The Journal of Pathology: Clinical Research

Decoding UTROSCT heterogeneity: systematic clinicopathological evaluation combined with molecular profiling

Abstract Uterine tumor resembling ovarian sex cord tumor (UTROSCT) constitutes an exceptionally rare histological subset of uterine mesenchymal neoplasms. While most cases have benign clinical behavior, a subset of UTROSCTs exhibits clinically aggressive behavior characterized by recurrence and metastasis. Here, we present a cohort of 25 UTROSCT cases molecularly confirmed by recurrent fusion gene detection, including ESR1::NCOA3 ( n  = 12), GREB1::NCOA1 ( n  = 6), ESR1::NCOA2 ( n  = 3), GREB1::NCOA2 ( n  = 2), GREB1::SS18 ( n  = 1), and GREB1::CTNNB1 ( n  = 1). Notably, six cases (6/25, 24%) demonstrated recurrence/metastasis: two cases showed intrauterine recurrence (harboring ESR1::NCOA3 and GREB1::NCOA1 fusions), while four developed extrauterine metastases (carrying ESR1::NCOA3 , ESR1::NCOA2, GREB1::NCOA1, and GREB1::NCOA2 fusions), with one fatality. To dissect the biological basis of UTROSCT aggressiveness, we performed integrated clinicopathologic, immunohistochemical, and molecular profiling. Multivariate analysis identified tumor size >5 cm, FIGO stage IB, and lymphovascular space invasion (LVSI) as independent predictors of recurrence/metastasis, whereas histologic features, proliferation index, and fusion gene subtypes lacked prognostic significance. Multi‐omics analysis of primary versus metastatic tumors revealed striking copy number variations (CNVs) exclusively in metastatic lesions. Specifically, heterozygous losses of SMARCB1 (2/4 metastatic cases) and ATRX (1/4 metastatic cases) were identified; both play critical roles in chromatin remodeling. These genetic alterations were conspicuously absent in primary tumors, suggesting their potential role in metastatic progression. Our findings represent the first demonstration of CNV‐driven oncogenic evolution in UTROSCTs, particularly implicating SWI/SNF complex dysregulation in metastatic competence.

Context is key: how diagnostic and prognostic markers are reshaping gynecologic pathology

Abstract The field of gynecologic pathology is undergoing a transformative change. The translation of molecular findings from large‐scale genomic studies into clinical practice has been facilitated by robust surrogate assays, particularly immunohistochemistry (IHC). These tools provide scalable molecular proxies with short turnaround times, enabling molecular subclassification and conclusive prognostic biomarker studies. This commentary highlights how prognostic markers now refine diagnoses in challenging borderline areas, where an abnormal result can help exclude benign or precursor entities. However, the utility of these markers is highly context‐dependent, modified by histotype and molecular subtype. Furthermore, the confident diagnosis of rare entities and the study of their precursors have been advanced by defining specific molecular and IHC profiles, opening new avenues for research and therapy. The integration of morphology with molecular features is increasing the robustness of diagnoses and dictating oncology management as never before.

Synchronous endometrial/cervical and ovarian/fallopian tube carcinoma: a genome‐wide mutation analysis

AbstractIn this study, we explored the genomic underpinnings of synchronous endometrial and ovarian/fallopian tube carcinoma (SEOC) and synchronous cervical and ovarian/fallopian tube carcinoma (SCOC), focusing on their clonal relationships to discern whether these malignancies represent dual primary tumors (DPTs) or have metastatic origins. We established a cohort comprising 54 SEOC patients and 7 SCOC patients. After selection, 17 patients (12 SEOC and 5 SCOC) underwent comprehensive analysis via whole‐exome sequencing. The study encompassed a diverse array of histological subtypes, including high‐grade serous carcinoma (HGSC) or uterine serous carcinoma (USC), endometrioid carcinoma exhibiting papillary/mucinous features, dedifferentiated carcinoma (DC), clear cell carcinoma (CCC), HPV‐associated cervical squamous cell carcinoma, and HPV‐independent cervical adenocarcinoma. Analysis revealed that 58.3% (7 of 12) of SEOC cases and all SCOC cases demonstrated shared mutations. This suggests a clonal relationship and supports a metastatic origin for these tumors. Notably, metastatic SEOC instances included co‐occurrences of USC and HGSC in both the endometrium and the ovaries/fallopian tubes, endometrial and ovarian CCC, concurrent endometrioid endometrial carcinoma (EEC) and endometrioid ovarian carcinoma (EOC) with mucinous metaplasia, as well as cases of endometrial DC with ovarian CCC, and both EEC and ovarian DC. Among the SEOC cases classified as metastatic, patients with high‐grade tumors and advanced ovarian stage succumbed to their disease, whereas the remainder survived without relapse. In the SCOC cohort, one patient died from the disease. The favorable survival outcomes across varied histotypes suggest that a stage upgrade may not be warranted. Given the favorable clinical outcomes observed, the term ‘trans‐tubal spread’ may be more appropriate than ‘metastasis’ in this context to prevent potential overtreatment. Directionality analysis revealed a bidirectional pattern of trans‐tubal spread between the uterus/cervix and ovary/fallopian tubes. The presence of dedifferentiated carcinoma confirms the manifestation of dedifferentiation during spread. These findings lend support to the trans‐tubal implantation hypothesis and contribute novel insights into the molecular mechanisms underlying tumor dissemination in SEOC and SCOC.

