Relevance of Molecular Profiling in Patients With Low-Grade Endometrial Cancer

Molecular Profiling; Endometrial CancerPerfil molecular; Càncer d'endometriPerfil molecular; Cáncer de endometrioImportance Patients with low-grade (ie, grade 1-2) endometrial cancer (EC) are characterized by their favorable prognosis compared with patients with high-grade (ie, grade 3) EC. With the implementation of molecular profiling, the prognostic relevance of tumor grading might lose attention. As most patients present with low-grade EC and have an excellent outcome, the value of molecular profiling for these patients is unclear. Objective To determine the association of molecular profiling with outcomes among patients with low-grade EC. Design, Setting, and Participants This retrospective cohort study included a multicenter international European cohort of patients diagnosed with EC between 1994 and 2018, with a median follow-up of 5.9 years. Molecular subgroups were determined by next-generation sequencing using single-molecule molecular inversion probes and by immunohistochemistry. Subsequently, tumors were classified as polymerase epsilon (POLE)-altered, microsatellite instable (MSI), tumor protein p53 (TP53)-altered, or no specific molecular profile (NSMP). Patients diagnosed with any histological subtypes and FIGO (International Federation of Gynecology and Obstetrics) stages of EC were included, but patients with early-stage EC (FIGO I-II) were only included if they had known lymph node status. Data were analyzed February 20 to June 16, 2022. Exposures Molecular testing of the 4 molecular subgroups. Main Outcomes and Measures The main outcome was disease-specific survival (DSS) within the molecular subgroups. Results A total of 393 patients with EC were included, with a median (range) age of 64.0 (31.0-86.0) years and median (range) body mass index (BMI; calculated as weight in kilograms divided by height in meters squared) of 29.1 (18.0-58.3). Most patients presented with early-stage (290 patients [73.8%]) and low-grade (209 patients [53.2%]) disease. Of all patients, 33 (8.4%) had POLE-altered EC, 78 (19.8%) had MSI EC, 72 (18.3%) had TP53-altered EC, and 210 (53.4%) had NSMP EC. Across all molecular subgroups, patients with low-grade EC had superior 5-year DSS compared with those with high-grade EC, varying between 90% to 100% vs 41% to 90% (P < .001). Multivariable analysis in the entire cohort including age, tumor grade, FIGO stage, lymphovascular space invasion, and the molecular subgroups as covariates found that only high-grade (hazard ratio [HR], 4.29; 95% CI, 2.15-8.53; P < .001), TP53-altered (HR, 1.76; 95% CI, 1.04-2.95; P = .03), and FIGO stage III or IV (HR, 4.26; 95% CI, 2.50-7.26; P < .001) disease were independently associated with reduced DSS. Conclusions and Relevance This cohort study found that patients with low-grade EC had an excellent prognosis independent of molecular subgroup. These findings do not support routine molecular profiling in patients with low-grade EC, and they demonstrate the importance of primary diagnostic tumor grading and selective profiling in low-grade EC to increase cost-effectiveness

In silico Approach for Validating and Unveiling New Applications for Prognostic Biomarkers of Endometrial Cancer

Bioinformática; Cáncer de endometrio; Biomarcador pronósticoBioinformàtica; Càncer d'endometri; Biomarcador pronòsticBioinformatics; Endometrial cancer; Prognostic biomarkerEndometrial cancer (EC) mortality is directly associated with the presence of prognostic factors. Current stratification systems are not accurate enough to predict the outcome of patients. Therefore, identifying more accurate prognostic EC biomarkers is crucial. We aimed to validate 255 prognostic biomarkers identified in multiple studies and explore their prognostic application by analyzing them in TCGA and CPTAC datasets. We analyzed the mRNA and proteomic expression data to assess the statistical prognostic performance of the 255 proteins. Significant biomarkers related to overall survival (OS) and recurrence-free survival (RFS) were combined and signatures generated. A total of 30 biomarkers were associated either to one or more of the following prognostic factors: histological type (n = 15), histological grade (n = 6), FIGO stage (n = 1), molecular classification (n = 16), or they were associated to OS (n = 11), and RFS (n = 5). A prognostic signature composed of 11 proteins increased the accuracy to predict OS (AUC = 0.827). The study validates and identifies new potential applications of 30 proteins as prognostic biomarkers and suggests to further study under-studied biomarkers such as TPX2, and confirms already used biomarkers such as MSH6, MSH2, or L1CAM. These results are expected to advance the quest for biomarkers to accurately assess the risk of EC patients.This research was funded by grants from the Instituto de Salud Carlos III (ISCIII) grant number PI17/02155, PI20/00644, and the IFI19/00029 to E.C.-d.l.R., the Ministerio de ciencia, Innovación y Universidades through a RETOS Colaboración (RTC-2017-6261-1), both co-financed by the European Regional Development Fund (FEDER); from Fundación Científica Asociación Española Contra el Cáncer (AECC) grant number GCTRA1804MATI and CIBERONC network grant number CB16/12/00328; and Grups Consolidats de la Generalitat de Catalunya (2017SGR1661). E.C. is supported by an Investigator Grant from AECC (INVES20051COLA). E.M.-G. was supported by Televie grant F5/20/5-TLV/DD

