Eugenia Fraile-Bethencourt

Objective: To systematically review the literature on the impact of integrated molecular classification of endometrial cancer on diagnosis, prognostic stratification, and clinical management. Methods: A systematic search was conducted in PubMed/MEDLINE, Web of Science, Scopus, and Cochrane Library (January 2013–December 2025), following PRISMA 2020 guidelines. We included original studies, systematic reviews, clinical guidelines, and cost-effectiveness analyses applying TCGA molecular classification or its surrogate classifiers (ProMisE, TransPORTEC). Data were extracted on subtype distribution, survival outcomes, hazard ratios, stage migration, and therapeutic implications. Results: Fifty studies were included. Of these, 13 provided data on molecular subtype distribution (comprising 3527 patients), 8 reported survival outcomes, and 7 reported hazard ratios. The weighted subtype distribution from the 13 studies was as follows: POLEmut 8.9%, MMRd 25.8%, p53abn 16.7%, and NSMP 48.2%. POLEmut was associated with an excellent prognosis (5-year overall survival >95%, hazard ratio 2.0). p53abn was the only independent risk factor for peritoneal (odds ratio 8.54) and vaginal (odds ratio 6.51) recurrence. Molecular classification modified risk group assignment in 6–33% of patients and improved prognostic discrimination of the 2023 FIGO staging system. Therapeutic implications include treatment de-escalation in POLEmut, immunotherapy in MMRd, and intensive chemotherapy in p53abn. Barriers to implementation include access to POLE sequencing and cost-effectiveness. Conclusions: Integrated molecular classification is an essential tool for prognostic stratification and treatment personalization in endometrial cancer. Molecular subtypes demonstrate clearly differentiated prognostic profiles and recurrence patterns, justifying their systematic incorporation into clinical practice and international guidelines.

Endothelial cells are highly responsive to environmental changes that allow them to adapt to intrinsic and extrinsic stimuli and switch their transcriptome accordingly to go back to vascular homeostasis. Our previous data demonstrated that small non-coding-RNAs respond quickly to genotoxic stressors and determined endothelial cell fate and DNA damage response. To further understand the contribution of non-coding-RNAs, we profiled differentially expressed long non-coding RNAs in response to genotoxic stress and compared them to pro-angiogenic growth factor signaling. We identified the Maternally expressed gene 9 (MEG9) as a cytoprotective lncRNA in the endothelium. Gain and Loss-of-function studies indicate that MEG9 prevents endothelial cells from cell death, suggesting that MEG9 responses to genotoxic stress can be an adaptive and protective mechanism. Consistent with this phenotype, the knockdown of MEG9 decreases growth factor-dependent angiogenesis in a 3D fibrin gel angiogenesis assay. Deletion of the MEG9 ortholog, Mirg, in mice results in increased vascular leak in Matrigel plugs and a sex and age-dependent decrease in platelets. Mechanistically, we observed that both MEG9 knockdown in vitro and Mirg -deleted mice in vivo activated common pathways, including apoptosis, clotting, and inflammation. Indeed, the proinflammatory adhesion molecule ICAM1 was significantly increased in human and mouse endothelial cells in a MEG9-dependent manner, supporting the increased vascular permeability observed on MEG9 deficient cells. Taken together, our findings illustrate how genotoxic stress responses through dynamic modulation of lncRNAs, such as MEG9, trigger adaptive mechanisms to maintain endothelial function, while loss of these molecules contributes to maladaptive responses and endothelial cell dysfunction.