Pathogenesis of peritoneal high‐grade serous carcinoma after risk‐reducing surgery: a systematic review

AbstractGermline BRCA1/2 pathogenic variant carriers have an increased risk for high‐grade serous carcinoma (HGSC) and are therefore advised to have risk‐reducing salpingo‐oophorectomy around the age of 40. However, a risk of 0.9% to develop peritoneal HGSC remains in these women, which increases to 27.5% when serous tubal intraepithelial carcinoma (STIC) is detected. The pathophysiological mechanism that leads to the development of peritoneal HGSC after salpingectomy or salpingo‐oophorectomy is still largely unknown. In this systematic review, we aim to provide insights into the pathogenic pathways of peritoneal HGSC after salpingectomy or salpingo‐oophorectomy. Therefore, we performed a systematic search for studies investigating pathophysiological mechanisms related to peritoneal HGSC in PubMed and EMBASE. A total of 49 articles were included in this study. Most evidence was found on mechanisms following a tubal origin, such as clonality between STIC and peritoneal HGSC as well as molecular similarities between fallopian tube (FT) epithelium and peritoneal HGSC. Additionally, FT epithelium was shown to adhere to the ovary and could therefore stay present after isolated salpingectomy. There might be a role for the endometrium, as it was observed that serous endometrial intraepithelial carcinoma (SEIC) has a clonal relationship with extra‐uterine HGSC. The role of the ovary seems limited, although some mouse models show a role for follicular fluid in the dissemination of malignant cells on the peritoneum. In conclusion, different mechanisms might be responsible for peritoneal HGSC development after bilateral salpingectomy or salpingo‐oophorectomy. Most available evidence supports the dissemination of precursor cells originating in the FT. Also, a possible role for the endometrium was found. An ovarian origin seems less likely; however, execution of oophorectomy does not seem obsolete in clinical practice as follicular fluid might promote dissemination and residual tubal tissue can be present on the ovary after salpingectomy.

Clinicopathological and epigenetic differences between primary neuroendocrine tumors and neuroendocrine metastases in the ovary

AbstractCurrently, the available literature provides insufficient support to differentiate between primary ovarian neuroendocrine tumors (PON) and neuroendocrine ovarian metastases (NOM) in patients. For this reason, patients with a well‐differentiated ovarian neuroendocrine tumor (NET) were identified through electronic patient records and a nationwide search between 1991 and 2023. Clinical characteristics were collected from electronic patient files. This resulted in the inclusion of 71 patients with NOM and 17 patients with PON. Histologic material was stained for Ki67, SSTR2a, CDX2, PAX8, TTF1, SATB2, ISLET1, OTP, PDX1, and ARX. DNA methylation analysis was performed on a subset of cases. All PON were unilateral and nine were found within a teratoma (PON‐T+). A total of 78% of NOM were bilateral, and none were associated with a teratoma. PON without teratomous components (PON‐T−) displayed a similar insular growth pattern and immunohistochemistry as NOM (p > 0.05). When compared with PON‐T+, PON‐T− more frequently displayed ISLET1 positivity and were larger, and patients were older at diagnosis (p < 0.05). Unsupervised analysis of DNA methylation profiles from tumors of ovarian (n = 16), pancreatic (n = 22), ileal (n = 10), and rectal (n = 7) origin revealed that four of five PON‐T− clustered together with NOM and ileal NET, whereas four of five PON‐T+ grouped with rectum NET. In conclusion, unilateral ovarian NET within a teratoma should be treated as a PON. Ovarian NET localizations without teratomous components have a molecular profile analogous to midgut NET metastases. For these patients, a thorough review of imaging should be performed to identify a possible undetected midgut NET and a corresponding follow‐up strategy may be recommended.

Assessing the impact of deep‐learning assistance on the histopathological diagnosis of serous tubal intraepithelial carcinoma (STIC) in fallopian tubes

AbstractIn recent years, it has become clear that artificial intelligence (AI) models can achieve high accuracy in specific pathology‐related tasks. An example is our deep‐learning model, designed to automatically detect serous tubal intraepithelial carcinoma (STIC), the precursor lesion to high‐grade serous ovarian carcinoma, found in the fallopian tube. However, the standalone performance of a model is insufficient to determine its value in the diagnostic setting. To evaluate the impact of the use of this model on pathologists' performance, we set up a fully crossed multireader, multicase study, in which 26 participants, from 11 countries, reviewed 100 digitalized H&E‐stained slides of fallopian tubes (30 cases/70 controls) with and without AI assistance, with a washout period between the sessions. We evaluated the effect of the deep‐learning model on accuracy, slide review time and (subjectively perceived) diagnostic certainty, using mixed‐models analysis. With AI assistance, we found a significant increase in accuracy (p < 0.01) whereby the average sensitivity increased from 82% to 93%. Further, there was a significant 44 s (32%) reduction in slide review time (p < 0.01). The level of certainty that the participants felt versus their own assessment also significantly increased, by 0.24 on a 10‐point scale (p < 0.01). In conclusion, we found that, in a diverse group of pathologists and pathology residents, AI support resulted in a significant improvement in the accuracy of STIC diagnosis and was coupled with a substantial reduction in slide review time. This model has the potential to provide meaningful support to pathologists in the diagnosis of STIC, ultimately streamlining and optimizing the overall diagnostic process.

