation profile different from the typical low-grade neoplasms and may be associated with aggressive behaviour.

Spatial Cancer-Immune Phenotypes Predict Shorter Recurrence-Free Survival in the No Specific Molecular Profile Molecular Subtype of Endometrial Carcinoma.

Compartmentation of the immune response into 3 main spatial cancer-immune phenotypes (SCIs) - inflamed, excluded, and desert - has been proposed as the main predictor of response to immune checkpoint inhibitors in solid tumors. The objective of the study was to define and characterize the SCI in a consecutive series of 213 endometrial carcinomas (ECs) by correlating it with molecular subtypes, clinicopathologic features, and prognosis. Immunohistochemistry (IHC) and next-generation sequencing were used to assign surrogate molecular EC subtypes: POLE mutant (POLE), mismatch repair deficient (MMRd), TP53 mutant (p53abn), and no specific molecular profile (NSMP). Immune cell markers (CD20, CD3, CD8, CD68, PD-L1) were assessed by IHC on whole sections and quantified by digital image analysis to define the 3 SCIs. ECs were stratified into 4 molecular subtypes: 17 (8.0%) POLE, 68 (31.9%) MMRd, 42 (19.7%) p53abn, and 86 (40.4%) NSMP. SCI determination showed 105 (49.3%) inflamed, 62 (29.1%) desert, and 46 (25.6%) excluded tumors. The inflamed phenotype was more prevalent in MMRd (64.7%) and POLE (76.5%) subtypes compared with NSMP (45.3%) and p53abn (21.4%). SCI revealed a strong correlation with disease-free survival in NSMP tumors: inflamed 96.2%, desert 83.2%, and excluded 40.5%. The SCI prognostic impact was also maintained in NSMP cases treated with adjuvant therapy resulting in a significant difference in recurrence between the inflamed and excluded phenotypes. To simplify SCI determination, a subset of immune cell markers was selected as appropriate to define the 3 SCI patterns: high intraepithelial CD8 for the inflamed phenotype; CD68, CD20, and PD-L1 to discriminate between desert and excluded tumors. The integration of SCI into molecular classification could be a promising opportunity to improve the prognostic risk stratification of patients and may guide the therapeutic approach, particularly in the NSMP subtype. Thus, the different patterns of immune response are a new prognostic parameter in the NSMP subtype.

Impact of immune, inflammatory and nutritional indices on outcome in patients with locally advanced cervical cancer treated with definitive (chemo)radiotherapy.

OBJECTIVE: Systemic immune, inflammatory, and nutritional indices are prognostic across multiple tumor sites. Comprehensive analysis of these markers in patients with locally advanced cervical cancer (LACC) treated with definitive (chemo)radiotherapy [(C)RT] is limited and may assist with future prognostication. METHODS: For this retrospective cohort study, patients with LACC treated with definitive (C)RT were identified from a comprehensive cancer center's clinicopathological database. Pre-treatment indices were derived including systemic immune-inflammation index (SII), platelet lymphocyte ratio (PLR), neutrophil lymphocyte ratio (NLR), monocyte lymphocyte ratio (MLR), albumin to alkaline phosphatase ratio (AAPR) and prognostic nutritional index (PNI). Univariate analysis was performed for PFS and OS. ROC curves were analyzed to determine optimal cut points. PFS and OS were assessed by the Kaplan-Meier method and Log-Rank test. Multivariate analysis was performed using Cox regression. RESULTS: 196 patients were identified: median follow-up 7 years (IQR 2-11). Higher SII (≤700 vs >700; p = 0.01), higher PLR (≤ 250 vs >250; p < 0.001) and higher NLR (≤ 5 vs >5; p = 0.003) were associated with worse PFS. Higher SII (≤700 vs >700: p = 0.02), higher PLR (≤ 250 vs >250: p < 0.001) and higher NLR (≤ 5 vs >5; p = 0.01) were associated with worse OS. On multivariate analysis, SII, NLR and PLR were independently associated with PFS. SII and PLR were independently associated with OS. CONCLUSIONS: SII and PLR were independently associated with PFS and OS in patients with LACC treated with definitive (C)RT. NLR was independently associated with PFS. High inflammatory state is associated with shorter survival suggesting this as a target for interventions if validated in future studies.

Ovarian carcinosarcomas: p53 status defines two distinct patterns of oncogenesis and outcomes.

