BACKGROUND:High-grade serous ovarian carcinoma (HGSOC) presents a significant therapeutic challenge. Late-stage disease is frequently associated with peritoneal carcinomatosis. The peritoneal metastases exhibit a unique tumour microenvironment (TME) distinct from the primary tumours and other metastatic sites. Understanding the critical influence of the extracellular matrix (ECM) in shaping the tumour phenotype is essential for the development of effective new therapies. METHODS:This study introduces a three-dimensional (3D) model of HGSOC peritoneal metastases using a porcine decellularised peritoneal-derived ECM scaffold, referred to as peritoneal matrix (PerMa). FINDINGS:We show that the decellularisation maintains the structural integrity and composition of ECM molecules. Comparative analysis reveals structural, compositional, and mechanical similarities between porcine and human peritoneal matrices, underscoring the porcine model's translational relevance for modelling human peritoneum physiology. The PerMa supports the 3D growth of HGSOC cell lines. The model enables the assessment of sensitivity to traditional chemotherapy and novel cell-based immunotherapy through confocal imaging and quantification of cell volume. INTERPRETATION:Our model offers a valuable platform for investigating peritoneal carcinomatosis in HGSOC, with the potential to contribute significantly to developing novel therapeutic approaches. FUNDING:Financial support was provided by the University of Bergen, Helse Vest RHF (F-12183-D10616, 779, 911182, 912035, and 912146), Helse Bergen HF (240222), the Norwegian Cancer Society (6833652 and 182735), the Research Council of Norway grants (250317, 326300, 223250, 262652, and 295910), the Novo Nordisk Foundation (NNF21OC0070381), the Kolbjørn Brambani Legat for Kreftforskning, the National Institute of Health (R01CA199646) and the Swedish Cancer Society (21 1888 Pj).

BACKGROUND:Effusion specimens are frequently encountered in cytopathology practice. As in surgical pathology, candidate markers for effusion diagnosis require thorough assessment prior to inclusion in diagnostic panels. This review discusses some of the currently used markers, with a focus on immunohistochemistry. SUMMARY:A literature search of relevant publications, with a focus on markers with an established diagnostic role. BRCA1-associated protein 1 (BAP1) and methylthioadenosine phosphorylase (MTAP) protein expression by immunohistochemistry has been shown to be useful in the differentiation of reactive mesothelial proliferations from mesothelioma, as well as in differentiating the latter from metastatic carcinoma, in multiple studies. Molecular analysis of CDKN2A, MTAP, BAP1, and NF2 status has been additionally applied to the differential diagnosis of mesothelioma from reactive mesothelium. Robust markers for the differentiation between mesothelioma and carcinoma, such as TAG-72/B72.3, claudin-4, calretinin and HEG1, are available, as are markers that support a specific origin for metastatic adenocarcinoma, such as PAX8, TTF1 and TRPS1. KEY MESSAGES:Effusion diagnosis remains central to staging, and consequently, prognosis of cancer patients. Effusions additionally constitute optimal material for predictive studies and thereby guide the choice of targeted therapy.

Genomic instability in high-grade serous tubo-ovarian carcinoma (HGSTOC) can generate fusion genes with potential roles in tumor evolution and clinical relevance. HGSTOCs commonly disseminate prior to diagnosis, but most studies analyze only one tumor sample per patient, limiting understanding of fusion gene dynamics. Here, RNA sequencing was performed on 108 tumor samples from multiple intra‑abdominal sites in 23 patients. A consensus approach involving three fusion callers identified 170 high-confidence fusion genes present in all samples from each patient, most of which were non‑recurrent and not in-frame.Protein kinases were significantly enriched among fusion partners, and commonly retained intact catalytic domains. Several of the kinase fusion genes may have oncogenic relevance, including AKAP8L::BRD4 (recurrent in three patients), GPBP1L1::MKNK1, SPAG16::ERBB4, UGP2::MAPK4, and STRN3::PRKD1. Four fusions involved homologous recombination-related genes, including FUT10::SPIDR, RAD54B::VIRMA, RAD54L::PLXNA2, and SMARCAD1::BOD1L1. Integration of these fusion genes with other somatic alterations in homologous recombination-related genes, deficiency signatures, and germline BRCA1/2 variants increased the involvement of the homologous recombination pathway to 15 HGSTOCs (65%). Overall, HGSTOC has a heterogeneous fusion gene landscape, and multi-site sampling with consensus calling offers a robust strategy for resolving fusion profiles.