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

AbstractMesonephric‐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.

PRMT5 promotes progression of endometrioid adenocarcinoma via ERα and cell cycle signaling pathways

AbstractProtein arginine methyltransferase 5 (PRMT5) has previously been reported to be upregulated in many malignant tumors. This study investigated the significance of PRMT5 in endometrial carcinoma (EC) and explored its function in tumorigenesis. Immunohistochemistry was performed to evaluate PRMT5 expression in 62 EC and 66 endometrial hyperplasia samples. The functions of PRMT5 were investigated by cell counting kit‐8, plate colony formation, wound healing, and transwell and flow cytometry assays. Quantitative reverse transcription‐polymerase chain reaction and western blotting were used to measure the expression of PRMT5, changes in estrogen receptor α (ERα), and related functional proteins. Coimmunoprecipitation was performed to examine the interaction of PRMT5 with ERα and its coactivator steroid receptor coactivator‐1 (SRC1). Compared to endometrial hyperplasia tissue, PRMT5 was overexpressed in endometrioid adenocarcinoma (EAC) but not overexpressed in mucinous EC. The main expression pattern of PRMT5 in EAC was cytoplasmic. However, the positive cases of endometrial hyperplasia showed both cytoplasmic and nuclear positivity in the endometrial glands or were mainly positive in stromal cells. Knockdown of PRMT5 significantly inhibited the growth and migration ability of EAC cells and promoted their apoptosis by regulating cyclin D1, c‐myc, p53, and Bcl2 proteins. Furthermore, PRMT5 could form a complex with ERα and SRC1 to promote the expression of ERα. In conclusion, PRMT5 plays a significant role in the progression of EAC by interacting with ERα and impacting the cell cycle signaling pathways.

Endometrial stromal sarcomas with BCOR‐rearrangement harbor MDM2 amplifications

AbstractRecently a novel subtype of endometrial stromal sarcoma (ESS) defined by recurrent genomic alterations involving BCOR has been described (HGESS‐BCOR). We identified a case of HGESS‐BCOR with a ZC3H7B‐BCOR gene fusion, which harbored an amplification of the MDM2 locus. This index case prompted us to investigate MDM2 amplification in four additional cases of HGESS‐BCOR. Tumors were analyzed for MDM2 amplification by array‐based profiling of copy number alterations (CNAs) and fluorescence in situ hybridization (FISH), as well as for MDM2 expression by immunohistochemistry (IHC). Additionally, a cohort of other mesenchymal uterine neoplasms, including 17 low‐grade ESS, 6 classical high‐grade ESS with YWHAE‐rearrangement, 16 uterine tumors resembling ovarian sex cord tumors, 7 uterine leiomyomas and 8 uterine leiomyosarcomas, was analyzed for CNAs in MDM2. Copy number profiling identified amplification of the 12q15 region involving the MDM2 locus in all five HGESS‐BCOR. Subsequent validation analyses of three tumors confirmed MDM2 amplification using MDM2 FISH. Accordingly, IHC showed MDM2 overexpression in all analyzed cases. None of the other uterine neoplasms in our series, including tumors that are in the histopathological differential diagnoses of HGESS‐BCOR, showed copy number gains of MDM2. Together, our results indicate that HGESS‐BCOR carries MDM2 amplifications, which has diagnostic implications and could potentially be used for targeted therapies in these clinically aggressive tumors.

Adult‐type granulosa cell tumor of the ovary: a FOXL2‐centric disease

AbstractAdult‐type granulosa cell tumors (aGCTs) account for 90% of malignant ovarian sex cord‐stromal tumors and 2–5% of all ovarian cancers. These tumors are usually diagnosed at an early stage and are treated with surgery. However, one‐third of patients relapse between 4 and 8 years after initial diagnosis, and there are currently no effective treatments other than surgery for these relapsed patients. As the majority of aGCTs (>95%) harbor a somatic mutation in FOXL2 (c.C402G; p.C134W), the aim of this study was to identify genetic mutations besides FOXL2 C402G in aGCTs that could explain the clinical diversity of this disease. Whole‐genome sequencing of 10 aGCTs and their matched normal blood was performed to identify somatic mutations. From this analysis, a custom amplicon‐based panel was designed to sequence 39 genes of interest in a validation cohort of 83 aGCTs collected internationally. KMT2D inactivating mutations were present in 10 of 93 aGCTs (10.8%), and the frequency of these mutations was similar between primary and recurrent aGCTs. Inactivating mutations, including a splice site mutation in candidate tumor suppressor WNK2 and nonsense mutations in PIK3R1 and NLRC5, were identified at a low frequency in our cohort. Missense mutations were identified in cell cycle‐related genes TP53, CDKN2D, and CDK1. From these data, we conclude that aGCTs are comparatively a homogeneous group of tumors that arise from a limited set of genetic events and are characterized by the FOXL2 C402G mutation. Secondary mutations occur in a subset of patients but do not explain the diverse clinical behavior of this disease. As the FOXL2 C402G mutation remains the main driver of this disease, progress in the development of therapeutics for aGCT would likely come from understanding the functional consequences of the FOXL2 C402G mutation.

