Análisis de la proteína de activación de fibroblastos a (FAP) en el carcinoma colorrectal

125 p.Los fibroblastos asociados al cáncer son uno de los componentes mayoritarios del microambiente tumoral. La expresión de uno de sus marcadores, la Proteína de Activación de Fibroblastos ¿ (FAP) recientemente se ha vinculado con un peor pronóstico en diferentes tipos de neoplasias, por ejemplo, el carcinoma renal de células claras. En esta tesis doctoral se estudia la influencia de dicha proteína en el carcinoma colorrectal, una de las neoplasias con mayor incidencia a nivel mundial. Para ello se analiza la expresión inmunohistoquímica de FAP en toda la secuencia evolutiva de la neoplasia (pólipo adenomatoso, tumor primario, metástasis locorregional y metástasis a distancia). Se corrobora su expresión positiva en el tumor infiltrante y se evidencia que, además de expresarse más en el subtipo histológico convencional, ejerce fundamentalmente su influencia carcinogénica en las fases iniciales del proceso a nivel del tumor primario. No se le atribuye, sin embargo, un papel pronóstico influyente en la supervivencia. El estudio de los niveles séricos de la fracción soluble de FAP (FAPs) permite observar que los niveles de la proteína son inferiores en pacientes de carcinoma colorrectal que en sujetos sanos. Es más, a menores valores de concentración sérica el pronóstico de la enfemedad parece ser peor. Se presenta, por tanto, a FAPs como un posible biomarcador en el diagnóstico y seguimiento del carcinoma colorrectal

(Pro)renin Receptor Expression Increases throughout the Colorectal Adenoma-Adenocarcinoma Sequence and It Is Associated with Worse Colorectal Cancer Prognosis

(Pro)renin receptor (PRR) is a protein that takes part in several signaling pathways such as Renin Angiotensin System and Wnt signalling. Its biological role has recently been related to cancer progression and in this study, we investigated its relevance in colorectal cancer (CRC). To that end, we analysed the immunohistochemical expression of PRR in adenomatous polyps and CRCs from the same patients (n = 42), and in primary tumours and nodal and liver metastases from advanced CRC patients (n = 294). In addition, the soluble fraction of PRR was measured by ELISA in plasma samples from 161 CRC patients. The results showed that PRR expression was gradually augmented along the uninvolved mucosa-adenoma-adenocarcinoma sequence. Besides, the stronger expression of PRR in primary tumours was markedly associated with local tumour extent and the onset of metastases. Moreover, PRR expression in both primary and distant metastases was associated with worse 5- and 10-year survival of CRC patients. Plasmatic PRR levels did not change with respect to controls and were not associated with CRC aggressiveness. These results suggest a key role of PRR in the development and progression of CRC and a potential use of this protein as a new prognostic biomarker and/or therapeutic target for this disease.We wish to thank Gangoiti Barrera's Foundation for it support to this research project

Altered expression of fibroblast activation protein-α (FAP) in colorectal adenoma-carcinoma sequence and in lymph node and liver metastases

Colorectal cancer (CRC) is a major health problem in elderly people because of its high incidence and high mortality rate. Despite early screening programs, more than half of CRC patients are diagnosed at advanced stages. Fibroblast activation protein-alpha (FAP) expression in cancer-associated fibroblasts (CAFs) has been associated with a higher risk of metastases and poor survival. Here, we have analyzed the immunohistochemical expression of FAP in 41 adenoma-carcinoma sequences. In addition, FAP expression was analyzed individually and in combination with beta-catenin (BCAT), CD44 and Cyclin-D1 expression in primary tumors and in their corresponding lymph node and liver metastases (n=294). Finally, soluble FAP (sFAP) levels in plasma from CRC patients (n=127) were also analyzed by ELISA. FAP was expressed only in CRC tissue and its expression level was found to be higher in tumors exhibiting deeper local invasion and poorer cancer cell differentiation. FAP and concomitant nuclear BCAT expression in cancer cells at the infiltrating front of primary tumors and in lymph node metastases was independently associated with 5- and 10-year cancer specific and disease-free survival. Moreover, lower sFAP levels correlated with poorer survival. These findings support the potential importance of FAP as a biomarker of CRC development and progression.This work was partially funded by the ELKARTEK 18/10 grant from the Basque Government