Background: Mesothelial proliferations range from reactive lesions to benign and malignant tumors, bearing witness to the plasticity of these cells. Their diagnosis often requires combination of morphological assessment, immunohistochemistry, and molecular testing. Summary: Considerable progress has been made in recent years in all the above aspects. The separation of reactive from neoplastic mesothelial proliferations has become more robust. Mesothelioma in situ is now recognized as precursor of invasive mesothelioma and better tools exist for its differentiation from benign mimics. Differentiating mesothelial tumors from other malignancies, including metastatic carcinomas, sarcomas, and other tumors, is now more easily achieved. Genetic conditions which predispose to development of mesothelioma at a young age have been identified and characterized. These issues are discussed in this review. Key messages: Mesothelial pathology is an evolving field and more knowledge of these processes is likely to be gained in coming years. The diagnosis of these tumors requires experts with subspecialty in cytology, thoracic, soft tissue/bone, and gynecologic pathology, cooperating with surgeons, radiologists, and oncologists at specialized centers.

INTRODUCTION:FAT1 is mutated in many types of cancer. The objective of this study was to analyze the diagnostic role of FAT1 protein in cancers affecting the serosal cavities. METHODS:FAT1 protein expression by immunohistochemistry was analyzed in 256 effusions (139 peritoneal, 116 pleural, 1 pericardial), consisting of 96 tubo-ovarian carcinomas, 56 breast carcinomas, 44 mesotheliomas, 28 uterine corpus and cervical carcinomas, 15 lung carcinomas, 13 gastrointestinal carcinomas, and 4 cancers of other origin. RESULTS:FAT1 expression was most common in gastrointestinal carcinomas (12/13; 92%), followed by lung (9/15; 60%), breast (31/56; 55%), and uterine cervical (4/8; 50%) carcinomas, with infrequent expression in other tumors, including tubo-ovarian carcinomas (5/96; 5%). Mesotheliomas were uniformly negative. Staining of a limited series of surgical specimens showed comparable results for patient-matched breast carcinomas (29/41; 71%), whereas tubo-ovarian carcinomas were more frequently positive (16/57; 28%), though often focally, compared to effusions. Eight epithelioid mesotheliomas were negative (0/8; 0%). Survival analysis for 42 breast carcinoma patients with effusion for whom clinical data were available showed no association with overall (p = 0.398) or disease-free (p = 0.255) survival. CONCLUSION:FAT1 is commonly expressed in carcinomas of gastrointestinal, lung, or breast origin but is rarely expressed in tubo-ovarian carcinoma effusions and is absent in mesothelioma. Whether this difference is of use in the diagnostic setting remains to be established.

The objective of this study was to analyze the expression and prognostic role of cancer-associated proteins in uterine leiomyosarcoma (uLMS). p53, DAXX, ATRX, HMGA2, IMP3, Stathmin, and phospho-Stathmin (p-Stathmin) protein expression by immunohistochemistry was analyzed in tissue microarrays from 244 uLMS. Expression was assessed for association with clinicopathologic parameters in 173 patients with available data. Tissue microarrays were informative in 230 cases. p53 was aberrant in 44% of tumors. DAXX, ATRX, HMGA2, IMP3, and Stathmin were expressed in 90%, 55%, 40%, 33%, and 97% uLMS, respectively. Cytoplasmic and nuclear p-Stathmin staining was seen in 77% and 68% of tumors, respectively. Stathmin expression was significantly related to higher mitotic count ( P < 0.001), a higher degree of atypia ( P = 0.006), and vascular invasion ( P = 0.016), whereas p-Stathmin expression was significantly related to advanced stage ( P < 0.001), higher mitotic count ( P < 0.001), and vascular invasion ( P = 0.001). In univariate survival analysis for 165 patients with informative tissue microarrays, aberrant p53 ( P = 0.026) and higher IMP3 ( P = 0.024), Stathmin ( P < 0.001), cytoplasmic p-Stathmin ( P < 0.001), and nuclear p-Stathmin ( P < 0.001) expression was associated with poor disease-specific survival. Clinicopathologic parameters significantly related to poor disease-specific survival were older age ( P = 0.006), extrauterine disease at diagnosis (International Federation of Gynecology and Obstetrics (FIGO) stage ≥2; P < 0.001), high mitotic count ( P = 0.02), and grade 2 to 3 atypia ( P = 0.017). In multivariate analysis, age ( P = 0.002), FIGO stage ( P < 0.001), and Stathmin expression ( P < 0.001) were independent prognosticators. Stathmin was the only prognosticator in a multivariate analysis limited to patients with FIGO stage I disease ( P = 0.013). In conclusion, Stathmin expression is strongly associated with poor survival in uLMS and may be a new prognostic marker in this malignancy.