Endothelial cells form a powerful interface between tissues and immune cells. In fact, one of the underappreciated roles of endothelial cells is to orchestrate immune attention to specific sites. Tumor endothelial cells have a unique ability to dampen immune responses and thereby maintain an immunosuppressive microenvironment. Recent approaches to trigger immune responses in cancers have focused on activating nucleic acid sensors, such as cGAS-STING, in combination with immunotherapies. In this review, we present a case for targeting nucleic acid-sensing pathways within the tumor vasculature to invigorate tumor-immune responses. We introduce two specific nucleic acid sensors—the DNA sensor TREX1 and the RNA sensor RIG-I—and discuss their functional roles in the vasculature. Finally, we present perspectives on how these nucleic acid sensors in the tumor endothelium can be targeted in an antiangiogenic and immune activation context. We believe understanding the role of nucleic acid-sensing in the tumor vasculature can enhance our ability to design more effective therapies targeting the tumor microenvironment by co-opting both vascular and immune cell types.

Hereditary breast and/or ovarian cancer is a highly heterogeneous disease with more than 10 known disease-associated genes. In the framework of the BRIDGES project (Breast Cancer Risk after Diagnostic Gene Sequencing), the RAD51C gene has been sequenced in 60,466 breast cancer patients and 53,461 controls. We aimed at functionally characterizing all the identified genetic variants that are predicted to disrupt the splicing process. Forty RAD51C variants of the intron-exon boundaries were bioinformatically analyzed, 20 of which were selected for splicing functional assays. To test them, a splicing reporter minigene with exons 2 to 8 was designed and constructed. This minigene generated a full-length transcript of the expected size (1062 nucleotides), sequence, and structure (Vector exon V1- RAD51C exons_2-8- Vector exon V2). The 20 candidate variants were genetically engineered into the wild type minigene and functionally assayed in MCF-7 cells. Nineteen variants (95%) impaired splicing, while 18 of them produced severe splicing anomalies. At least 35 transcripts were generated by the mutant minigenes: 16 protein-truncating, 6 in-frame, and 13 minor uncharacterized isoforms. According to ACMG/AMP-based standards, 15 variants could be classified as pathogenic or likely pathogenic variants: c.404G > A, c.405-6T > A, c.571 + 4A > G, c.571 + 5G > A, c.572-1G > T, c.705G > T, c.706-2A > C, c.706-2A > G, c.837 + 2T > C, c.905-3C > G, c.905-2A > C, c.905-2_905-1del, c.965 + 5G > A, c.1026 + 5_1026 + 7del, and c.1026 + 5G > T.

Activation of acid sphingomyelinase (SMPD1) and the generation of ceramide is a critical regulator of apoptosis in response to cellular stress including radiation. Endothelial SMPD1 has been shown to regulate tumor responses to radiation therapy. We show here that the SMPD1 gene is regulated by a microRNA (miR), miR-15a, in endothelial cells (ECs). Standard low dose radiation (2 Gy) upregulates miR-15a and decreases SMPD1 levels. In contrast, high dose radiation (10 Gy and above) decreases miR-15a and increases SMPD1. Ectopic expression of miR-15a decreases both mRNA and protein levels of SMPD1. Mimicking the effects of high dose radiation with a miR-15a inhibitor decreases cell proliferation and increases active Caspase-3 & 7. Mechanistically, inhibition of miR-15a increases inflammatory cytokines, activates caspase-1 inflammasome and increases Gasdermin D, an effector of pyroptosis. Importantly, both systemic and vascular-targeted delivery of miR-15a inhibitor decreases angiogenesis and tumor growth in a CT26 murine colorectal carcinoma model. Taken together, our findings highlight a novel role for miR mediated regulation of SMPD1 during radiation responses and establish proof-of-concept that this pathway can be targeted with a miR inhibitor.

