Irene Carretero‐Barrio

Single‐nucleus RNA sequencing identifies a novel tenogenic heterologous differentiation in endometrial carcinosarcomas: implications for diagnosis and tumor classification

Abstract Carcinosarcomas (CSs) are aggressive biphasic tumors characterized by epithelial and mesenchymal components, whose histogenesis and differentiation dynamics remain poorly understood. We present single‐nucleus RNA sequencing (snRNA‐seq) analysis of six CSs (five endometrial and one ovarian) and two normal endometrial samples, profiling over 96,298 cells. By integrating transcriptomic data with inferred copy number variations (CNVs), immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and in situ hybridization (ISH) validation, we resolved the complex cellular architecture of these tumors, identified lineage‐specific programs, and revealed unexpected differentiation trajectories. snRNA‐seq was used to further refine the histopathological classification of three cases by uncovering heterologous differentiation not previously recognized: one rhabdomyogenic, one osteogenic, and, notably, one exhibiting a novel tenogenic program, defined by the expression of SCX , MKX , and TNMD . All CSs displayed a prominent mesenchymal compartment comprising both undifferentiated fibroblast‐like cells and distinct lineage committed populations, including rhabdomyoblasts (Rhab), tenoblasts (Teno), osteoblasts (Osteo), and chondroblasts (Chond). In some tumors, multiple mesenchymal identities co‐existed, and in others, differentiation gradients (e.g. immature versus mature rhabdomyoblasts) were observed. These patterns underscore the cellular plasticity and multilineage potential of the sarcomatous component. Furthermore, the expression of specialized interface markers ( COL22A1 , NCAM1 , ACAN , CHRNG , MUSK ) suggests that some tumors use structured developmental programs reminiscent of the muscle–tendon junction, enthesis, or neuromuscular junction. CNV analysis revealed tumor‐specific genomic alterations with clonal and subclonal patterns linked to differentiation state, which were validated by FISH. Altogether, this study demonstrates that CSs are not static biphasic tumors but rather complex ecosystems with extensive developmental plasticity. Our findings redefine their classification and support the use of single‐nucleus approaches to uncover hidden differentiation trajectories in highly heterogeneous cancers, including the discovery of a previously unreported tenogenic lineage. Our results challenge the diagnosis of homologous CS when only morphological criteria are applied. © 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Histologic and Molecular Type Changes in Endometrial Cancer Recurrences in Comparison With Their Corresponding Primary Tumors

In this study, molecular alterations in endometrial carcinoma (EC) recurrences were analyzed. We aimed to identify genes implicated in tumor progression and to evaluate whether histologic and molecular type shifting occurs in recurrences. Thus, we analyzed 50 samples corresponding to 24 primary ECs (15 low-grade endometrioid endometrial carcinomas [LG-EECs] and 9 high-grade endometrial carcinomas) and their corresponding 26 recurrences. These were studied by immunohistochemistry, next-generation sequencing, and MLH1 promoter methylation. We observed shared mutations in all primary tumors and their recurrences, indicating a clonal relationship between both lesions. Most morphologic and molecular changes associated with progression were found in LG-EEC. In this group, 6 patients (40%) presented additional mutations in the recurrence. These mutations more frequently affected genes of the PI3K/AKT/PTEN pathway, implicating this pathway not only in tumor initiation but also in progression. In addition, 2 patients (13%) in which the primary tumor belonged to the nonspecific molecular profile subtype, shifted to the mismatch repair deficient (MMRd) subtype after the acquisition of MLH1 promoter methylation in the recurrence lesions. In 3 patients (20%) with MMRd, there was a change from LG-EEC to G3-EEC. One TP53-mutated LG-EEC transformed into an undifferentiated carcinoma in a mediastinal lymph node metastasis after losing the expression of SMARCA2 while preserving SMARCA4 and SMARCB1. Morphologic and molecular changes in EC recurrences, especially dedifferentiation and the acquisition of MMRd, should be considered for a correct diagnosis and treatment. MMRd should be tested in metastatic lesions, if available, in patients with primary tumors reported to be of a molecular subtype different from MMRd.

