Potential Targets' Analysis Reveals Dual PI3K/mTOR Pathway Inhibition as a Promising Therapeutic Strategy for Uterine Leiomyosarcomas-an ENITEC Group Initiative

Purpose: Uterine sarcomas are rare and heterogeneous tumors characterized by an aggressive clinical behavior. Their high rates of recurrence and mortality point to the urgent need for novel targeted therapies and alternative treatment strategies. However, no molecular prognostic or predictive biomarkers are available so far to guide choice and modality of treatment. Experimental Design: We investigated the expression of several druggable targets (phospho-S6(S240) ribosomal protein, PTEN, PDGFR-alpha, ERBB2, and EGFR) in a large cohort of human uterine sarcoma samples (288), including leiomyosarcomas, low-grade and high-grade endometrial stromal sarcomas, undifferentiated uterine sarcomas, and adenosarcomas, together with 15 smooth muscle tumors of uncertain malignant potential (STUMP), 52 benign uterine stromal tumors, and 41 normal uterine tissues. The potential therapeutic value of the most promising target, p-S6(S240), was tested in patient-derived xenograft (PDX) leiomyosarcoma models. Results: In uterine sarcomas and STUMPs, S6S240 phosphorylation (reflecting mTOR pathway activation) was associated with higher grade (P = 0.001) and recurrence (P = 0.019), as shown by logistic regression. In addition, p-S6(S240) correlated with shorter progression-free survival (P = 0.034). Treatment with a dual PI3K/mTOR inhibitor significantly reduced tumor growth in 4 of 5 leiomyosarcoma PDX models (with tumor shrinkage in 2 models). Remarkably, the 4 responding models showed basal p-S6(S240) expression, whereas the nonresponding model was scored as negative, suggesting a role for p-S6(S240) in response prediction to PI3K/mTOR inhibition. Conclusions: Dual PI3K/mTOR inhibition represents an effective therapeutic strategy in uterine leiomyosarcoma, and p-S6(S240) expression is a potential predictive biomarker for response to treatment. (C)2017 AACR.Peer reviewe

Molecular profiling of circulating tumor cells links plasticity to the metastatic process in endometrial cancer

Background About 20% of patients diagnosed with endometrial cancer (EC) are considered high-risk with unfavorable prognosis. In the framework of the European Network for Individualized Treatment in EC (ENITEC), we investigated the presence and phenotypic features of Circulating Tumor Cells (CTC) in high-risk EC patients. Methods CTC isolation was carried out in peripheral blood samples from 34 patients, ranging from Grade 3 Stage IB to Stage IV carcinomas and recurrences, and 27 healthy controls using two methodologies. Samples were subjected to EpCAM-based immunoisolation using the CELLection™ Epithelial Enrich kit (Invitrogen, Dynal) followed by RTqPCR analysis. The phenotypic determinants of endometrial CTC in terms of pathogenesis, hormone receptor pathways, stem cell markers and epithelial to mesenchymal transition (EMT) drivers were asked. Kruskal-Wallis analysis followed by Dunn’s post-test was used for comparisons between groups. Statistical significance was set at p < 0.05. Results EpCAM-based immunoisolation positively detected CTC in high-risk endometrial cancer patients. CTC characterization indicated a remarkable plasticity phenotype defined by the expression of the EMT markers ETV5, NOTCH1, SNAI1, TGFB1, ZEB1 and ZEB2. In addition, the expression of ALDH and CD44 pointed to an association with stemness, while the expression of CTNNB1, STS, GDF15, RELA, RUNX1, BRAF and PIK3CA suggested potential therapeutic targets. We further recapitulated the EMT phenotype found in endometrial CTC through the up-regulation of ETV5 in an EC cell line, and validated in an animal model of systemic dissemination the propensity of these CTC in the accomplishment of metastasis. Conclusions Our results associate the presence of CTC with high-risk EC. Gene-expression profiling characterized a CTC-plasticity phenotype with stemness and EMT features. We finally recapitulated this CTC-phenotype by over-expressing ETV5 in the EC cell line Hec1A and demonstrated an advantage in the promotion of metastasis in an in vivo mouse model of CTC dissemination and homing

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

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

Viṣṇu-Nārāyaṇa

The contributions collected in this volume deal with the complex history of the Indian deity Viṣṇu-Nārāyana. This conception of God evolved in various traditions in India, especially in South India, during the first millennium CE. The history of this development is reconstructed here by various means, including philological exegesis, the history of ideas, and iconographic evidence

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