Background: Patients with high-grade serous carcinoma (HGSC) are commonly diagnosed at late disease stages and after primary tumors have disseminated in the peritoneum. The overexpression of tight junction proteins has been associated with poor prognosis in this setting, potentially reflecting the tumors adaptive changes in the disease cascade. Methods: By performing immunohistochemistry in a large single-center cohort of a total of 705 HGSC, we test the hypothesis that the protein expression of PReferentially expressed Antigen of MElanoma (PRAME) contains prognostic, predictive or clinically translatable information. We further examine its co-expression with tight junction proteins. Results: We confirmed the nuclear expression of PRAME in 442 (63 %) of specimens with comparable expression levels in peritoneal and pleural effusions (p = 0.72), and in effusions versus surgical specimens (p = 0.339). In effusions, any degree of expression of PRAME was significantly associated with suboptimal debulking surgery during primary treatment (p = 0.034). In surgical specimens, higher expression of PRAME was significantly linked to more advanced FIGO stage (p = 0.021). PRAME expression was not associated with other clinicopathologic factors as age, CA125 levels, chemoresistance or survival, but correlated with PRAME mRNA levels. Significant correlation was found between expression levels of PRAME and the tight junction protein Occludin (p = 0.002). Conclusion: Taken together, our study confirms PRAME to be expressed in the majority of HGSC effusions and surgical samples. The association of high levels of PRAME expression with incomplete surgical resection status and advanced stage disease may suggest PRAME expression as adaptative mechanism during disease dissemination. This finding warrants confirmation in independent series.

Accurate identification of tumor-specific markers is vital for developing chimeric antigen receptor (CAR)-based therapies. While cell surface antigens are seldom cancer-restricted, their post-translational modifications (PTMs), particularly aberrant carbohydrate structures, offer attractive alternatives. Among these, the sialyl-Tn (STn) antigen stands out for its prevalent presence in various epithelial tumors. Although monoclonal antibodies (mAbs) against STn have been developed, their clinical application has been hindered by concerns regarding specificity. Herein, we describe AM52.1, a mAb with unprecedented specificity for STn and lack of reactivity with healthy tissues. The single-chain variable fragment (scFv) of AM52.1 was assembled into a second-generation CAR scaffold. AM52.1CAR T cells efficiently targeted STn-expressing cancer cell lines and patient-derived organoids (PDOs), while sparing STn-negative cells. In further preclinical models, AM52.1CAR T cells robustly controlled gastric and tubo-ovarian tumors, as well as colorectal cancer mucinous peritoneal metastases, highlighting their strong therapeutic potential for targeting and managing complex solid tumors.