A relevant fraction of BRCA2 variants is associated with splicing alterations and with an increased risk of hereditary breast and ovarian cancer (HBOC). In this work, we have carried out a thorough study of variants from BRCA2 exons 14 and 15 reported at mutation databases. A total of 294 variants from exons 14 and 15 and flanking intronic sequences were analysed with the online splicing tools NNSplice and Human Splicing Finder. Fifty-three out of these 294 variants were selected as candidate splicing variants. All variants but one, were introduced into the minigene MGBR2_ex14-20 (with exons 14 to 20) by site-directed mutagenesis and assayed in MCF-7 cells. Twelve of the remaining 52 variants (23.1%) impaired splicing at different degrees, yielding from 5% to 100% of aberrant transcripts. Nine variants affected the natural acceptor or donor sites of both exons and three affected putative enhancers or silencers. Fluorescent capillary electrophoresis revealed at least ten different anomalous transcripts: ▼(E14q5), ∆ (E14p10), ∆(E14p246), ∆(E14q256), ∆(E14), ∆(E15p12), ∆(E15p13), ∆(E15p83), ∆(E15) and a 942-nt fragment of unknown structure. All transcripts, except for ∆(E14q256) and ∆(E15p12), are expected to truncate the BRCA2 protein. Nine variants induced severe splicing aberrations with more than 90% of abnormal transcripts. Thus, according to the guidelines of the American College of Medical Genetics and Genomics, eight variants should be classified as pathogenic (c.7008-2A>T, c.7008-1G>A, c.7435+1G>C, c.7436-2A>T, c.7436-2A>G, c.7617+1G>A, c.7617+1G>T and c.7617+2T>G), one as likely pathogenic (c.7008-3C>G) and three remain as variants of uncertain clinical significance or VUS (c.7177A>G, c.7447A>G and c.7501C>T). In conclusion, functional assays by minigenes constitute a valuable strategy to primarily check the splicing impact of DNA variants and their clinical interpretation. While bioinformatics predictions of splice site variants were accurate, those of enhancer or silencer variants were poor (only 3/23 spliceogenic variants) which showed weak impacts on splicing (~5 to 16% of aberrant isoforms). So, the Exonic Splicing Enhancer and Silencer (ESE and ESS, respectively) prediction algorithms require further improvement.

Splicing disruption is a common mechanism of gene inactivation associated with germline variants of susceptibility genes. To study the role of BRCA2 mis‐splicing in hereditary breast/ovarian cancer (HBOC), we performed a comprehensive analysis of variants from BRCA2 exons 2–9, as well as the initial characterization of the regulatory mechanisms of such exons. A pSAD‐based minigene with exons 2–9 was constructed and validated in MCF‐7 cells, producing the expected transcript (1016‐nt/V1‐BRCA2_exons_2–9‐V2). DNA variants from mutational databases were analyzed by NNSplice and Human Splicing Finder softwares. To refine ESE‐variant prediction, we mapped the regulatory regions through a functional strategy whereby 26 exonic microdeletions were introduced into the minigene and tested in MCF‐7 cells. Thus, we identified nine spliceogenic ESE‐rich intervals where ESE‐variants may be located. Combining bioinformatics and microdeletion assays, 83 variants were selected and genetically engineered in the minigene. Fifty‐three changes impaired splicing: 28 variants disrupted the canonical sites, four created new ones, 10 abrogated enhancers, eight created silencers and three caused a double‐effect. Notably, nine spliceogenic‐ESE variants were located within ESE‐containing intervals. Capillary electrophoresis and sequencing revealed more than 23 aberrant transcripts, where exon skipping was the most common event. Interestingly, variant c.67G>A triggered the usage of a noncanonical GC‐donor 4‐nt upstream. Thirty‐six variants that induced severe anomalies (>60% aberrant transcripts) were analyzed according to the ACMG guidelines. Thus, 28 variants were classified as pathogenic, five as likely pathogenic and three as variants of uncertain significance. Interestingly, 13 VUS were reclassified as pathogenic or likely pathogenic variants. In conclusion, a large fraction of BRCA2 variants (∼64%) provoked splicing anomalies lending further support to the high prevalence of this disease‐mechanism. The low accuracy of ESE‐prediction algorithms may be circumvented by functional ESE‐mapping that represents an optimal strategy to identify spliceogenic ESE‐variants. Finally, systematic functional assays by minigenes depict a valuable tool for the initial characterization of splicing anomalies and the clinical interpretation of variants. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

On the Back Cover: This cover image, by Gemma Montalban et al., is based on the Brief Report Characterization of spliceogenic variants located in regions linked to high levels of alternative splicing: BRCA2 c.7976+5G > T as a case study, Pages 1155–1160. DOI: 10.1002/humu.23583. Cover Funding: Grant/Award Number: PI15/00355. Spanish Instituto de Salud Carlos III (ISCIII) funding, an initiative of the Spanish Ministry of Economy and Innovation partially supported by European Regional Development FEDER Funds.

