Identification of miRNAs and Their Target Genes Associated with Sunitinib Resistance in Clear Cell Renal Cell Carcinoma Patients.

Sunitinib has greatly improved the survival of clear cell renal cell carcinoma (ccRCC) patients in recent years. However, 20-30% of treated patients do not respond. To identify miRNAs and genes associated with a response, comparisons were made between biopsies from responder and non-responder ccRCC patients. Using integrated transcriptomic analyses, we identified 37 miRNAs and 60 respective target genes, which were significantly associated with the NF-kappa B, PI3K-Akt and MAPK pathways. We validated expression of the miRNAs (miR-223, miR-155, miR-200b, miR-130b) and target genes (FLT1, PRDM1 and SAV1) in 35 ccRCC patients. High levels of miR-223 and low levels of FLT1, SAV1 and PRDM1 were associated with worse overall survival (OS), and combined miR-223 + SAV1 levels distinguished responders from non-responders (AUC = 0.92). Using immunohistochemical staining of 170 ccRCC patients, VEGFR1 (FLT1) expression was associated with treatment response, histological grade and RECIST (Response Evaluation Criteria in Solid Tumors) score, whereas SAV1 and BLIMP1 (PRDM1) were associated with metachronous metastatic disease. Using in situ hybridisation (ISH) to detect miR-155 we observed higher tumoural cell expression in non-responders, and non-tumoural cell expression with increased histological grade. In summary, our preliminary analysis using integrated miRNA-target gene analyses identified several novel biomarkers in ccRCC patients that surely warrant further investigation.

The Expression of Alamandine Receptor MrgD in Clear Cell Renal Cell Carcinoma Is Associated with a Worse Prognosis and Unfavorable Response to Antiangiogenic Therapy.

Renal cell carcinoma (RCC) ranks among the most prevalent malignancies in Western countries, marked by its notable heterogeneity, which contributes to an unpredictable clinical trajectory. The insufficiency of dependable biomarkers adds complexity to assessing this tumor progression. Imbalances of several components of the intrarenal renin-angiotensin system (iRAS) significantly impact patient prognoses and responses to first-line immunotherapies. In this study, we analyzed the immunohistochemical expression of the Mas-related G-protein-coupled receptor D (MrgD), which recognizes the novel RAS peptide alamandine (ALA), in a series of 87 clear cell renal cell (CCRCCs), 19 papillary (PRCC), 7 chromophobe (ChRCC) renal cell carcinomas, and 11 renal oncocytomas (RO). MrgD was expressed in all the renal tumor subtypes, with a higher mean staining intensity in the PRCCs, ChRCCs, and ROs. A high expression of MrgD at the tumor center and at the infiltrative front of CCRCC tissues was significantly associated with a high histological grade, large tumor diameter, local invasion, and locoregional node and distant metastasis. Patients with worse 5-year cancer-specific survival and a poorer response to antiangiogenic tyrosine-kinase inhibitors (TKIs) showed higher MrgD expression at the center of their primary tumors. These findings suggest a possible role of MrgD in renal carcinogenetic processes. Further studies are necessary to unveil its potential as a novel biomarker for CCRCC prognosis and response to frontline therapies.

Clear Cell Renal Cell Carcinoma: A Test Bench for Investigating Tumor Complexity.

Clear cell renal cell carcinoma (CCRCC), by far the most common renal cancer subtype, is an aggressive tumor variant, serving in recent years as a prolific test bench in cancer research [...].

Fibroblast Activation Protein-α (FAP) Identifies Stromal Invasion in Colorectal Neoplasia.