Folate receptor alpha ( FRα ) expression in tubo‐ovarian and endometrial tumors: a study of 923 cases

Abstract Folate receptor alpha (FRα) is a promising therapeutic target due to its high expression in several tumor types and its rare expression in healthy tissue. Recently, the antibody‐drug conjugate mirvetuximab soravtansine has been approved for treatment of advanced platinum‐resistant high‐grade serous carcinoma (HGSC). Immunohistochemical expression of FRα has been extensively studied in HGSC, but most studies conducted before the clinical studies targeting FRα used variable antibodies and scoring criteria, which makes comparison of older literature data with recent studies difficult. Moreover, the data regarding its expression in other types of ovarian and other female genital tract tumors are limited or absent. In our study, we focused on immunohistochemical expression in 923 tubo‐ovarian and endometrial tumors (assessed on tissue microarrays), using standardized scoring criteria and the VENTANA FOLR1 CDx assay. The results of our study showed the highest FRα expression in serous carcinomas, specifically HGSC (45% positive cases), followed by low‐grade serous carcinoma (25%), endometrial serous carcinoma (11%), and serous borderline tumor (10%). Endometrioid and clear cell ovarian carcinomas showed rare positivity (2% and 1%, respectively). All other tumors examined were negative, including mucinous ovarian tumors, sex cord‐stromal tumors, endometrial endometrioid carcinomas, undifferentiated and dedifferentiated carcinomas, and endometrial clear cell carcinomas. In conclusion, these results confirm that FRα expression in HGSC and LGSC reaches similar values compared to published data, and is present in a minority of endometrial serous carcinomas. In other ovarian and endometrial tumors examined, FRα expression is absent or rare.

ATM immunohistochemistry as an effective screening method for POLE variants among endometrial carcinomas lacking mismatch repair deficiency and p53 abnormalities

Abstract The molecular classification of endometrial carcinomas (ECs) is now integrated into clinical practice. However, identification of polymerase‐ε ( POLE ) variants remains reliant on DNA sequencing, which limits broader implementation. Given the strong prognostic value of pathogenic POLE mutations and the established efficacy of immunohistochemistry (IHC) for detecting mismatch repair (MMR) deficiency and p53 abnormalities, there is a clear need for IHC‐based screening strategies to identify patients likely to carry POLE variants and prioritize them for confirmatory sequencing. In this study, we analyzed 24 cases with POLE pathogenic mutations ( POLE mut ECs), 3 with benign POLE variants, and 32 matched cases with no specific molecular profile (NSMP) from a cohort of 378 ECs. IHC evaluation of the ataxia telangiectasia mutated (ATM) protein revealed that POLE ‐mutated ECs (with pathogenic or benign POLE variants) exhibited significantly higher frequencies of non‐diffuse positive staining patterns, including null, heterogeneous positive, and subclonal loss, compared with NSMP cases. Targeted next‐generation sequencing of all exons across 474 cancer‐related genes in the 27 POLE ‐mutated ECs and 20 NSMP cases with ATM non‐diffuse positive staining patterns confirmed that POLE ‐mutated ECs typically had high tumor mutational burden and were enriched for ATM truncating variants. ATM molecular alterations, including various variant subtypes and multisite mutations, also closely correlated with these staining patterns. Based on these findings, we refined the ATM IHC interpretation framework to integrate staining patterns with sequencing data for improved molecular correlation. Specifically, the null and subclonal loss patterns showed high specificity (96.9%), positive predictive value (94.1%), and accuracy (79.7%) for identifying POLE variants. Notably, the null pattern appeared exclusively in ECs with pathogenic POLE mutations. These results suggest that ATM IHC staining is an effective screening tool for identifying patients who may benefit from confirmatory POLE sequencing among those lacking MMR deficiency or p53 abnormalities.

Spatial expression of the FGFR2b splice isoform and its prognostic significance in endometrioid endometrial carcinoma

AbstractEndometrial carcinoma (EC) is the most common gynecological malignancy and fibroblast growth factor receptor 2 (FGFR2) is a frequently dysregulated receptor tyrosine kinase. FGFR2b and FGFR2c are the two main splice isoforms of FGFR2 and are normally localized in epithelial and mesenchymal cells, respectively. Previously, we demonstrated that FGFR2c mRNA expression was associated with aggressive tumor characteristics, shorter progression‐free survival (PFS), and disease‐specific survival (DSS) in endometrioid ECs (EECs). The objectives of this study were to investigate the spatial expression of FGFR2b in normal and hyperplasia with and without atypia of human endometrium and to assess the prognostic significance of FGFR2b expression in EC. FGFR2b and FGFR2c mRNA expression was evaluated in normal (proliferative [n = 10], secretory [n = 15], and atrophic [n = 10] endometrium), hyperplasia with and without atypia (n = 19) as well as two patient cohorts of EC samples (discovery [n = 78] and Vancouver [n = 460]) using isoform‐specific BaseScope RNA in situ hybridization assays. Tumors were categorized based on FGFR2 isoform expression (one, both, or neither) and categories were correlated with clinicopathologic markers, molecular subtypes, and clinical outcomes. The FGFR2b splice isoform was exclusively expressed in the epithelial compartment of normal endometrium and hyperplasia without atypia. We observed FGFR2c expression at the basalis layer of glands in 33% (3/9) of hyperplasia with atypia. In patients with EEC, FGFR2b+/FGFR2c− expression was found in 48% of the discovery cohort and 35% of the validation Vancouver cohort. In univariate analyses, tumors with FGFR2b+/FGFR2c− expression had longer PFS (hazard ratio [HR] 0.265; 95% CI 0.145–0.423; log‐rank p < 0.019) and DSS (HR 0.31; 95% CI 0.149–0.622; log‐rank p < 0.001) compared to tumors with FGFR2b−/FGFR2c+ expression in the large EEC Vancouver cohort. In multivariable Cox regression analyses, tumors with FGFR2b+/FGFR2c− expression were significantly associated with longer DSS (HR 0.37; 95% CI 0.153–0.872; log‐rank p < 0.023) compared to FGFR2b−/FGFR2c+ tumors. In conclusion, FGFR2b+/FGFR2c− expression is associated with favorable clinicopathologic markers and clinical outcomes suggesting that FGFR2b could play a role in tailoring the management of EEC patients in the clinic if these findings are confirmed in an independent cohort.