Modeling ANXA2-overexpressing circulating tumor cells homing and high throughput screening for metastasis impairment in endometrial carcinomas

Cèl·lules tumorals circulants; Micrometàstasi; DaunorrubicinaCélulas tumorales circulantes; Micrometástasis; DaunorrubicinaCirculating tumor cells; Micrometastasis; DaunorubicinEndometrial cancer (EC) is the most common neoplasm of the female reproductive tract in the developed world. Patients usually are diagnosed in early stage having a good prognosis. However, up to 20–25% of patients are diagnosed in advanced stages and have a higher risk of recurrence, making the prognosis worse. Previously studies identified ANXA2 as a predictor of recurrent disease in EC even in low risk patients. Furthermore, Circulating Tumor Cells (CTC) released from the primary tumor into the bloodstream, are plasticity entities responsible of the process of metastasis, becoming into an attractive clinical target. In this work we validated ANXA2 expression in CTC from high-risk EC patients. After that, we modelled in vitro and in vivo the tumor cell attachment of ANXA2-expressing CTC to the endothelium and the homing for the generation of micrometastasis. ANXA2 overexpression does not provide an advantage in the adhesion process of CTC, but it could be playing an important role in more advanced steps, conferring a greater homing capacity. We also performed a high-throughput screening (HTS) for compounds specifically targeting ANXA2, and selected Daunorubicin as candidate hit. Finally, we validated Daunorubicin in a 3D transendothelial migration system and also in a in vivo model of advanced EC, demonstrating the ability of Daunorubicin to inhibit the proliferation of ANXA2-overexpressing tumor cells.This work was supported by grants from the Instituto de Salud Carlos III ( ISCIII ), grant PI17/01919 and PI20/00969 , co-financed by the European Regional Development Fund (FEDER); from Fundación Científica de la Asociación Española Contra el Cáncer (AECC), Grupos Clínicos Coordinados 2018; Xunta de Galicia ( ED431C 2018/21 ); Ministry of Economy and Competiveness (Innopharma Project) and from CIBERONC ( CB16/12/00328 ); Carolina Herrero is supported by a predoctoral i-PFIS fellowship from Instituto de Salud Carlos III ( IFI17/00047 )

EV-associated miRNAs from pleural lavage as potential diagnostic biomarkers in lung cancer

Taqman OpenArray technology; Lung cancer diagnosis; BiomarkersTecnología Taqman OpenArray; Diagnóstico de cáncer de pulmón; BiomarcadoresTecnologia Taqman OpenArray; Diagnòstic del càncer de pulmó; BiomarcadorsLung cancer is the leading cause of cancer-related deaths among men and women in the world, accounting for the 25% of cancer mortality. Early diagnosis is an unmet clinical issue. In this work, we focused to develop a novel approach to identify highly sensitive and specific biomarkers by investigating the use of extracellular vesicles (EVs) isolated from the pleural lavage, a proximal fluid in lung cancer patients, as a source of potential biomarkers. We isolated EVs by ultracentrifuge method from 25 control pleural fluids and 21 pleural lavages from lung cancer patients. Analysis of the expression of EV-associated miRNAs was performed using Taqman OpenArray technology through which we could detect 288 out of the 754 miRNAs that were contained in the OpenArray. The differential expression analysis yielded a list of 14 miRNAs that were significantly dysregulated (adj. p-value < 0.05 and logFC lower or higher than 3). Using Machine Learning approach we discovered the lung cancer diagnostic biomarkers; miRNA-1-3p, miRNA-144-5p and miRNA-150-5p were found to be the best by accuracy. Accordance with our finding, these miRNAs have been related to cancer processes in previous studies. This results opens the avenue to the use of EV-associated miRNA of pleural fluids and lavages as an untapped source of biomarkers, and specifically, identifies miRNA-1-3p, miRNA-144-5p and miRNA 150-5p as promising biomarkers of lung cancer diagnosis.The sample collection was supported by IRBLleida BIOBANK (B.0000682) and Plataforma biobancos PT17/0015/0027.EC hold a postdoctoral fellowship from the Departament de Salut of the Generalitat de Catalunya (SLT002/16/00274). This study was supported by: “Discovery, validation and implementation of biomarkers for Precision Oncology” (ISCIII PIE15/00029), CIBERONC (CB16/12/00231 and CB16/12/00328), Grups consolidats de la Generalitat de Catalunya (2017SGR1368 and 2017SGR1661) and Asociación Española contra el Cancer (GCTRA1804MATI)