The increasing detection of colorectal adenomas and early adenocarcinomas (ADCs) in the context of nationwide screening programs has led to a significant increase in the incidence of inconclusive diagnoses in which histopathologic analysis of endoscopic biopsies does not allow pathologists to provide a reliable diagnosis of stromal invasion. The objective of this study was to analyze the discriminative capacity of the immunohistochemical expression of fibroblast activation protein-α (FAP) in distinguishing colorectal adenomas with low-grade dysplasia (LGD) and high-grade dysplasia (HGD) from invasive intestinal-type ADCs. The study analyzed the first endoscopic biopsies from a series of patients classified as inconclusive or conclusive for stromal invasion based on the pathologic report. In total, 30 ADCs, 52 HGDs, and 15 LGDs were included in the study. FAP expression was detected in 23/30 ADCs and was negative in all adenomas with either LGD or HGD features (100% specificity and 76.7% sensitivity, area under the curve=0.883, CI=0.79-0.98). Considering these findings, we conclude that FAP is a potentially useful tool for helping pathologists identify invasive lesions in colorectal endoscopic biopsies, avoiding unnecessary biopsy repetitions.

The expression pattern of pyruvate dehydrogenase kinases predicts prognosis and correlates with immune exhaustion in clear cell renal cell carcinoma.

Renal cancer cells constitute a paradigm of tumor cells with a glycolytic reprogramming which drives metabolic alterations favouring cell survival and transformation. We studied the expression and activity of pyruvate dehydrogenase kinases (PDK1-4), key enzymes of the energy metabolism, in renal cancer cells. We analysed the expression, subcellular distribution and clinicopathological correlations of PDK1-4 by immunohistochemistry of tumor tissue microarray samples from a cohort of 96 clear cell renal cell carcinoma (ccRCC) patients. Gene expression analysis was performed on whole tumor tissue sections of a subset of ccRCC samples. PDK2 and PDK3 protein expression in tumor cells correlated with lower patient overall survival, whereas PDK1 protein expression correlated with higher patient survival. Gene expression analysis revealed molecular association of PDK2 and PDK3 expression with PI3K signalling pathway, as well as with T cell infiltration and exhausted CD8 T cells. Inhibition of PDK by dichloroacetate in human renal cancer cell lines resulted in lower cell viability, which was accompanied by an increase in pAKT. Together, our findings suggest a differential role for PDK enzymes in ccRCC progression, and highlight PDK as actionable metabolic proteins in relation with PI3K signalling and exhausted CD8 T cells in ccRCC.

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.

(Pro)renin Receptor Is a Novel Independent Prognostic Marker in Invasive Urothelial Carcinoma of the Bladder.

(Pro)renin receptor (PRR) is being investigated in several malignancies as it activates pathogenic pathways that contribute to cell proliferation, immunosuppressive microenvironments, and acquisition of aggressive neoplastic phenotypes. Its implication in urothelial cancer (UC) has not been evaluated so far. We retrospectively evaluate the prognostic role of PRR expression in a series of patients with invasive UC treated with radical cystectomy and other clinical and histopathological parameters including p53, markers of immune-checkpoint inhibition, and basal and luminal phenotypes evaluated by tissue microarray. Cox regression analyses using stepwise selection evaluated candidate prognostic factors and disease-specific survival. PRR was expressed in 77.3% of the primary tumors and in 70% of positive lymph nodes. PRR expression correlated with age (p = 0.006) and was associated with lower preoperatively hemoglobin levels. No other statistical association was evidenced with clinical and pathological variables (gender, ASA score, Charlson comorbidity index, grade, pT, pN) or immunohistochemical expressions evaluated (CK20, GA-TA3, CK5/6, CD44, PD-L1, PD-1, B7-H3, VISTA, and p53). PRR expression in primary tumors was associated with worse survival (log-rank, p = 0.008). Cox regression revealed that PRR expression (HR 1.85, 95% CI 1.22-2.8), pT (HR 7.02, 95% CI 2.68-18.39), pN (HR 2.3, 95% CI 1.27-4.19), and p53 expression (HR 1.95, 95% CI 1.1-3.45) were independent prognostic factors in this series. In conclusion, we describe PRR protein and its prognostic role in invasive UC for the first time. Likely mechanisms involved are MAPK/ERK activation, Wnt/ß-catenin signaling, and v-ATPAse function.

GeromiRs Are Downregulated in the Tumor Microenvironment during Colon Cancer Colonization of the Liver in a Murine Metastasis Model.