Intra‐ and extra‐cranial BCOR‐ITD tumours are separate entities within the BCOR‐rearranged family

AbstractBCOR‐ITD tumours form an emerging family of aggressive entities with an internal tandem duplication (ITD) in the last exon of the BCOR gene. The family includes cerebral tumours, termed central nervous system BCOR‐ITD (CNS BCOR‐ITD), and sarcomatous types described in the kidney as clear cell sarcoma of the kidney (CCSK), in the endometrium as high‐grade endometrial stromal sarcoma, and in the bone and soft tissue as undifferentiated round cell sarcoma or primitive myxoid mesenchymal tumour of infancy. Based on a series of 33 retrospective cases, including 10 CNS BCOR‐ITD and 23 BCOR‐ITD sarcomas, we interrogated the homogeneity of the entity regarding clinical, radiological, and histopathological findings, and molecular signatures. Whole‐transcriptomic sequencing and DNA methylation profiling were used for unsupervised clustering. BCOR‐ITD tumours mostly affected young children with a median age at diagnosis of 2.1 years (range 0–62.4). Median overall survival was 3.9 years and progression‐free survival was 1.4 years. This dismal prognosis is shared among tumours in all locations except CCSK. Histopathological review revealed marked differences between CNS BCOR‐ITD and BCOR‐ITD sarcomas. These two groups were consistently segregated by unsupervised clustering of expression (n = 22) and DNA methylation (n = 21) data. Proximity between the two groups may result from common somatic changes within key pathways directly related to the novel activity of the ITD itself. Conversely, comparison of gene signatures with single‐cell RNA‐Seq atlases suggests that the distinction between BCOR‐ITD sarcomas and CNS BCOR‐ITD may result from differences in cells of origin.

SWI/SNF‐deficiency defines highly aggressive undifferentiated endometrial carcinoma

AbstractDedifferentiated/undifferentiated endometrial carcinoma (DDEC/UEC) is an endometrial cancer characterized by the presence of histologically undifferentiated carcinoma. Genomic inactivation of core switch/sucrose nonfermentable (SWI/SNF) complex proteins was recently identified in approximately two‐thirds of DDEC/UEC. The aim of this study was to delineate the clinical behavior of SWI/SNF‐deficient DDEC/UEC in comparison to SWI/SNF‐intact DDEC/UEC. The study cohort consisted of 56 SWI/SNF‐deficient DDEC/UEC (2 POLE‐mutated), which showed either SMARCA4 (BRG1) loss, ARID1A/1B co‐loss, or SMARCB1 (INI1) loss in the undifferentiated tumor, and 26 SWI/SNF‐intact DDEC/UEC (4 POLE‐mutated). The average age at diagnosis was 61 years for patients with SWI/SNF‐deficient tumors and 64 years for SWI/SNF‐intact tumors. Mismatch repair (MMR) protein deficiency was seen in 66% of SWI/SNF‐deficient and 50% of SWI/SNF‐intact tumors. At initial presentation, 55% of patients with SWI/SNF‐deficient tumors had extrauterine disease spread in contrast to 38% of patients with SWI/SNF‐intact tumors. The 2‐year disease specific survival (DSS) for stages I and II disease was 65% for SWI/SNF deficient tumors relative to 100% for SWI/SNF‐intact tumors (p = 0.042). For patients with stages III and IV disease, the median survival was 4 months for SWI/SNF‐deficient tumors compared to 36 months for SWI/SNF‐intact tumors (p = 0.0003). All six patients with POLE‐mutated tumors, including one with stage IV SWI/SNF‐deficient tumor were alive with no evidence of disease. Among the patients with advanced stage SWI/SNF‐deficient tumors, 68% (21 of 31) received adjuvant or neoadjuvant chemotherapy (platinum/taxane‐based) and all except the patient with a POLE‐mutated tumor (20 of 21) experienced disease progression either during chemotherapy or within 4 months after its completion. These findings show that core SWI/SNF‐deficiency defines a highly aggressive group of undifferentiated cancer characterized by rapid disease progression that is refractory to conventional platinum/taxane‐based chemotherapy. This underscores the importance of accurate clinical recognition of this aggressive tumor and the need to consider alternative systemic therapy for these tumors.