The amount of preoperative endometrial tissue surface in relation to final endometrial cancer classification

Diagnosis; Endometrial carcinoma; Endometrial samplingDiagnóstico; Carcinoma de endometrio; Muestreo endometrialDiagnòstic; Carcinoma endometrial; Mostreig endometrialObjective To evaluate whether the amount of preoperative endometrial tissue surface is related to the degree of concordance with final low- and high-grade endometrial cancer (EC). In addition, to determine whether discordance is influenced by sampling method and impacts outcome. Methods A retrospective cohort study within the European Network for Individualized Treatment of Endometrial Cancer (ENITEC). Surface of preoperative endometrial tissue samples was digitally calculated using ImageJ. Tumor samples were classified into low-grade (grade 1–2 endometrioid EC (EEC)) and high-grade (grade 3 EEC + non-endometroid EC). Results The study cohort included 573 tumor samples. Overall concordance between pre- and postoperative diagnosis was 60.0%, and 88.8% when classified into low- and high-grade EC. Upgrading (preoperative low-grade, postoperative high-grade EC) was found in 7.8% and downgrading (preoperative high-grade, postoperative low-grade EC) in 26.7%. The median endometrial tissue surface was significantly lower in concordant diagnoses when compared to discordant diagnoses, respectively 18.7 mm2 and 23.5 mm2 (P = 0.022). Sampling method did not influence the concordance in tumor classification. Patients with preoperative high-grade and postoperative low-grade showed significant lower DSS compared to patients with concordant low-grade EC (P = 0.039). Conclusion The amount of preoperative endometrial tissue surface was inversely related to the degree of concordance with final tumor low- and high-grade. Obtaining higher amount of preoperative endometrial tissue surface does not increase the concordance between pre- and postoperative low- and high-grade diagnosis in EC. Awareness of clinically relevant down- and upgrading is crucial to reduce subsequent over- or undertreatment with impact on outcome

Noninvasive detection of microsatellite instability in patients with endometrial cancer

Endometrial cancer; Liquid biopsy; Uterine aspirateCàncer d'endometri; Biòpsia líquida; Aspirat uteríCáncer de endometrio; Biopsia líquida; Aspirado uterinoThe analysis of mismatch repair proteins in solid tissue is the standard of care (SoC) for the microsatellite instability (MSI) characterization in endometrial cancer (EC). Uterine aspirates (UAs) or circulating-DNA (cfDNA) samples capture the intratumor heterogeneity and provide a more comprehensive and dynamic molecular diagnosis. Thus, MSI analysis by droplet-digital PCR (ddPCR) in UAs and cfDNA can provide a reliable tool to characterize and follow-up the disease. The UAs, paraffin-embedded tumor tissue (FFPE) and longitudinal plasma samples from a cohort of 90 EC patients were analyzed using ddPCR panel and compared to the SoC. A high concordance (96.67%) was obtained between the analysis of MSI markers in UAs and the SoC. Three discordant cases were validated as unstable by ddPCR on FFPE samples. Besides, a good overall concordance (70.27%) was obtained when comparing the performance of the ddPCR assay on UAs and cfDNA in high-risk tumors. Importantly, our results also evidenced the value of MSI analysis to monitor the disease evolution. MSI evaluation in minimally invasive samples shows great accuracy and sensitivity and provides a valuable tool for the molecular characterization and follow-up of endometrial tumors, opening new opportunities for personalized management of EC.Centro de Investigación Biomédica en Red de Cáncer, Grant/Award Numbers: CB16/12/00295, CB16/12/00328; Fundación Científica Asociación Española Contra el Cáncer, Grant/Award Numbers: FC_AECC PROYE19036MOR, 2018-AECC, INVES20051COLA; Fundación Instituto de Investigación Sanitaria de Santiago de Compostela; Instituto de Salud Carlos III and FEDER, Grant/Award Numbers: CM19/00087, CP20/00119, PI20/00969, PI20/01566, PI21/00990; Spanish Ministry of Economy and Innovation, Grant/Award Number: PID2019-104644RB-I0

The ERK5/NF-κB signaling pathway targets endometrial cancer proliferation and survival