Objectives: Ovarian carcinosarcoma (OCS) is a rare and lethal type of ovarian cancer. Despite its incredibly poor prognosis, it has received little research attention. In this study, we aim to evaluate the molecular features of OCS and elucidate their clinical significance. Study methods: We examined 30 OCS by immunohistochemistry (IHC) and targeted panel sequencing collected from a single institution (2003-2013) as the initial molecularly characterized cohort (Cohort A). From November 2016 to April 2023, we collected an additional 67 OCS cases from three institutions across British Columbia and Alberta as the contemporary cohort (Cohort B) for clinical correlation. The Kaplan-Meier method was used to estimate overall and progression-free survival, and differences in survival rates were compared using the log-rank test. All tests were two-sided. A p-value of less than 0.05 was considered statistically significant. Results: The majority of OCS (82%) in the initial Cohort A were p53-mutated, and the carcinomatous component displayed the histological and molecular features of a high-grade tubo-ovarian serous carcinoma (HGSC-like). In a minority of OCS, the epithelial components were characteristics of endometrioid or clear cell carcinomas, and IHC staining was wild type for p53. In the contemporary Cohort B, we observed the same histological findings related to the p53 IHC staining pattern. The median overall survival of the p53-mutated HGSC-like OCS (47 patients) was significantly higher (43.5 months) compared with that of the p53 wild-type OCS (10 patients, 8.8 months; P < 0.01). Pathogenic BRCA1/2 germline/somatic mutations were observed in 7 patients (17.5%) of HGSC-like OCS, and all these patients were alive at 3 years from diagnosis compared to a 51% 3-year survival among the patients with BRCA1/2 wild-type HGSC-like OCS (33 patients) (p = 0.022). Majority of patients (6/7) with BRCA1/2-mutated OCS received poly (ADP-ribose) polymerase inhibitor as maintenance therapy in this cohort. Conclusions: Most OCSs have a morphologic and molecular profile resembling HGSC; however, some OCSs display a molecular profile that suggests origin through non-serous oncogenic pathways. This molecular distinction has both prognostic and treatment (predictive) implications. These findings underscore the importance of routine p53 IHC testing on all OCS and BRCA1/2 testing on p53-mutated OCS.

Preclinical 3D model screening reveals digoxin as an effective therapy for a rare and aggressive type of endometrial cancer.

OBJECTIVE: Dedifferentiated endometrial carcinoma (DDEC) characterized by SWItch/Sucrose Non-Fermentable (SWI/SNF) complex inactivation is a highly aggressive type of endometrial cancer without effective systemic therapy options. Its uncommon nature and aggressive disease trajectory pose significant challenges for therapeutic progress. To address this obstacle, we focused on developing preclinical models tailored to this tumor type and established patient tumor-derived three-dimensional (3D) spheroid models of DDEC. METHODS: High-throughput drug repurposing screens were performed on in vitro 3D spheroid models of DDEC cell lines (SMARCA4-inactivated DDEC-1 and ARID1A/ARID1B co-inactivated DDEC-2). The dose-response relationships of the identified candidate drugs were evaluated in vitro, followed by in vivo evaluation using xenograft models of DDEC-1 and DDEC-2. RESULTS: Drug screen in 3D models identified multiple cardiac glycosides including digoxin and digitoxin as candidate drugs in both DDEC-1 and DDEC-2. Subsequent in vitro dose-response analyses confirmed the inhibitory activity of digoxin and digitoxin with both drugs showing lower IC50 in DDEC cells compared to non-DDEC endometrial cancer cells. In in vivo xenograft models, digoxin significantly suppressed the growth of DDEC tumors at clinically relevant serum concentrations. CONCLUSION: Using biologically precise preclinical models of DDEC derived from patient tumor samples, our study identified digoxin as an effective drug in suppressing DDEC tumor growth. These findings provide compelling preclinical evidence for the use of digoxin as systemic therapy for SWI/SNF-inactivated DDEC, which may also be applicable to other SWI/SNF-inactivated tumor types.

Learning generalizable AI models for multi-center histopathology image classification.

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.

The association between body mass index and molecular subtypes in endometrial carcinoma.