Introduction: Grainyhead-like 2 (GRHL2) regulates epithelial-to-mesenchymal transition (EMT) in cancer. This study analyzed the expression and prognostic role of GRHL2 in high-grade serous carcinoma (HGSC). METHODS:GRHL2 protein expression by immunohistochemistry was analyzed in 411 HGSC (198 effusions, 213 surgical specimens). Expression score was generated by combination of staining extent and intensity and was assessed for association with clinicopathologic parameters and survival. RESULTS:GRHL2 expression was significantly higher in effusions compared to surgical specimens in both analysis of all specimens (p < 0.001) and patient-matched tumors (n = 39 patients; p < 0.001). Expression was additionally higher in post-chemotherapy compared to chemo-naive effusions (p < 0.001). Higher GRHL2 score in effusions was associated with a trend for shorter overall survival (OS; p = 0.088). Higher GRHL2 score in surgical specimens was significantly related to nonoptimal (>0 cm) debulking (p = 0.004), non-complete chemoresponse at diagnosis (p = 0.003), primary chemoresistance (p = 0.045), and shorter OS (p = 0.038) and progression-free survival (PFS; p = 0.024). Both OS and PFS findings remained significant in Cox multivariate analysis (OS: p = 0.048; PFS: p = 0.04). CONCLUSION:GRHL2 is overexpressed in HGSC effusions compared to solid lesions, possibly reflecting altered EMT status. However, high expression in solid lesions is associated with chemoresistance and poor survival, possibly due to mediation of tumor cell migration and invasion. .

OBJECTIVE:Lymphovascular invasion can predict nodal spread and recurrence in endometrioid endometrial cancer; however, the impact of lymphovascular invasion quantification on local versus distant recurrence in surgically staged patients has not yet been established. METHODS:This multicenter, retrospective cohort study included surgically staged patients with International Federation of Obstetrics and Gynecology 2009 stage I node-negative endometrioid endometrial cancer. Patients were treated between January 2012 and December 2019 at 2 tertiary cancer centers. Staging included a total hysterectomy and lymph node assessment. The extent of lymphovascular invasion was defined using the World Health Organization criteria as focal (<5 vessels involved on at least 1 pathology slide) or substantial (≥5 vessels involved). Recurrence and death were considered as events. A competing risk analysis was performed and controlled for multicenter clustering. RESULTS:Overall, 1555 patients met the inclusion criteria: 65 (4.2%) had substantial invasion, 119 (7.7%) had focal, and 1371 (88.2%) had no invasion. The median follow-up was 61.5 months (range; 0.8-133.9). There were 173 evaluable events among the 1554 patients: 56 local recurrences, 43 distant recurrences, and 74 deaths without recurrence. Deep (>50%) myoinvasion and grade 3 histology were more frequently observed in patients with substantial myoinvasion. Overall, 323 patients (20.8%) received adjuvant therapy. The 5-year cumulative incidence failure rates for any recurrence were 6.0% for no, 19.5% for focal, and 19.0% for substantial invasion. Compared to no lymphovascular invasion, substantial invasion was associated with an increased risk of distant recurrence (adjusted HR 2.29, 95% CI 1.17 to 4.46). CONCLUSIONS:In patients with surgical stage I endometrioid endometrial cancer, the focal and substantial lymphovascular invasion was associated with a 3-fold increased risk of cumulative incidence failure versus no lymphovascular invasion. Patients with substantial invasion had more deeply invasive and grade 3 tumors and appeared to experience more distant than local recurrences. These findings challenge the International Federation of Obstetrics and Gynecology 2023 staging classification that combines no lymphovascular invasion and focal lymphovascular invasion into a single risk category.

OBJECTIVE:At present, no reliable blood-based biomarkers have been established for patients with endometrial cancer. Liquid biopsies, which can detect circulating tumor DNA (ctDNA), provide a non-invasive way to assess prognosis, monitor tumor evolution and treatment response. We aimed to examine the feasibility and performance of ctDNA as a prognostic tool in a multi-center cohort of EC patients with matched tumor samples. METHODS:Blood plasma samples were collected preoperatively from 83 patients at three European cancer centers. Circulating cell-free DNA (cfDNA) was isolated and analyzed using the Oncomine™ Pan-Cancer cell-free assay. Tumor tissue from 56 of the 83 patients was subjected to whole-exome sequencing, and clinical data were collected for oncological outcome assessment. RESULTS:The mean input of cfDNA was 8.17 ng (range 1.47-29.12 ng). Sixteen (19.3 %) patients were considered ctDNA positive with mutations in one or more genes. Most alterations detected in plasma were concordant with mutations found in the matched tumor for the paired cases. The preoperative presence of ctDNA was associated with a significantly higher rate of recurrence (37.5 % vs 11.9 %, P = 0.024). Although eight of the 14 (57 %) patients with recurrence were negative for ctDNA at diagnosis, positive ctDNA status remained an independent predictor of recurrence also when controlling for other known histopathologic risk factors (HR 5.49, 95 % CI 1.5-20, P = 0.010). CONCLUSIONS:Our results demonstrated the feasibility of using an off-the-shelf gene panel to detect ctDNA in patients with endometrial cancer. ctDNA positivity was significantly associated with worse oncological outcomes.