Selection of endometrial carcinomas for p53 immunohistochemistry based on nuclear features

AbstractThe World Health Organization endorses molecular subclassification of endometrial endometrioid carcinomas (EECs). Our objectives were to test the sensitivity of tumor morphology in capturing p53 abnormal (p53abn) cases and to model the impact of p53abn on changes to ESGO/ESTRO/ESP (European Society of Gynaecological Oncology/European Society for Radiotherapy and Oncology/European Society of Pathology) risk stratification. A total of 292 consecutive endometrial carcinoma resections received at Foothills Medical Centre, Calgary, Canada (2019–2021) were retrieved and assigned to ESGO risk groups with and without p53 status. Three pathologists reviewed the representative H&E‐stained slides, predicted the p53 status, and indicated whether p53 immunohistochemistry (IHC) would be ordered. Population‐based survival for endometrial carcinomas diagnosed during 2008–2016 in Alberta was obtained from the Alberta Cancer Registry. The cohort consisted mostly of grade 1/2 endometrioid carcinomas (EEC1/2; N = 218, 74.6%). One hundred and fifty‐two EEC1/2 (52.1% overall) were stage IA and 147 (50.3%) were low risk by ESGO. The overall prevalence of p53abn and subclonal p53 was 14.5 and 8.3%, respectively. The average sensitivity of predicting p53abn among observers was 83.6%. Observers requested p53 IHC for 39.4% with 98.5% sensitivity to detect p53abn (99.6% negative predictive value). Nuclear features including smudged chromatin, pleomorphism, atypical mitoses, and tumor giant cells accurately predicted p53abn. In 7/292 (2.4%), p53abn upgraded ESGO risk groups (2 to intermediate risk, 5 to high risk). EEC1/2/stage IA patients had an excellent disease‐specific 5‐year survival of 98.5%. Pathologists can select cases for p53 testing with high sensitivity and low risk of false negativity. Molecular characterization of endometrial carcinomas has great potential to refine ESGO risk classification for a small subset but offers little value for approximately half of endometrial carcinomas, namely, EEC1/2/stage IA cases.

Exploring prognostic biomarkers in pathological images of colorectal cancer patients via deep learning

AbstractHematoxylin and eosin (H&E) whole slide images provide valuable information for predicting prognostic outcomes in colorectal cancer (CRC) patients. However, extracting prognostic indicators from pathological images is challenging due to the subtle complexities of phenotypic information. We trained a weakly supervised deep learning model on data from 640 CRC patients in the prostate, lung, colorectal, and ovarian (PLCO) cancer screening trial dataset and validated it using data from 522 CRC patients in the cancer genome atlas (TCGA) dataset. We created the colorectal cancer risk score (CRCRS) to assess patient prognosis, visualized the pathological phenotype of the risk score using Grad‐CAM, and employed multiomics data from the TCGA CRC cohort to investigate the potential biological mechanisms underlying the risk score. The overall survival analysis revealed that the CRCRS served as an independent prognostic indicator for both the PLCO cohort (p < 0.001) and the TCGA cohort (p < 0.001), with its predictive efficacy remaining unaffected by the clinical staging system. Additionally, satisfactory chemotherapeutic benefits were observed in stage II/III CRC patients with high CRCRS but not in those with low CRCRS. A pathomics nomogram constructed by integrating the CRCRS with the tumor‐node‐metastasis (TNM) staging system enhanced prognostic prediction accuracy compared with using the TNM staging system alone. Noteworthy features of the risk score were identified, such as immature tumor mesenchyme, disorganized gland structures, small clusters of cancer cells associated with unfavorable prognosis, and infiltrating inflammatory cells associated with favorable prognosis. The TCGA multiomics data revealed potential correlations between the CRCRS and the activation of energy production and metabolic pathways, the tumor immune microenvironment, and genetic mutations in APC, SMAD2, EEF1AKMT4, EPG5, and TANC1. In summary, our deep learning algorithm identified the CRCRS as a prognostic indicator in CRC, providing a significant approach for prognostic risk stratification and tailoring precise treatment strategies for individual patients.

Expression of B7‐H3 and TIM‐3 in gastric‐type endocervical adenocarcinoma: prevalence, association with PD‐L1 expression, and prognostic significance

AbstractGastric‐type endocervical adenocarcinoma (GEA) is the second most common subtype of endocervical adenocarcinoma and has a poor prognosis. Anti‐programmed death‐1 and anti‐programmed death‐ligand 1 (PD‐L1) inhibitors have emerged as a major treatment option for GEA; however, data on the expression of other immune checkpoints in GEA are limited. We analyzed the expression of T‐cell immunoglobulin and mucin‐domain containing‐3 (TIM‐3) and B7 homolog 3 protein (B7‐H3) in 58 GEA and investigated their prognostic significance as well as association with PD‐L1 expression and other known prognostic factors. Applying the tumor proportion score (TPS) with a cutoff of 1%, B7‐H3 and TIM‐3 were present in 48.3% and 17.2% of cases, respectively. Applying the combined positive score (CPS) with a cutoff of 1, TIM‐3 expression was present in 70.7% of cases. Moreover, the expression of three checkpoints (B7‐H3, TIM‐3, and PD‐L1) was incompletely overlapping. Patients with B7‐H3 positive tumors (by TPS) or TIM‐3 positive tumors (by TPS) had significantly worse recurrence‐free survival (RFS) and overall survival (OS) (log‐rank). Using CPS, patients with TIM‐3 positive tumors showed significantly worse RFS (log‐rank). Similarly, B7‐H3 positivity (by TPS) and TIM‐3 positivity (by TPS) were associated with worse RFS and OS in univariate analysis. TIM‐3 positivity (by CPS) was associated with worse RFS in univariate analysis and the final Cox multivariate analysis. In conclusion, our results show that (1) B7‐H3 and TIM‐3 are frequently expressed in GEA and their expression overlaps incompletely with PD‐L1; and (2) both B7‐H3 and TIM‐3 are independent negative prognostic markers in GEA.