Apoptosis; Endometrial cancer; Map kinaseApoptosis; Cáncer endometrial; Mapa quinasaApoptosi; Càncer d'endometri; Mapa quinasaEndometrial cancer (EC) is the most common type of gynecologic cancer in women of developed countries. Despite surgery combined with chemo-/radiotherapy regimens, overall survival of patients with high-risk EC tumors is poor, indicating a need for novel therapies. The MEK5-ERK5 pathway is activated in response to growth factors and to different stressors, including oxidative stress and cytokines. Previous evidence supports a role for the MEK5-ERK5 pathway in the pathology of several cancers. We investigated the role of ERK5 in EC. In silico analysis of the PanCancer Atlas dataset showed alterations in components of the MEK5-ERK5 pathway in 48% of EC patients. Here, we show that ERK5 inhibition or silencing decreased EGF-induced EC cell proliferation, and that genetic deletion of MEK5 resulted in EC impaired proliferation and reduced tumor growth capacity in nude mice. Pharmacologic inhibition or ERK5 silencing impaired NF-kB pathway in EC cells and xenografts. Furthermore, we found a positive correlation between ERK5 and p65/RELA protein levels in human EC tumor samples. Mechanistically, genetic or pharmacologic impairment of ERK5 resulted in downregulation of NEMO/IKKγ expression, leading to impaired p65/RELA activity and to apoptosis in EC cells and xenografts, which was rescued by NEMO/IKKγ overexpression. Notably, ERK5 inhibition, MEK5 deletion or NF-kB inhibition sensitized EC cells to standard EC chemotherapy (paclitaxel/carboplatin) toxicity, whereas ERK5 inhibition synergized with paclitaxel to reduce tumor xenograft growth in mice. Together, our results suggest that the ERK5-NEMO-NF-κB pathway mediates EC cell proliferation and survival. We propose the ERK5/NF-κB axis as new target for EC treatment.Open Access Funding provided by Universitat Autonoma de Barcelona. The JM Lizcano research group was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO, grant SAF2015-64237-R), and the Spanish Ministry of Science and Innovation (grant PID2019-107561RB-I00), and co-funded by the European Regional Development Fund (ERDF)

Intratumor genetic heterogeneity and clonal evolution to decode endometrial cancer progression

Endometrial cancer; Clonal evolution; MutationCáncer endometrial; Evolución clonal; MutaciónCàncer d'endometri; Evolució clonal; MutacióAnalyzing different tumor regions by next generation sequencing allows the assessment of intratumor genetic heterogeneity (ITGH), a phenomenon that has been studied widely in some tumor types but has been less well explored in endometrial carcinoma (EC). In this study, we sought to characterize the spatial and temporal heterogeneity of 9 different ECs using whole-exome sequencing, and by performing targeted sequencing validation of the 42 primary tumor regions and 30 metastatic samples analyzed. In addition, copy number alterations of serous carcinomas were assessed by comparative genomic hybridization arrays. From the somatic mutations, identified by whole-exome sequencing, 532 were validated by targeted sequencing. Based on these data, the phylogenetic tree reconstructed for each case allowed us to establish the tumors’ evolution and correlate this to tumor progression, prognosis, and the presence of recurrent disease. Moreover, we studied the genetic landscape of an ambiguous EC and the molecular profile obtained was used to guide the selection of a potential personalized therapy for this patient, which was subsequently validated by preclinical testing in patient-derived xenograft models. Overall, our study reveals the impact of analyzing different tumor regions to decipher the ITGH in ECs, which could help make the best treatment decision.We thank all those at the Translational Research Laboratory of the MD Anderson Cancer Center Madrid for their invaluable help with this study. Tissue samples were obtained with the support of the MD Anderson Foundation Biobank (record number B.0000745, ISCIII National Biobank Record), the “Xarxa Catalana de Bancs de Tumors” and “Plataforma de Biobancos” ISCIII (PT13/0010/0014, B.000609). This study has been supported by the Spanish Ministry of Economy and Innovation (PID2019-104644RB-I00 (GMB), the Instituto de Salud Carlos III (ISCIII, CIBERONC, CB16/12/00295 - GMB-, CB16/12/00328 -EC, AGM- and CB16/12/00231 -XMG- [all partly supported by FEDER funds]) and by the AECC Scientific Foundation (FC_AECC PROYE19036MOR -GMB- and Coordinated groups 2018 -XMG, AGM, GMB-). SO is funded by an AECC-postdoctoral grant (2020). JSR-F and BW are funded in part by the Breast Cancer Research Foundation and in part by the NIH/NCI P50 CA247749 01 grant. Research reported in this publication was supported in part by a Cancer Center Support Grant of the NIH/NCI (Grant No. P30CA008748; MSK). We thank the Eurofins Megalab laboratory for helping us to perform the analysis of DNA HPV detection