The objective of the present study was to characterize the molecular features of endometrial carcinomas with ambiguous histology. Eighteen carcinomas that could not be conclusively typed based on morphology and immunohistochemistry underwent analysis of mismatch repair (MMR) status, microsatellite status, and whole-exome sequencing. None of the tumors had pathogenic POLE mutation. Twelve tumors (67%) were microsatellite stable, and 6 (33%) had microsatellite instability. Fourteen tumors (78%) harbored TP53 mutations, and 2 (11%) had mutations in MMR genes. Eleven carcinomas (61%) were classified as copy number high and 7 (39%) as MSI-hypermutated, the latter including 3 tumors with TP53 mutation who concomitantly had MSI or mutation in a MMR gene. Other mutations that were found in > 1 tumor affected MUC16 (7 tumors), PIK3CA (6 tumors), PPP2R1A (6 tumors), ARID1A (5 tumors), PTEN (5 tumors), FAT1 (4 tumors), FAT4 (3 tumors), BRCA2 (2 tumors), ERBB2 (2 tumors), FBXW7 (2 tumors), MET (2 tumors), MTOR (2 tumors), JAK1 (2 tumors), and CSMD3 (2 tumors). At the last follow-up (median = 68.6 months), 8 patients had no evidence of disease, 1 patient was alive with disease, 8 patients were dead of disease, and 1 patient died of other cause. In conclusion, based on this series, the molecular landscape of endometrial carcinomas with ambiguous histology is dominated by TP53 mutations and the absence of POLE mutations, with heterogeneous molecular profile with respect to other genes. A high proportion of these tumors is clinically aggressive.

Carcinosarcoma (CS) is an uncommon and clinically aggressive malignancy. The objective of the present study was to characterize the molecular features of CS at various anatomic locations, including serous effusions. Specimens (n = 32) consisted of 25 biopsies/surgical resection specimens and 7 serous effusions (6 peritoneal, 1 pleural) from 25 patients. Fresh-frozen cell pellets and surgical specimens underwent targeted next-generation sequencing covering 50 unique genes. A total of 31 mutations were found in 25 of the 32 tumors studied, of which 1 had 3 mutations, 4 had 2 different mutations, and 20 had a single mutation. The most common mutations were in TP53 (n = 25 in 24 tumors; 1 tumor with 2 different mutations), with less common mutations found in RB1 (n = 2), MET (n = 1), KRAS (n = 1), PTEN (n = 1), and KIT (n = 1). Patient-matched specimens harbored the same TP53 mutation. Tumors with no detected mutations were more common in serous effusion specimens (3/7; 43%) compared with surgical specimens (4/25; 16%). In conclusion, the molecular landscape of CS is dominated by TP53 mutations, reinforcing the observation that the majority of these tumors develop from high-grade serous carcinoma. Whether CS cells in serous effusions differ from their counterparts in solid lesions remains uncertain.

The European Society of Gynaecological Oncology, the European Society for Medical Oncology (ESMO) and the European Society of Pathology held a consensus conference (CC) on ovarian cancer on 15-16 June 2022 in Valencia, Spain. The CC panel included 44 experts in the management of ovarian cancer and pathology, an ESMO scientific advisor and a methodologist. The aim was to discuss new or contentious topics and develop recommendations to improve and harmonise the management of patients with ovarian cancer. Eighteen questions were identified for discussion under four main topics: (i) pathology and molecular biology, (ii) early-stage disease and pelvic mass in pregnancy, (iii) advanced stage (including older/frail patients) and (iv) recurrent disease. The panel was divided into four working groups (WGs) to each address questions relating to one of the four topics outlined above, based on their expertise. Relevant scientific literature was reviewed in advance. Recommendations were developed by the WGs and then presented to the entire panel for further discussion and amendment before voting. This manuscript focuses on the recommendation statements that reached a consensus, their voting results and a summary of evidence supporting each recommendation.