Bases moleculares del carcinoma endometrial. Invasión, diseminación y recurrencia

El carcinoma endometrial (CE) es el cáncer ginecológico más frecuente en los países desarrollados (Sankaranarayanan & Ferlay 2006) y representa la cuarta neoplasia en mujeres, después del cáncer de mama, pulmón y colon (Siegel et al. 2012). Normalmente se diagnostica en estadios tempranos, cuando la enfermedad está confinada en el útero, por lo que suele presentar un buen pronóstico. Sin embargo, en torno a un 20% de las pacientes presenta enfermedad avanzada al diagnóstico, lo que va asociado a un descenso en la supervivencia. En este contexto, el estudio de las células del tumor primario, con capacidad de invadir y colonizar localmente o a distancia, y de las células tumorales circulantes (CTC), fuente potencial de recurrencias, permitirá determinar nuevas dianas para controlar la aparición y desarrollo de metástasis. En nuestro estudio caracterizamos el programa transcripcional asociado a ETV5 en el frente de invasión de los carcinomas endometriales y determinamos la importancia de la regulación del factor neurotrófico BDNF. La sobreexpresión de ETV5 genera un fenotipo mesenquimal y un aumento en las capacidades de migración e invasión celular, a través de la participación del eje BDNF‐TrkBERK1/ 2. Además, el patrón de diseminación masivo que promueve ETV5 en un modelo in vivo, se revierte con el silenciamiento de BDNF. El fenotipo invasivo dirigido por ETV5 en el frente de invasión muestra una continuidad en el perfil molecular encontrado en las CTC de las pacientes con CE, a través de un patrón clásico de genes implicados en el proceso de transición epitelio‐mesénquima. La expresión de genes asociados con características de célula madre en las CTC sugiere potenciales dianas terapéuticas. Entre un 13‐17% de las mujeres con CE sufren una recurrencia, un tercio de ellas en regiones locales y dos tercios en lugares a distancia (Smith et al. 2007). En nuestro trabajo hemos identificado a ANXA2 como biomarcador de enfermedad recurrente demostrando su valor predictivo de recurrencia incluso en pacientes con carcinomas endometriales de bajo riesgo. La validación funcional de ANXA2 tanto in vitro como in vivo sugiere un papel principal en el proceso de diseminación y metástasis en el CE

Biomimetic device and foreign body reaction cooperate for efficient tumour cell capture in murine advanced ovarian cancer

Metastasis is facilitated by the formation of pre-metastatic niches through the remodelling of the extracellular matrix (ECM) promoted by haematopoietic and stromal cells. The impact of these primed sites is pronounced for intraperitoneal metastases, where the cavity-exposed ECM supports the attachment of the disseminating tumour cells. Likewise, implantation of biomaterial scaffolds influences metastatic progression systemically through a foreign body reaction (FBR). In this study, we integrated the concept of creating an artificial niche to capture tumour cells actively disseminating in the peritoneal cavity with a therapeutic strategy modulating the interactions of metastatic cells with the ECM. The aim was to transform a disseminated disease into a focal disease. For this, we designed and developed a ‘biomimetic’ ECM composed of a nonresorbable three-dimensional scaffold with collagen coating and characterized the FBR to the implanted biomaterial. We also analysed the safety of the implanted devices and their ability to capture tumour cells in different murine preclinical models of advanced ovarian cancer. Implantation of the biomimetic devices resulted in an initial inflammatory reaction that transformed progressively into a fibrous connective tissue response. The adhesive capabilities of the scaffold were improved with the ancillary effect of the FBR and showed clinical utility in terms of the efficacy of capture of tumour cells, disease focalization and survival benefit. These results demonstrated the performance and safety of this ‘biomimetic’ ECM in preclinical models of advanced ovarian cancer. Translated into the clinical setting, this new therapeutic strategy represents the possibility for control of peritoneal carcinomatosis upon primary ovarian debulking surgery and to expand the percentage of patients who are candidates for second rescue surgeries at the time of relapse.This work was supported by Nasasbiotec

ETV5 transcription program links BDNF and promotion of EMT at invasive front of endometrial carcinomas