Cancer is a phenomenon broadly related to ageing in various ways such as cell cycle deregulation, metabolic defects or telomerases dysfunction as principal processes. Although the tumor cell is the main actor in cancer progression, it is not the only element of the disease. Cells and the matrix surrounding the tumor, called the tumor microenvironment (TME), play key roles in cancer progression. Phenotypic changes of the TME are indispensable for disease progression and a few of these transformations are produced by epigenetic changes including miRNA dysregulation. In this study, we found that a specific group of miRNAs in the liver TME produced by colon cancer called geromiRs, which are miRNAs related to the ageing process, are significantly downregulated. The three principal cell types involved in the liver TME, namely, liver sinusoidal endothelial cells, hepatic stellate (Ito) cells and Kupffer cells, were isolated from a murine hepatic metastasis model, and the miRNA and gene expression profiles were studied. From the 115 geromiRs and their associated hallmarks of aging, which we compiled from the literature, 75 were represented in the used microarrays, 26 out of them were downregulated in the TME cells during colon cancer colonization of the liver, and none of them were upregulated. The histone modification hallmark of the downregulated geromiRs is significantly enriched with the geromiRs miR-15a, miR-16, miR-26a, miR-29a, miR-29b and miR-29c. We built a network of all of the geromiRs downregulated in the TME cells and their gene targets from the MirTarBase database, and we analyzed the expression of these geromiR gene targets in the TME. We found that Cercam and Spsb4, identified as prognostic markers in a few cancer types, are associated with downregulated geromiRs and are upregulated in the TME cells.

High-Resolution Human Kidney Molecular Histology by Imaging Mass Spectrometry of Lipids.

For many years, traditional histology has been the gold standard for the diagnosis of many diseases. However, alternative and powerful techniques have appeared in recent years that complement the information extracted from a tissue section. One of the most promising techniques is imaging mass spectrometry applied to lipidomics. Here, we demonstrate the capabilities of this technique to highlight the architectural features of the human kidney at a spatial resolution of 10 µm. Our data demonstrate that up to seven different segments of the nephron and the interstitial tissue can be readily identified in the sections according to their characteristic lipid fingerprints and that such fingerprints are maintained among different individuals (n = 32). These results set the foundation for further studies on the metabolic bases of the diseases affecting the human kidney.

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.

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.

Effectiveness of a structured group intervention based on pain neuroscience education for patients with fibromyalgia in primary care: A multicentre randomized open-label controlled trial.

BACKGROUND: There has been increased interest in pain neuroscience education (PNE) as a therapeutic approach for the management of fibromyalgia (FM). METHODS: A multicentre randomized, open-label, controlled trial was conducted to assess the effectiveness of a structured group intervention based on PNE in patients with FM. A total of 139 patients were included in the study and randomized to the intervention group (7 group sessions of education in neurobiology of pain) or to the control group (treatment as usual only). The primary outcome was the improvement of functional status and pain measured with the Fibromyalgia Impact Questionnaire (FIQ), and secondary outcomes were the reduction in the impact of pain and other symptoms (catastrophizing, anxiety and depression) and number of patients reaching no worse than moderate functional impairment (FIQ score <39). Differences between groups were calculated by linear mixed-effects (intention-to-treat approach) and mediational models through path analyses. RESULTS: At 1 year, improvements in FIQ scores were higher in the intervention group with moderate or high effect size, and decreases of ≥20% in 69.1% of patients (20.9% in the control group) and of ≥50% in 39.7% (4.5% in the control group). Also, 52.9% of patients had a FIQ <39 points (13.4% in the control group). CONCLUSIONS: In this sample of patients with FM, the improvement in quality of life and control of symptoms obtained by adding a PNE intervention showed promising results, equalling or surpassing previously reported outcomes. SIGNIFICANCE: A structured group intervention based on pain neuroscience education for 1 year in patients with fibromyalgia was associated with significant amelioration of the impact of the disease on scores of the Fibromyalgia Impact Questionnaire, the Health Assessment Questionnaire, the Hospital Anxiety and Depression Scale, the Pain Catastrophizing Scale and the Polysymptomatic Distress Scale as compared with only treatment as usual. These findings are clinically relevant considering the challenges posed by fibromyalgia to clinicians and patients alike.