Objective: This study aims to investigate the relationship between body mass index (BMI) and molecular subtypes of endometrial carcinoma using an immunohistochemistry (IHC)-based classification approach. Methods: We analyzed a consecutive series of endometrial cancer cases undergoing surgical staging in southern Alberta (2019-2021). Molecular classification was determined through IHC-based molecular typing, incorporating p53 and mismatch repair (MMR), and further characterized with the addition of ER and PR. BMI associations with molecular classification were assessed using t-tests. Hormone receptor status was further examined in a separate cohort of MMRd endometrial cancer patients undergoing surgical staging at Foothills Medical Centre (Alberta, Canada). Results: Among 289 cases, comprising various histological subtypes, the pNSMP subtype exhibited the highest average BMI (33.93 kg/m2) compared to the p53 abnormal subtype (30.40 kg/m2, p = 0.02). The MMRd subtype had an average BMI of 33.22 kg/m2. While there were no significant BMI differences between FIGO grade 1 and grade 2/3 tumours in the pNSMP or MMRd, a trend toward higher BMI in grade 1 tumours versus grade 2/3 tumours in the MMRd was observed (p = 0.13). A separate cohort of 53 MMRd endometrial carcinomas revealed that FIGO grade 1 tumours were associated with higher BMI (p < 0.05) and more frequent ER/PR expression compared to grade 2/3 tumours (p < 0.05). Conclusions: This study suggests an association between obesity and NSMP endometrial carcinoma. The relationship between BMI and low-grade MMRd endometrial carcinomas with increased ER/PR expression warrants further exploration.

Mesonephric-type adenocarcinomas of the ovary: prevalence, diagnostic reproducibility, outcome, and value of PAX2.

Mesonephric-type (or -like) adenocarcinomas (MAs) of the ovary are an uncommon and aggressive histotype. They appear to arise through transdifferentiation from Müllerian lesions creating diagnostic challenges. Thus, we aimed to develop a histologic and immunohistochemical (IHC) approach to optimize the identification of MA over its histologic mimics, such as ovarian endometrioid carcinoma (EC). First, we screened 1,537 ovarian epithelial neoplasms with a four-marker IHC panel of GATA3, TTF1, ER, and PR followed by a morphological review of EC to identify MA in retrospective cohorts. Interobserver reproducibility for the distinction of MA versus EC was assessed in 66 cases initially without and subsequently with IHC information (four-marker panel). Expression of PAX2, CD10, and calretinin was evaluated separately, and survival analyses were performed. We identified 23 MAs from which 22 were among 385 cases initially reported as EC (5.7%) and 1 as clear cell carcinoma. The interobserver reproducibility increased from fair to substantial (κ = 0.376-0.727) with the integration of the four-marker IHC panel. PAX2 was the single most sensitive and specific marker to distinguish MA from EC and could be used as a first-line marker together with ER/PR and GATA3/TTF1. Patients with MA had significantly increased risk of earlier death from disease (hazard ratio = 3.08; 95% CI, 1.62-5.85; p < 0.0001) compared with patients with EC, when adjusted for age, stage, and p53 status. A diagnosis of MA has prognostic implications for stage I disease, and due to the subtlety of morphological features in some tumors, a low threshold for ancillary testing is recommended.

Molecular Surrogate Subtypes of Ovarian and Peritoneal Low-grade Serous Carcinoma.

Low-grade serous carcinoma (LGSC) is an uncommon histotype of ovarian carcinoma, accounting for ~3% of cases. There is evidence that survival of peritoneal LGSC (pLGSC) is longer than that of ovarian LGSC (oLGSC). Key molecular alterations of LGSC have been established, including loss of CDKN2A and PR expression, MAPK pathway alterations, and loss of USP9X expression. We hypothesized that LGSC could be subclassified into clinically applicable molecular subtypes by a few surrogate tests similar to endometrioid carcinomas using a hierarchical decision tree based on the strength of the prognostic associations of the individual alterations. Our study included 71 LGSCs. Immunohistochemistry for CDKN2A, ER, PR, NF1, and USP9X and sequencing for KRAS , NRAS , and BRAF were performed. Our data showed the co-occurrence of key molecular alterations, and despite suggestive trends, hierarchical molecular subtyping did not provide significantly different stratification of patients according to survival in this cohort. We confirmed that patients diagnosed with pLGSC have a longer survival than high-stage oLGSC, with the intriguing observation that normal CDKN2A and PR status were associated with excellent survival in pLGSC. Therefore, CDKN2A and PR status might aid in the classification of indeterminate implants, where abnormal findings favor pLGSC over noninvasive implants. Molecular subtypes should be further evaluated in larger cohorts for their prognostic and potentially predictive value.

POLE-mutated Endometrial "Carcinosarcoma".

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."

Prevalence and Prognostic Significance of PIK3CA Mutation and CNV Status and Phosphorylated AKT Expression in Patients With Cervical Cancer Treated With Primary Surgery.