Myometrial infiltration represents a main clinical determinant of endometrial carcinomas (EC) presenting as aggressive high-grade deeply invasive neoplasms, substantially associated with risk of recurrence and death. The up-regulation of ETV5 transcription factor linked to the promotion of epithelial to mesenchymal transition is considered as a basic mechanism underlying the initial steps of EC invasion. In this work, we aimed to investigate the transcription program of tumor invasion regulated by ETV5. We performed a comparative Chip-on-chip analysis at invasive front and superficial area of human EC. ETV5 specific binding to promoter regions of genes related to cellular migration, adhesion and invasion at deep invasion tumor areas highlighted the relevance of neural networks associated with cellular plasticity. Interestingly, brain-derived neurotrophic factor (BDNF) demonstrated a principal role orchestrating ETV5-mediated epithelial-to-mesenchymal transition in endometrial cancer. Impairment of the BDNF/tropomyosin-related kinase B (TrkB)/extracellular signal-regulated kinase axis in endometrial cancer cell lines reversed the aggressive and invasive phenotype promoted by the up-regulation of ETV5 at the invasive front of EC. Likewise, BDNF directly impacted on the efficiency of ETV5 promoted metastasis in a mice model of endometrial distant dissemination. These results translate the recognized role of BDNF/TrkB on neural plasticity into a relevant cancer metastasis event; suggest common mechanisms shared by neural development and tumor invasion; and offer new therapeutic opportunities specifically directed against disseminated disease in endometrial cancer

PrediCTC, liquid biopsy in precision oncology: a technology transfer experience in the Spanish health system.

Purpose: Management of metastatic disease in oncology includes monitoring of therapy response principally by imaging techniques like CT-Scan. In addition to some limitations, the irruption of liquid biopsy and its application in personalized medicine has encouraged the development of more efficient technologies for prognosis and follow-up of patients in advanced disease. Methods: PrediCTC constitutes a panel of genes for the assessment of Circulating Tumor Cells (CTC) in metastatic colorectal cancer patients, with demonstrated improved efficiency compared to CT-Scan for the evaluation of early therapy response in a multicenter prospective study. In this work, we designed and developed a technology transfer strategy to define the market opportunity for an eventual implementation of PrediCTC in the clinical practice. Results: This included the definition of the regulatory framework, the analysis of the regulatory roadmap needed for CE mark, a benchmarking study, the design of a product development strategy, a revision of intellectual property, a cost-effectiveness study and an expert panel consultation. Conclusion: The definition and analysis of an appropriate technology transfer strategy and the correct balance among regulatory, financial and technical determinants are critical for the transformation of a promising technology into a viable technology, and for the decision of implementing liquid biopsy in the monitoring of therapy response in advanced disease.pre-print533 K

A logistic model for the detection of circulating tumour cells in human metastatic colorectal cancer

The accuracy in the diagnosis of metastatic colorectal cancer (mCRC) represents one of the challenges in the clinical management of patients. The detection of circulating tumour cells (CTC) is becoming a promising alternative to current detection techniques, as it focuses on one of the players of the metastatic disease and it should provide with more specific and sensitive detection rates. Here, we describe an improved method of detection of CTC from mCRC patients by combining immune-enrichment, optimal purification of RNA from very low cell numbers, and the selection of accurate PCR probes. As a result, we obtained a logistic model that combines GAPDH and VIL1 normalized to CD45 rendering powerful results in the detection of CTC from mCRC patients (AUROC value 0.8599). We further demonstrated the utility of this model at the clinical setting, as a reliable prognosis tool to determine progression-free survival in mCRC patients. Overall, we developed a strategy that ameliorates the specificity and sensitivity in the detection of CTC, resulting in a robust and promising logistic model for the clinical management of metastatic colorectal cancer patients.Ministerio de Sanidad, Consumo y Bienestar Social. Beca número: CP08/00142Programa Comisión Europea Fondo Europeo de Desarollo Regional (FEDER

Circulating Tumor Cells Characterization Revealed TIMP1 as a Potential Therapeutic Target in Ovarian Cancer