The Prognostic Significance of Tumor-Infiltrating Lymphocytes, PD-L1, BRCA Mutation Status and Tumor Mutational Burden in Early-Stage High-Grade Serous Ovarian Carcinoma—A Study by the Spanish Group for Ovarian Cancer Research (GEICO)

Early stages are under-represented in studies on the molecular and immune features of high-grade serous ovarian carcinoma (HGSOC), and specific studies focused on early-stage HGSOC are required for a better prognostic stratification and to personalize chemotherapy. The aim of this study was to determine the prognostic significance of CD8+ and CD4+ tumor-infiltrating lymphocytes (TILs), tumoral cell PD-L1 expression, BRCA mutational status and tumor mutation burden (TMB) in early-stage HGSOC. A retrospective study was performed on stage I and II HGSOC from the Molecular Reclassification of Early Stages of Ovarian Cancer (RECLAMO) cohort from the Spanish Group of Ovarian Cancer Research (GEICO). Centralized histological typing was performed based on morphological and immunohistochemical features. Intraepithelial (i) and stromal (s) CD8+ and CD4+ T cells and PD-L1 were evaluated on tissue microarrays by immunohistochemistry. BRCA1 and BRCA2 mutation status and TMB were analyzed in tumor DNA using next-generation sequencing. The study included 124 tumors. High iCD8+ (>20 TILs/core), low/intermediate CD4+ (<20 TILs/core) and high CD8+/CD4+ ratio (>35/core) were associated with favorable outcomes. Tumor cell PD-L1 expression (TPS ≥ 1) was present in only 8% of tumors. In total, 11 (16%) and 6 (9%) out of 69 HGSOC tested carried pathogenic or likely pathogenic BRCA1 or BRCA2 mutations, respectively. Median TMB of 40 tumors analyzed was 5.04 mutations/Mb and only 6 tumors had 10 or more mutations/Mb. BRCA status and TMB were not associated with TILs or prognosis. When compared with studies on advanced HGSOC, our results suggested that prognostic variables differed according to stage and that more studies focused on early stages of HGSOC are needed to better stratify these tumors.

Immune‐related gene expression signatures: a step forward in the stratification of patients with ovarian clear cell carcinoma †

Abstract Clear cell carcinoma of the ovary (CCC) is one of the five major histological types of ovarian cancer and presents specific clinicopathological features, such as a higher prevalence in the Asian population and a poor response to conventional chemotherapy. In a recent publication in The Journal of Pathology , Heong, Tan, Miwa et al demonstrated the heterogeneity of the immune landscape of CCC. They reported the immune signatures observed in a large cohort of CCC, including tumours from both Asian and Caucasian women. The authors analysed three cohorts from Europe, Japan, and Singapore, with a total of 246 tumours, and evaluated 730 immune‐related genes using NanoString technology. The study revealed four main transcriptional subtypes characterised by the expression of specific sets of genes: PD1‐high (11%), CTLA4‐high (29%), antigen‐presentation (42%), and a pro‐angiogenic subtype (18%). The two main conclusions of the study were: (1) that CCCs in women of Asian and Caucasian descent share significant molecular similarities, since all four molecular signatures are present in all the cohorts analysed, without any evident differences in frequency; and (2) that the PD1‐high and CTLA4‐high subtypes were associated with worse clinical outcomes and may be useful when stratifying treatment in early‐stage tumours. The immune signature could represent a promising biomarker of immunotherapy response if future prospective studies confirm it. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

MDM4 amplification as a potential predictor of risk of recurrence in early-stage low-grade endometrial carcinoma

This study aimed to assess the frequency of MDM4 amplification in endometrial carcinomas and its association with clinicopathological features, particularly relapse risk in early-stage low-grade endometrioid endometrial carcinoma. We conducted a case-control study including 110 stage I to II low-grade endometrioid endometrial carcinomas (39 relapsing and 71 non-relapsing tumors) and an additional cohort of 82 high-grade endometrial carcinomas. An independent retrospective cohort of 194 early-stage low-grade endometrioid endometrial carcinomas was used to validate the results. MDM4 amplification was analyzed by fluorescent in situ hybridization. Gene expression of MDM4 and GADD45A was measured by quantitative real-time polymerase chain reaction. Immunohistochemistry assessed hormone receptor status, p53, and mismatch repair proteins. POLE and CTNNB1 mutations were evaluated by Sanger sequencing. MDM4 amplification was detected in 31 of 186 tumors (16.7%): 14 of 104 low-grade endometrioid endometrial carcinomas (13.5%) and 17 of 82 high-grade endometrial carcinomas (20.7%). Among low-grade endometrioid endometrial carcinomas, amplification was significantly more frequent in relapsing tumors (11/38, 28.9%) than in non-relapsing ones (3/66, 4.5%) (p <.01). Concordant MDM4 amplification status was observed between biopsies and hysterectomy samples. In some relapsing cases, amplification emerged during disease progression. Multi-variable Cox regression identified MDM4 amplification as an independent predictor of relapse, alongside lymphovascular space invasion, tumor necrosis, and myometrial invasion. In the independent cohort, MDM4 amplification was associated with progression-free survival. Our study suggested that MDM4 amplification is a potential predictor of the risk of recurrence in early-stage low-grade endometrioid endometrial carcinoma that can be easily evaluated in endometrial samples.