Currently, there are limited and conflicting reports on the prognostic utility of PIK3CA and associated pathway markers for cervical cancers treated with primary surgical management. Moreover, current studies are lacking complete characterization of adjuvant treatment with RT and/or chemotherapy. We aimed to document the prevalence, clinicopathologic, adjuvant treatment details, and prognostic value of PI3K/AKT pathway mutations and copy number variation and phosphorylated AKT status in patients with cervical cancers treated with primary surgery. A clinicopathologic review was performed on a retrospective cohort of 185 patients with cervical cancer, treated with primary surgery at a single tertiary institution. Next-generation sequencing and digital PCR was used to determine PI3K/AKT pathway mutational status and PIK3CA copy number variation, respectively, and fluorescent immunohistochemistry measured phosphorylated AKT expression. In all, 179 of 185 (96.8%) of tumors were successfully sequenced; 48 (26.8%) were positive for PI3K/AKT pathway mutations-the majority (n=37, 77.1%) PIK3CA mutations. PIK3CA mutation was associated with pathologically positive lymph nodes [12 (32%) vs. 22 (16%); P =0.022] and indication for postoperative chemoradiotherapy [17 (45.9%) vs. 32 (22.5%); P =0.004]. On multivariable analysis, PIK3CA status was not associated with overall survival ( P =0.103) or progression-free survival ( P =0.240) at 5 yrs, nor was PIK3CA copy number variation status. phosphorylated AKT ≤ median significantly predicted for progression-free survival [multivariable hazard ratio 0.39 (0.17-0.89; P =0.025)] but not overall survival ( P =0.087). The correlation of PIK3CA with pathologic positive lymph node status yet lack of association with survival outcomes may be due to the use of adjuvant postoperative therapy. PIK3CA assessment before radical hysterectomy may help identify patients with a higher risk of node-positive disease.

TP53 as a Diagnostic Aid in the Distinction of Ovarian Mucinous Borderline Tumors From Mucinous Carcinoma.

Ovarian mucinous borderline tumors (MBTs) are clinically managed as benign neoplasms while the management of ovarian mucinous carcinomas (MC) is dependent on tumor stage. Despite the standardization of sampling of ovarian mucinous neoplasms, limited interobserver reproducibility between MBT and MC persists. Based on our recent finding that abnormal TP53 expression is associated with unfavorable outcome in MBT, we hypothesized that TP53 status might improve the reproducible distinction of MBT from MC. A virtual slide set of 85 consecutive ovarian mucinous neoplasms received at a single institution, with each case represented by 3 full sections, were reviewed by 3 pathologists in 2 iterations. The initial assessment was based solely on morphologic review, while the second iteration was performed with knowledge of TP53 status. The reproducibility of a trinary categorization (MBT, MBT with intraepithelial carcinoma [IEC], MC) significantly improved from a κ of 0.60 based on the initial morphologic assessment to a κ of 0.76 (t-test, P =0.0042) after consideration of TP53 immunohistochemistry (IHC) results. Six out of 85 patients died of disease, and in 2 of them, at least 1 pathologist assessed MBT with IEC and not MC even after integration of TP53 IHC. With the integration of TP53 IHC, substantial interobserver agreement for MBT and MC can be reached, particularly in cases with an uncertain degree of confluent growth. TP53 IHC can also be used to highlight and support the presence of IEC in MBT, however, discordances remained in 2 cases with adverse outcome.

Dedifferentiated and Undifferentiated Ovarian Carcinoma: An Aggressive and Molecularly Distinct Ovarian Tumor Characterized by Frequent SWI/SNF Complex Inactivation.