Many BRCA1 and BRCA2 (BRCA1/2) genetic variants have been studied at mRNA level and linked to hereditary breast and ovarian cancer due to splicing alteration. In silico tools are reliable when assessing variants located in consensus splice sites, but we may identify variants in complex genomic contexts for which bioinformatics is not precise enough. In this study, we characterize BRCA2 c.7976+5G > T variant located in intron 17 which has an atypical donor site (GC). This variant was identified in three unrelated Spanish families and we have detected exon 17 skipping as the predominant transcript occurring in carriers. We have also detected several isoforms (Δ16‐18, Δ17,18, Δ18 and ▼17q²²⁴) at different expression levels among carriers and controls. This study remarks the challenge of interpreting genetic variants when multiple alternative isoforms are present, and that caution must be taken when using in silico tools to identify potential spliceogenic variants located in GC‐AG introns. This article is protected by copyright. All rights reserved

Genetic testing of BRCA1 and BRCA2 identifies a large number of variants of uncertain clinical significance whose functional and clinical interpretations pose a challenge for genetic counseling. Interestingly, a relevant fraction of DNA variants can disrupt the splicing process in cancer susceptibility genes. We have tested more than 200 variants throughout 19 BRCA2 exons mostly by minigene assays, 54% of which displayed aberrant splicing, thus confirming the utility of this assay to check genetic variants in the absence of patient RNA. Our goal was to investigate BRCA2 exon 16 with a view to characterizing spliceogenic variants recorded at the mutational databases. Seventy-two different BIC and UMD variants were analyzed with NNSplice and Human Splicing Finder, 12 of which were selected because they were predicted to disrupt essential splice motifs: canonical splice sites (ss; eight variants) and exonic/intronic splicing enhancers (four variants). These 12 candidate variants were introduced into the BRCA2 minigene with seven exons (14–20) by site-directed mutagenesis and then transfected into MCF-7 cells. Seven variants (six intronic and one missense) induced complete abnormal splicing patterns: c.7618-2A>T, c.7618-2A>G, c.7618-1G>C, c.7618-1G>A, c.7805G>C, c.7805+1G>A, and c.7805+3A>C, as well as a partial anomalous outcome by c.7802A>G. They generated at least 10 different transcripts: Δ16p₄₄ (alternative 3’ss 44-nt downstream; acceptor variants), Δ16 (exon 16-skipping; donor variants), Δ16p₅₅ (alternative 3’ss 55-nt downstream), Δ16q₄ (alternative 5’ss 4-nt upstream), Δ16q₁₀₀ (alternative 5’ss 4-nt upstream), ▾16q²⁰ (alternative 5’ss 20-nt downstream), as well as minor (Δ16p₉₃ and Δ16,17p₆₉) and uncharacterized transcripts of 893 and 954 nucleotides. Isoforms Δ16p₄₄, Δ16, Δ16p₅₅, Δ16q₄, Δ16q₁₀₀, and ▾16q²⁰ introduced premature termination codons which presumably inactivate BRCA2. According to the guidelines the American College of Medical Genetics and Genomics these eight variants could be classified as pathogenic or likely pathogenic whereas the Evidence-based Network for the Interpretation of Germline Mutant Alleles rules suggested seven class 4 and one class 3 variants. In conclusion, our study highlights the relevance of splicing functional assays by hybrid minigenes for the clinical classification of genetic variations. Hence, we provide new data about spliceogenic variants of BRCA2 exon 16 that are directly correlated with breast cancer susceptibility.