Background: Recent studies showed a relevant role of hematogenous spread in ovarian cancer and the interest of circulating tumor cells (CTCs) monitoring as a prognosis marker. The aim of the present study was the characterization of CTCs from ovarian cancer patients, paying special attention to cell plasticity characteristics to better understand the biology of these cells. Methods: CTCs isolation was carried out in 38 patients with advanced high-grade serous ovarian cancer using in parallel CellSearch and an alternative EpCAM-based immunoisolation followed by RT-qPCR analysis to characterize these cells. Results: Epithelial CTCs were found in 21% of patients, being their presence higher in patients with extraperitoneal metastasis. Importantly, this population was characterized by the expression of epithelial markers as MUC1 and CK19, but also by genes associated with mesenchymal and more malignant features as TIMP1, CXCR4 and the stem markers CD24 and CD44. In addition, we evidenced the relevance of TIMP1 expression to promote tumor proliferation, suggesting its interest as a therapeutic target. Conclusions: Overall, we evidenced the utility of the molecular characterization of EpCAM+ CTCs from advanced ovarian cancer patients to identify biomarkers with potential applicability for disseminated disease detection and as therapeutic targets such as TIMP1Part of this research was supported by CIBERONC funds (CB16/12/00328)S

Predicting Outcome and Therapy Response in mCRC Patients Using an Indirect Method for CTCs Detection by a Multigene Expression Panel: A Multicentric Prospective Validation Study

Colorectal cancer (CRC) is one of the major causes of cancer-related deaths. Early detection of tumor relapse is crucial for determining the most appropriate therapeutic management. In clinical practice, computed tomography (CT) is routinely used, but small tumor changes are difficult to visualize, and reliable blood-based prognostic and monitoring biomarkers are urgently needed. The aim of this study was to prospectively validate a gene expression panel (composed of GAPDH, VIL1, CLU, TIMP1, TLN1, LOXL3 and ZEB2) for detecting circulating tumor cells (CTCs) as prognostic and predictive tool in blood samples from 94 metastatic CRC (mCRC) patients. Patients with higher gene panel expression before treatment had a reduced progression-free survival (PFS) and overall-survival (OS) rates compared with patients with low expression (p = 0.003 and p ≤ 0.001, respectively). Patients with increased expression of CTCs markers during treatment presented PFS and OS times of 8.95 and 11.74 months, respectively, compared with 14.41 and 24.7 for patients presenting decreased expression (PFS; p = 0.020; OS; p ≤ 0.001). Patients classified as non-responders by CTCs with treatment, but classified as responders by CT scan, showed significantly shorter survival times (PFS: 8.53 vs. 11.70; OS: 10.37 vs. 24.13; months). In conclusion, our CTCs detection panel demonstrated efficacy for early treatment response assessment in mCRC patients, and with increased reliability compared to CT scan.ACIS (Axencia de Coñecemento en Saude); SERGAS. Cofinanced ERDF Funds 2007–201

Genomic profiling of uterine aspirates and cfDNA as an integrative liquid biopsy strategy in endometrial cancer

The incidence and mortality of endometrial cancer (EC) have risen in recent years, hence more precise management is needed. Therefore, wecombined di erent types of liquid biopsies to better characterize the genetic landscape of EC in a non-invasive and dynamic manner. Uterine aspirates (UAs) from 60 patients with EC were obtained during surgery and analyzed by next-generation sequencing (NGS). Blood samples, collected at surgery, were used for cell-free DNA (cfDNA) and circulating tumor cell (CTC) analyses. Finally, personalized therapies were tested in patient-derived xenografts (PDXs) generated from the UAs. NGS analyses revealed the presence of genetic alterations in 93% of the tumors. Circulating tumor DNA (ctDNA) was present in 41.2% of cases, mainly in patients with high-risk tumors, thus indicating a clear association with a more aggressive disease. Accordingly, the results obtained during the post-surgery follow-up indicated the presence of ctDNA in three patients with progressive disease. Moreover, 38.9% of patients were positive for CTCs at surgery. Finally, the e cacy of targeted therapies based on the UA-specific mutational landscape was demonstrated in PDX models. Our study indicates the potential clinical applicability of a personalized strategy based on a combination of different liquid biopsies to characterize and monitor tumor evolution, and to identify targeted therapiesThis work was supported by grants and support from the Instituto de Salud Carlos III (ISCIII) and FEDER (PI17/01919, PI17/02071), CIBERONC (CB16/12/00328), and the AECC (Grupos Estables de Investigacion 2018-AECC) to A.G.-M. and M.A.; Instituto de Salud Carlos III (ISCIII) and FEDER (PI16/00134), CIBERONC (CB16/12/00295), and the AECC (Grupos Estables de Investigacion 2018-AECC) to G.M.-B.; and the AECC to L.M.-R

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