Dedifferentiated and undifferentiated ovarian carcinomas (DDOC/UDOC) are rare neoplasms defined by the presence of an undifferentiated carcinoma. In this study, we detailed the clinical, pathological, immunohistochemical, and molecular features of a series of DDOC/UDOC. We collected a multi-institutional cohort of 23 DDOC/UDOC and performed immunohistochemistry for core switch/sucrose nonfermentable (SWI/SNF) complex proteins (ARID1A, ARID1B, SMARCA4, and SMARCB1), mismatch repair (MMR) proteins, and p53. Array-based genome-wide DNA methylation and copy number variation analyses were performed on a subset of cases with comparison made to a previously reported cohort of undifferentiated endometrial carcinoma (UDEC), small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), and tubo-ovarian high-grade serous carcinoma (HGSC). The age of all 23 patients with DDOC/UDOC ranged between 22 and 71 years (with an average age of 50 years), and a majority of them presented with extraovarian disease (16/23). Clinical follow-up was available for 19 patients. Except for 2 patients, the remaining 17 patients died from disease, with rapid disease progression resulting in mortality within a year in stage II-IV settings (median disease-specific survival of 3 months). Eighteen of 22 cases with interpretable immunohistochemistry results showed loss of expression of core SWI/SNF protein(s) that are expected to result in SWI/SNF complex inactivation as 10 exhibited coloss of ARID1A and ARID1B, 7 loss of SMARCA4, and 1 loss of SMARCB1. Six of 23 cases were MMR-deficient. Two of 20 cases exhibited mutation-type p53 immunoreactivity. Methylation profiles showed coclustering of DDOC/UDOC with UDEC, which collectively were distinct from SCCOHT and HGSC. However, DDOC/UDOC showed an intermediate degree of copy number variation, which was slightly greater, compared with SCCOHT but much less compared with HGSC. Overall, DDOC/UDOC, like its endometrial counterpart, is highly aggressive and is characterized by frequent inactivation of core SWI/SNF complex proteins and MMR deficiency. Its molecular profile overlaps with UDEC while being distinct from SCCOHT and HGSC.

Mesonephric-like adenocarcinoma harbours characteristic copy number variations and a distinct DNA methylation signature closely related to mesonephric adenocarcinoma of the cervix.

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.

Infiltrative pattern of invasion is independently associated with shorter survival and desmoplastic stroma markers FAP and THBS2 in mucinous ovarian carcinoma.

AIMS: Mucinous ovarian carcinoma (MOC) is a rare ovarian cancer histotype with generally good prognosis when diagnosed at an early stage. However, MOC with the infiltrative pattern of invasion has a worse prognosis, although to date studies have not been large enough to control for covariables. Data on reproducibility of classifying the invasion pattern are limited, as are molecular correlates for infiltrative invasion. We hypothesized that the invasion pattern would be associated with an aberrant tumour microenvironment. METHODS AND RESULTS: Four subspecialty pathologists assessed interobserver reproducibility of the pattern of invasion in 134 MOC. Immunohistochemistry on fibroblast activation protein (FAP) and THBS2 was performed on 98 cases. Association with survival was tested using Cox regression. The average interobserver agreement for the infiltrative pattern was moderate (kappa 0.60, agreement 86.3%). After reproducibility review, 24/134 MOC (18%) were determined to have the infiltrative pattern and this was associated with a higher risk of death, independent of FIGO stage, grade, and patient age in a time-dependent manner (hazard ratio [HR] = 10.2, 95% confidence interval [CI] 3.0-34.5). High stromal expression of FAP and THBS2 was more common in infiltrative MOC (FAP: 60%, THBS2: 58%, both P < 0.001) and associated with survival (multivariate HR for FAP: 1.5 [95% CI 1.1-2.1] and THBS2: 1.91 [95% CI 1.1-3.2]). CONCLUSIONS: The pattern of invasion should be included in reporting for MOC due to the strong prognostic implications. We highlight the histological features that should be considered to improve reproducibility. FAP and THBS2 are associated with infiltrative invasion in MOC.

Survey of NF1 inactivation by surrogate immunohistochemistry in ovarian carcinomas.

OBJECTIVE: Inhibition of the MAPK pathway by MEK inhibitors (MEKi) is currently a therapeutic standard in several cancer types, including ovarian low-grade serous carcinoma (LGSC). A common MAPK pathway alteration in tubo-ovarian high-grade serous carcinoma (HGSC) is the genomic inactivation of neurofibromin 1 (NF1). The primary objectives of our study were to survey the prevalence of NF1 inactivation in the principal ovarian carcinoma histotype as well as to evaluate its associations with clinico-pathological parameters and key biomarkers including BRCA1/2 status in HGSC. METHODS: A recently commercialized NF1 antibody (clone NFC) was orthogonally validated on an automated immunohistochemistry (IHC) platform and IHC was performed on tissue microarrays containing 2140 ovarian carcinoma cases. Expression was interpreted as loss/inactivated (complete or subclonal) versus normal/retained. RESULTS: Loss of NF1 expression was detected in 250/1429 (17.4%) HGSC including 11% with subclonal loss. Survival of NF1-inactivated HGSC patients was intermediate between favorable BRCA1/2 mutated HGSC and unfavorable CCNE1 high-level amplified HGSC. NF1 inactivation was mutually exclusive with CCNE1 high-level amplifications, co-occurred with RB1 loss and occurred at similar frequencies in BRCA1/2 mutated versus wild-type HGSC. NF1 loss was found in 21/286 (7.3%) endometrioid carcinomas with a favorable prognostic association (p = 0.048), and in 4/64 (5.9%) LGSC, mutually exclusive with other driver events. CONCLUSIONS: NF1 inactivation occurs in a significant subset of BRCA1/2 wild-type HGSC and a subset of LGSC. While the functional effects of NF1 inactivation need to be further characterized, this signifies a potential therapeutic opportunity to explore targeting NF1 inactivation in these tumors.