Combined CCNE1 high‐level amplification and overexpression is associated with unfavourable outcome in tubo‐ovarian high‐grade serous carcinoma

AbstractCCNE1 amplification is a recurrent alteration associated with unfavourable outcome in tubo‐ovarian high‐grade serous carcinoma (HGSC). We aimed to investigate whether immunohistochemistry (IHC) can be used to identify CCNE1 amplification status and to validate whether CCNE1 high‐level amplification and overexpression are prognostic in HGSC. A testing set of 528 HGSC samples stained with two optimised IHC assays (clones EP126 and HE12) was subjected to digital image analysis and visual scoring. DNA and RNA chromogenic in situ hybridisation for CCNE1 were performed. IHC cut‐off was determined by receiver operating characteristics (ROC). Survival analyses (endpoint ovarian cancer specific survival) were performed and validated in an independent validation set of 764 HGSC. Finally, combined amplification/expression status was evaluated in cases with complete data (n = 1114). CCNE1 high‐level amplification was present in 11.2% of patients in the testing set and 10.2% in the combined cohort. The optimal cut‐off for IHC to predict CCNE1 high‐level amplification was 60% positive tumour cells with at least 5% strong staining cells (sensitivity 81.6%, specificity 77.4%). CCNE1 high‐level amplification and overexpression were associated with survival in the testing and validation set. Combined CCNE1 high‐level amplification and overexpression was present in 8.3% of patients, mutually exclusive to germline BRCA1/2 mutation and significantly associated with a higher risk of death in multivariate analysis adjusted for age, stage and cohort (hazard ratio = 1.78, 95 CI% 1.38–2.26, p < 0.0001). CCNE1 high‐level amplification combined with overexpression identifies patients with a sufficiently poor prognosis that treatment alternatives are urgently needed. Given that this combination is mutually exclusive to BRCA1/2 germline mutations, a predictive marker for PARP inhibition, CCNE1 high‐level amplification combined with overexpression may serve as a negative predictive test for sensitivity to PARP inhibitors.

An analysis of HER2 amplification in cervical adenocarcinoma: correlation with clinical outcomes and the International Endocervical Adenocarcinoma Criteria and Classification

AbstractFew studies have explored HER2 status in cervical adenocarcinoma, particularly in the gastric‐type adenocarcinoma (GAC), a nonhuman‐papillomavirus‐related subtype with poor clinical outcomes. In this study, we investigated HER2 expression and amplification by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in 209 well annotated cervical adenocarcinomas diagnosed using the International Endocervical Adenocarcinoma Criteria and Classification. IHC identified HER2 protein expression in 57.4% (123/209) of adenocarcinomas, of which 62 were IHC 1+ (negative), 38 2+ (equivocal) and 23 3+ (positive). HER2 amplification was found in 13 cases (6.2%) including 10 with IHC 3+ and 3 with IHC 2+. Among all the major histotypes of cervical adenocarcinoma, HER2 amplification was most common in GAC cases with a frequency of 14.7% (5/34). Moreover, HER2 amplification was more frequently associated with 2018 International Federation of Gynecology & Obstetrics (FIGO) stage III/IV, perineural involvement and ovarian spread (p < 0.05) while IHC 3+ was more common in patients with lymphovascular invasion and ovarian involvement (p < 0.05). Survival analysis indicated that FIGO stage III/IV, GAC, and p53 overexpression were associated with poor disease‐specific survival and tumor recurrence (p < 0.05). In conclusion, HER2 amplification was present in a subset of adenocarcinomas, and more common in GAC, pointing to a potential benefit from trastuzumab treatment. HER2 overexpression does not identify gene amplification status in cervical adenocarcinoma; therefore, FISH is suggested for both IHC positive and equivocal cases. Further investigation on more cases with longer follow‐up times is required to consolidate these findings.

p53 and ovarian carcinoma survival: an Ovarian Tumor Tissue Analysis consortium study

AbstractOur 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.

Longitudinal analysis of a secondary BRCA2 mutation using digital droplet PCR

AbstractDevelopment of resistance to platinum and poly(ADP‐ribose) polymerase inhibitors via secondary BRCA gene mutations that restore functional homologous recombination has been observed in a number of cancer types. Here we report a case of somatic BRCA2 mutation in a patient with high grade serous ovarian carcinoma. A secondary mutation predicted to restore the BRCA2 open reading frame was detected at low frequency (2.3%) in whole exome sequencing of a peritoneal biopsy at disease progression after treatment that included carboplatin and olaparib. We used digital droplet PCR (ddPCR) to verify the presence and frequency of this mutation in the biopsy sample at progression and also used this approach to assess the presence of the secondary mutation in preceding biopsies at diagnosis and first relapse. We found no evidence for the secondary mutation being present prior to the final progression biopsy, suggesting that this mutation was acquired late in the course of treatment. ddPCR provides a sensitive and specific technique to investigate the presence of low frequency mutations in a time series of biopsies.