Genomic Validation of Endometrial Cancer Patient-Derived Xenograft Models as a Preclinical Tool

Bioinformatics; Endometrial cancer; Molecular markerBioinformática; Cáncer endometrial; Marcador molecularBioinformàtica; Càncer d'endometri; Marcador molecularEndometrial cancer (EC) is the second most frequent gynecological cancer worldwide. Although improvements in EC classification have enabled an accurate establishment of disease prognosis, women with a high-risk or recurrent EC face a dramatic situation due to limited further treatment options. Therefore, new strategies that closely mimic the disease are required to maximize drug development success. Patient-derived xenografts (PDXs) are widely recognized as a physiologically relevant preclinical model. Hence, we propose to molecularly and histologically validate EC PDX models. To reveal the molecular landscape of PDXs generated from 13 EC patients, we performed histological characterization and whole-exome sequencing analysis of tumor samples. We assessed the similarity between PDXs and their corresponding patient’s tumor and, additionally, to an extended cohort of EC patients obtained from The Cancer Genome Atlas (TCGA). Finally, we performed functional enrichment analysis to reveal differences in molecular pathway activation in PDX models. We demonstrated that the PDX models had a well-defined and differentiated molecular profile that matched the genomic profile described by the TCGA for each EC subtype. Thus, we validated EC PDX’s potential to reliably recapitulate the majority of histologic and molecular EC features. This work highlights the importance of a thorough characterization of preclinical models for the improvement of the success rate of drug-screening assays for personalized medicine.This research was funded by grants from the Instituto de Salud Carlos III (ISCIII) grant number PI17/02071, PI20/01566, and from the Ministerio de ciencia, Innovación y Universidades through a RETOS Colaboración (RTC-2017-6261-1), both co-financed by the European Regional Development Fund (FEDER); from Fundación Científica Asociación Española Contra el Cáncer (AECC) grant number GCTRA1804MATI, Biomedical Research Center Network (CIBERONC) grant number CB16/12/00328 and Generalitat de Catalunya, grant number 2017SGR1661. B.V.-M. is supported by a predoctoral fellowship (PERIS-SLT017/20/000183) from Generalitat de Catalunya. E.C. is supported by an Investigator Grant from AECC (INVES20051COLA)

Immunohistochemical biomarkers are prognostic relevant in addition to the ESMO-ESGO-ESTRO risk classification in endometrial cancer

Endometrial carcinoma; Immunohistochemistry; OutcomeCarcinoma de endometrio; Inmunohistoquímica; ResultadoCarcinoma d'endometri; Immunohistoquímica; ResultatObjective Pre-operative immunohistochemical (IHC) biomarkers are not incorporated in endometrial cancer (EC) risk classification. We aim to investigate the added prognostic relevance of IHC biomarkers to the ESMO-ESGO-ESTRO risk classification and lymph node (LN) status in EC. Methods Retrospective multicenter study within the European Network for Individualized Treatment of Endometrial Cancer (ENITEC), analyzing pre-operative IHC expression of p53, L1 cell-adhesion molecule (L1CAM), estrogen receptor (ER) and progesterone receptor (PR), and relate to ESMO-ESGO-ESTRO risk groups, LN status and outcome. Results A total of 763 EC patients were included with a median follow-up of 5.5-years. Abnormal IHC expression was present for p53 in 112 (14.7%), L1CAM in 79 (10.4%), ER- in 76 (10.0%), and PR- in 138 (18.1%) patients. Abnormal expression of p53/L1CAM/ER/PR was significantly related with higher risk classification groups, and combined associated with the worst outcome within the ‘high and advanced/metastatic’ risk group. In multivariate analysis p53-abn, ER/PR- and ESMO-ESGO-ESTRO ‘high and advanced/metastatic’ were independently associated with reduced disease-specific survival (DSS). Patients with abnormal IHC expression and lymph node metastasis (LNM) had the worst outcome. Patients with LNM and normal IHC expression had comparable outcome with patients without LNM and abnormal IHC expression. Conclusion The use of pre-operative IHC biomarkers has important prognostic relevance in addition to the ESMO-ESGO-ESTRO risk classification and in addition to LN status. For daily clinical practice, p53/L1CAM/ER/PR expression could serve as indicator for surgical staging and refine selective adjuvant treatment by incorporation into the ESMO-ESGO-ESTRO risk classification