Subclonal p53 immunostaining in the diagnosis of endometrial carcinoma molecular subtype.

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.

Establishment and validation of preclinical models of SMARCA4-inactivated and ARID1A/ARID1B co-inactivated dedifferentiated endometrial carcinoma.

OBJECTIVE: Dedifferentiated endometrial cancer (DDEC) is an uncommon and clinically highly aggressive subtype of endometrial cancer characterized by genomic inactivation of SWItch/Sucrose Non-Fermentable (SWI/SNF) complex protein. It responds poorly to conventional systemic treatment and its rapidly progressive clinical course limits the therapeutic windows to trial additional lines of therapies. This underscores a pressing need for biologically accurate preclinical tumor models to accelerate therapeutic development. METHODS: DDEC tumor from surgical samples were implanted into immunocompromised mice for patient-derived xenograft (PDX) and cell line development. The histologic, immunophenotypic, genetic and epigenetic features of the patient tumors and the established PDX models were characterized. The SMARCA4-deficienct DDEC model was evaluated for its sensitivity toward a KDM6A/B inhibitor (GSK-J4) that was previously reported to be effective therapy for other SMARCA4-deficient cancer types. RESULTS: All three DDEC models exhibited rapid growth in vitro and in vivo, with two PDX models showing spontaneous development of metastases in vivo. The PDX tumors maintained the same undifferentiated histology and immunophenotype, and exhibited identical genomic and methylation profiles as seen in the respective parental tumors, including a mismatch repair (MMR)-deficient DDEC with genomic inactivation of SMARCA4, and two MMR-deficient DDECs with genomic inactivation of both ARID1A and ARID1B. Although the SMARCA4-deficient cell line showed low micromolecular sensitivity to GSK-J4, no significant tumor growth inhibition was observed in the corresponding PDX model. CONCLUSIONS: These established patient tumor-derived models accurately depict DDEC and represent valuable preclinical tools to gain therapeutic insights into this aggressive tumor type.

The Prognostic Effect of Immune Cell Infiltration Depends on Molecular Subtype in Endometrioid Ovarian Carcinomas.

PURPOSE: Endometrioid ovarian carcinoma (ENOC) is the second most-common type of ovarian carcinoma, comprising 10%-20% of cases. Recently, the study of ENOC has benefitted from comparisons to endometrial carcinomas including defining ENOC with four prognostic molecular subtypes. Each subtype suggests differential mechanisms of progression, although tumor-initiating events remain elusive. There is evidence that the ovarian microenvironment may be critical to early lesion establishment and progression. However, while immune infiltrates have been well studied in high-grade serous ovarian carcinoma, studies in ENOC are limited. EXPERIMENTAL DESIGN: We report on 210 ENOC, with clinical follow-up and molecular subtype annotation. Using multiplex IHC and immunofluorescence, we examine the prevalence of T-cell lineage, B-cell lineage, macrophages, and populations with programmed cell death protein 1 or programmed death-ligand 1 across subtypes of ENOC. RESULTS: Immune cell infiltrates in tumor epithelium and stroma showed higher densities in ENOC subtypes with known high mutation burden (POLEmut and MMRd). While molecular subtypes were prognostically significant, immune infiltrates were not (overall survival P > 0.2). Analysis by molecular subtype revealed that immune cell density was prognostically significant in only the no specific molecular profile (NSMP) subtype, where immune infiltrates lacking B cells (TILB minus) had inferior outcome (disease-specific survival: HR, 4.0; 95% confidence interval, 1.1-14.7; P < 0.05). Similar to endometrial carcinomas, molecular subtype stratification was generally superior to immune response in predicting outcomes. CONCLUSIONS: Subtype stratification is critical for better understanding of ENOC, in particular the distribution and prognostic significance of immune cell infiltrates. The role of B cells in the immune response within NSMP tumors warrants further study.