STING pathway expression in low‐grade serous carcinoma of the ovary: an unexpected therapeutic opportunity?

AbstractOvarian carcinoma histotypes are distinct diseases with variable clinical outcomes and response to treatment. There is a need for new subtype‐specific treatment modalities, especially for women with widespread and chemo‐resistant disease. Stimulator of interferon genes (STING) is a part of the cGAS–STING pathway that mediates innate immune defence against infectious DNA‐containing pathogens and also detects tumour‐derived DNA and generates intrinsic antitumour immunity. The STING signalling pathway is suppressed by several mechanisms in a variety of malignant diseases and, in some cancers that may be a requirement for cellular transformation. The aim of this study was to use immunohistochemistry to evaluate STING protein expression across normal tissue, paratubal and ovarian cysts, and ovarian tumour histotypes including ovarian carcinomas. Herein, we show that the fallopian tube ciliated cells express STING protein, whereas the secretory cells are negative. STING expression differs among ovarian cancer histotypes; low‐grade serous ovarian carcinomas and serous borderline tumours have uniform high STING expression, while high‐grade serous and endometrioid carcinomas have heterogeneous expression, and clear cell and mucinous carcinomas show low expression. As low‐grade serous carcinomas are known to be genomically stable and typically lack a prominent host immune response, the consistently high STING expression is unexpected. High STING expression may reflect pathway activation or histogenesis and the mechanisms may be different in different ovarian carcinoma histotypes. Further studies are needed to determine whether the STING signalling pathway is active and whether these tumours would be candidates for therapeutic interventions that trigger innate immunity activation.

Homologous recombination deficiency is inversely correlated with microsatellite instability and identifies immunologically cold tumors in most cancer types

AbstractHomologous recombination deficiency (HRD) leads to DNA double‐strand breaks and can be exploited by the use of poly (ADP‐ribose) polymerase (PARP) inhibitors to induce synthetic lethality. Extending the original therapeutic concept, the role of HRD is currently being investigated in clinical trials testing immune checkpoint blockers alone or in combination with PARP inhibitors, but the relationship between HRD and immune cell context in cancer is incompletely understood. We analyzed the association between immune cell composition, gene expression, and HRD in 9,041 tumors of 32 solid cancer types from The Cancer Genome Atlas (TCGA). The numbers of genomic scars were quantified by the HRD sum score (HRDsum) including loss of heterozygosity, large‐scale state transitions, and telomeric allelic imbalance. The T‐cell inflamed gene expression profile correlated weakly, but significantly positively, with HRDsum across cancer types (ρ = 0.17). Within individual cancer types, a significantly positive correlation was observed only in breast cancer, ovarian cancer, and four other cancer types, but not in the remaining 26 cancer types. HRDsum and tumor mutational burden (TMB) correlated significantly positively across cancer types (ρ = 0.42) and within 18 cancer types. HRDsum and a proliferation metagene correlated significantly positively across cancer types (ρ = 0.52) and within 20 cancer types. Mismatch repair deficiency and HRD as well as proofreading deficiency showed a high level of exclusivity. High HRD scores were associated with an immunologically activated tumor microenvironment only in a minority of cancer types. Our data favor the combination of genetic markers, complex genomic markers (including HRDsum and TMB), and other molecular markers (including proliferation scores) for a precise and comprehensive read‐out of the tumor biology and an individually tailored treatment.

High cyclin E1 protein, but not gene amplification, is prognostic for basal‐like breast cancer

AbstractBasal‐like breast cancer (BLBC) has a greater overlap in molecular features with high‐grade serous ovarian cancer (HGSOC) than with other breast cancer subtypes. Similarities include BRCA1 mutation, high frequency of TP53 mutation, and amplification of CCNE1 (encoding the cyclin E1 protein) in 6–34% of cases, and these features can be used to group patients for targeted therapies in clinical trials. In HGSOC, we previously reported two subsets with high levels of cyclin E1: those in which CCNE1 is amplified, have intact homologous recombination (HR), and very poor prognosis; and a CCNE1 non‐amplified subset, with more prevalent HR defects. Here, we investigate whether similar subsets are identifiable in BLBC that may allow alignment of patient grouping in clinical trials of agents targeting cyclin E1 overexpression. We examined cyclin E1 protein and CCNE1 amplification in a cohort of 76 BLBCs and validated the findings in additional breast cancer datasets. Compared to HGSOC, CCNE1 amplified BLBC had a lower level of amplification (3.5 versus 5.2 copies) and lower relative cyclin E1 protein, a lack of correlation of amplification with expression, and no association with polyploidy. BLBC with elevated cyclin E1 protein also had prevalent HR defects, and high‐level expression of the cyclin E1 deubiquitinase ubiquitin‐specific protease 28 (USP28). Using a meta‐analysis across multiple studies, we determined that cyclin E1 protein overexpression but not amplification is prognostic in BLBC, while both cyclin E1 overexpression and amplification are prognostic in HGSOC. Overall CCNE1 gene amplification is not equivalent between BLBC and HGSOC. However, high cyclin E1 protein expression can co‐occur with HR defects in both BLBC and HGSOC, and is associated with poor prognosis in BLBC.