Clinical Implications of (Pro)renin Receptor (PRR) Expression in Renal Tumours

(1) Background: Renal cancer is one of the most frequent malignancies in Western countries, with an unpredictable clinical outcome, partly due to its high heterogeneity and the scarcity of reliable biomarkers of tumour progression. (Pro)renin receptor (PRR) is a novel receptor of the renin–angiotensin system (RAS) that has been associated with the development and progression of some solid tumours by RAS-dependent and -independent mechanisms. (2) Methods: In this study, we analysed the immunohistochemical expression of PRR at the centre and border in a series of 83 clear-cell renal cell (CCRCCs), 19 papillary (PRCC) and 7 chromophobe (ChRCC) renal cell carcinomas, and the benign tumour renal oncocytoma (RO, n = 11). (3) Results: PRR is expressed in all the tumour subtypes, with higher mean staining intensity in ChRCCs and ROs. A high expression of PRR at the tumour centre and at the infiltrative front of CCRCC tissues is significantly associated with high grade, tumour diameter, local invasion and stage, and with high mortality risk by UCLA integrated staging system (UISS) scale. (4) Conclusions: These findings indicate that PRR is associated with the development and progression of renal tumours. Its potential as a novel biomarker for RCC diagnosis/prognosis and as a promising therapeutic target should be taken into account in the future.The work was funded by the Basque Government (ELKARTEK KK2018-00090 and KK-2020/00069)

An Integrative Omics Approach Reveals Involvement of BRCA1 in Hepatic Metastatic Progression of Colorectal Cancer

(1) Background & Aims: The roles of different cells in the tumor microenvironment (TME) are critical to the metastatic process. The phenotypic transformation of the liver cells is one of the most important stages of the hepatic metastasis progression of colorectal cancer (CRC). Our aim was to identify the major molecules (i.e., genes, miRNAs and proteins) involved in this process. (2) Methods: We isolated and performed whole-genome analysis of gene, miRNA, and protein expression in three types of liver cells (Ito cells, Kupffer cells, and liver sinusoidal endothelial cells) from the TME of a murine model of CRC liver metastasis. We selected the statistically significant differentially expressed molecules using the Student’s t-test with Benjamini-Hochberg correction and performed functional statistically-significant enrichment analysis of differentially expressed molecules with hypergeometric distribution using the curated collection of molecular signatures, MSigDB. To build a gene-miRNA-protein network centered in Brca1, we developed a software package (miRDiana) that collects miRNA targets from the union of the TargetScan, MicroCosm, mirTarBase, and miRWalk databases. This was used to search for miRNAs targeting Brca1. We validated the most relevant miRNAs with real-time quantitative PCR. To investigate BRCA1 protein expression, we built tissue microarrays (TMAs) from hepatic metastases of 34 CRC patients. (3) Results: Using integrated omics analyses, we observed that the Brca1 gene is among the twenty transcripts simultaneously up-regulated in all three types of TME liver cells during metastasis. Further analysis revealed that Brca1 is the last BRCA1-associated genome surveillance complex (BASC) gene activated in the TME. We confirmed this finding in human reanalyzing transcriptomics datasets from 184 patients from non-tumor colorectal tissue, primary colorectal tumor and colorectal liver metastasis of the GEO database. We found that the most probable sequence of cell activation during metastasis is Endothelial→Ito→Kupffer. Immunohistochemical analysis of human liver metastases showed the BRCA1 protein was co-localized in Ito, Kupffer, and endothelial cells in 81.8% of early or synchronous metastases. However, in the greater part of the metachronous liver metastases, this protein was not expressed in any of these TME cells. (4) Conclusions: These results suggest a possible role of the co-expression of BRCA1 in Ito, Kupffer, and sinusoidal endothelial cells in the early occurrence of CRC liver metastases, and point to BRCA1 as a potential TME biomarker.D.G. and M.J.A.-B. have been supported by Grants DFG113/18 from Diputación Foral de Gipuzkoa (DFG), Spain, Ministry of Economy and Competitiveness, Spain, MINECO Grant BFU2016-77987-P and Instituto de Salud Carlos III (AC17/00012) Grant co-funded by the European Union (Eracosysmed/H2020 Grant Agreement No. 643271) and European Union (H2020-FETOPEN, Project 899417). D.G., M.J.A.-B. and I.B. have been supported by Grants Health Department of the Basque Government (Spain), RIS3 call, Exp. No. 2020333039 and 2020333001

Soluble PD-L1 Is an Independent Prognostic Factor in Clear Cell Renal Cell Carcinoma

(1). Background: Immunohistochemical (IHC) evaluation of programmed death-1 (PD-1) and its ligand (PD-L1) is being used to evaluate advanced malignancies with potential response to immune checkpoint inhibitors. We evaluated both plasma and tissue expression of PD-1 and PD-L1 in the same cohort of patients, including non-metastatic and metastatic clear cell renal cell carcinoma (CCRCC). Concomitant plasma and tissue expression of PD-1 and PD-L1 was evaluated with emphasis on diagnostic and prognostic implications. (2) Methods: we analyzed PD-1 and PD-L1 IHC expression in tumor tissues and soluble forms (sPD-1 and sPD-L1) in plasma from 89 patients with CCRCC, of which 23 were metastatic and 16 received systemic therapy. The primary endpoint was evaluation of overall survival using Kaplan-Meier analysis and the Cox regression model. Plasma samples from healthy volunteers were also evaluated. (3) Results: Interestingly, sPD-1 and sPD-L1 levels were lower in cancer patients than in controls. sPD-1 and sPD-L1 levels and their counterpart tissue expression both at the tumor center and infiltrating front were not associated. Higher expression of both PD-1 and PD-L1 were associated with tumor grade, necrosis and tumor size. PD-1 was associated to tumor stage (pT) and PD-L1 to metastases. sPD-1 and sPD-L1 were not associated with clinico-pathological parameters, although both were higher in patients with synchronous metastases compared to metachronous ones and sPD-L1 was also higher for metastatic patients compared to non-metastatic patients. sPD-1 was also associated with the International Metastatic Renal Cell Cancer Database Consortium (IMDC) prognostic groups in metastatic CCRCC and also to the Morphology, Attenuation, Size and Structure (MASS) response criteria in metastatic patients treated with systemic therapy, mainly tyrosine-kinase inhibitors. Regarding prognosis, PD-L1 immunostaining at the tumor center with and without the tumor front was associated with worse survival, and so was sPD-L1 at a cut-off >793 ng/mL. Combination of positivity at both the tissue and plasma level increased the level of significance to predict prognosis. (4) Conclusions: Our findings corroborate the role of PD-L1 IHC to evaluate prognosis in CCRCC and present novel data on the usefulness of plasma sPD-L1 as a promising biomarker of survival in this neoplasia.The work was funded by the Basque Government (ELKARTEK KK2018-00090 and KK-2020/00069)

Using the Synergy between HPLC-MS and MALDI-MS Imaging to Explore the Lipidomics of Clear Cell Renal Cell Carcinoma

Lipid imaging mass spectrometry (LIMS) has been tested in several pathological contexts, demonstrating its ability to segregate and isolate lipid signatures in complex tissues, thanks to the technique’s spatial resolution. However, it cannot yet compete with the superior identification power of high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS), and therefore, very often, the latter is used to refine the assignment of the species detected by LIMS. Also, it is not clear if the differences in sensitivity and spatial resolution between the two techniques lead to a similar panel of biomarkers for a given disease. Here, we explore the capabilities of LIMS and HPLC-MS to produce a panel of lipid biomarkers to screen nephrectomy samples from 40 clear cell renal cell carcinoma patients. The same set of samples was explored by both techniques, and despite the important differences between them in terms of the number of detected and identified species (148 by LIMS and 344 by HPLC-MS in negative-ion mode) and the presence/absence of image capabilities, similar conclusions were reached: using the lipid fingerprint, it is possible to set up classifiers that correctly identify the samples as either healthy or tumor samples. The spatial resolution of LIMS enables extraction of additional information, such as the existence of necrotic areas or the existence of different tumor cell populations, but such information does not seem determinant for the correct classification of the samples, or it may be somehow compensated by the higher analytical power of HPLC-MS. Similar conclusions were reached with two very different techniques, validating their use for the discovery of lipid biomarkers.The work was funded by the Basque Government (IT971-16, IT1162-19, and ELKARTEK KK2018-00090) and has been developed as a Ph.D. project of LMS, who is the recipient of a Predoctoral Fellowship from the Spanish Government (BES- 2016-078721) . The authors are grateful to SGiker Lipidomic Service (UPV/EHU, MICINN, GV/EG, ESF) for the expert advice and technical and human support in MALDI and HPLC- MS analysis

Análisis de la proteína de activación de fibroblastos a (FAP) en el carcinoma colorrectal

125 p.Los fibroblastos asociados al cáncer son uno de los componentes mayoritarios del microambiente tumoral. La expresión de uno de sus marcadores, la Proteína de Activación de Fibroblastos ¿ (FAP) recientemente se ha vinculado con un peor pronóstico en diferentes tipos de neoplasias, por ejemplo, el carcinoma renal de células claras. En esta tesis doctoral se estudia la influencia de dicha proteína en el carcinoma colorrectal, una de las neoplasias con mayor incidencia a nivel mundial. Para ello se analiza la expresión inmunohistoquímica de FAP en toda la secuencia evolutiva de la neoplasia (pólipo adenomatoso, tumor primario, metástasis locorregional y metástasis a distancia). Se corrobora su expresión positiva en el tumor infiltrante y se evidencia que, además de expresarse más en el subtipo histológico convencional, ejerce fundamentalmente su influencia carcinogénica en las fases iniciales del proceso a nivel del tumor primario. No se le atribuye, sin embargo, un papel pronóstico influyente en la supervivencia. El estudio de los niveles séricos de la fracción soluble de FAP (FAPs) permite observar que los niveles de la proteína son inferiores en pacientes de carcinoma colorrectal que en sujetos sanos. Es más, a menores valores de concentración sérica el pronóstico de la enfemedad parece ser peor. Se presenta, por tanto, a FAPs como un posible biomarcador en el diagnóstico y seguimiento del carcinoma colorrectal