The Activation of Prothrombin Seems to Play an Earlier Role than the Complement System in the Progression of Colorectal Cancer: A Mass Spectrometry Evaluation.

Colorectal cancer (CRC) is the second cause of death in men and the third in women. This work deals with the study of the low molecular weight protein fraction of sera from patients who underwent surgery for CRC and who were followed for several years thereafter. MALDI-TOF MS was used to identify serum peptidome profiles of healthy controls, non-metastatic CRC patients and metastatic CRC patients. A multiple regression model was applied to signals preliminarily selected by SAM analysis to take into account the age and gender differences between the groups. We found that, while a signal m/z 2021.08, corresponding to the C3f fragment of the complement system, appears significantly increased only in serum from metastatic CRC patients, a m/z 1561.72 signal, identified as a prothrombin fragment, has a significantly increased abundance in serum from non-metastatic patients as well. The findings were also validated by a bootstrap resampling procedure. The present results provide the basis for further studies on large cohorts of patients in order to confirm C3f and prothrombin as potential serum biomarkers. Thus, new and non-invasive tests might be developed to improve the classification of colorectal cancer.

Altered Tissue and Plasma Levels of Fibroblast Activation Protein-α (FAP) in Renal Tumours.

(1) Background: Renal cell carcinoma (RCC) is a heterogeneous and complex disease with only partial response to therapy, high incidence of metastasis and recurrences, and scarce reliable biomarkers indicative of progression and survival. Cancer-associated fibroblasts (CAFs) play an important role supporting and promoting renal cancer progression. (2) Methods: In this study, we analysed fibroblast activation protein-α (FAP) immunohistochemical expression and its soluble isoform (sFAP) in tumour tissues and plasma from 128 patients with renal tumours. (3) Results: FAP is expressed in the cell surface of CAFs of the tumour centre and infiltrating front from clear cell renal cell carcinomas (CCRCC, n = 89), papillary renal cell carcinomas (PRCC, n = 21), and chromophobe renal cell carcinomas (ChRCC, n = 8), but not in the benign tumour renal oncocytoma (RO, n = 10). A high expression of FAP and low levels sFAP are significantly associated with high tumour diameter, high grade, and high pT stage, lymph node invasion, development of early metastases, and worse 5-year cancer specific survival of CCRCC patients. (4) Conclusions: These findings corroborate the potential usefulness of FAP immunohistochemistry and plasma sFAP as a biomarker of CCRCC progression and point to CAF-related proteins as promising immunohistochemical biomarkers for the differential diagnosis of ChRCC and RO.

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.

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-α (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 ß-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.

The role of cancer-associated fibroblasts in renal cell carcinoma. An example of tumor modulation through tumor/non-tumor cell interactions.

Cancer-associated fibroblasts (CAF) are a cellular compartment of the tumor microenvironment (TME) with critical roles in tumor development. Fibroblast activation protein-α (FAP) is one of the proteins expressed by CAF and its immunohistochemical detection in routine practice is associated with tumor aggressiveness and shorter patient survival. For these reasons, FAP seems a good prognostic marker in many malignant neoplasms, including renal cell carcinoma (RCC). The start point of this Perspective paper is to review the role of CAF in the modulation of renal cell carcinoma evolution. In this sense, CAF have demonstrated to develop important protumor and/or antitumor activities. This apparent paradox suggests that some type of temporally or spatially-related specialization is present in this cellular compartment during tumor evolution. The end point is to remark that tumor/non-tumor cell interactions, in particular the symbiotic tumor/CAF connections, are permanent and ever-changing crucial phenomena along tumor lifetime. Interestingly, these interactions may be responsible of many therapeutic failures.

Altered expression of renin-angiotensin system receptors throughout colorectal adenoma-adenocarcinoma sequence.

Background and Objective: Colorectal cancer (CRC) is a major health problem in developed countries. Adenomatous lesions in the large bowel are the main precursors of CRC and the adenoma-adenocarcinoma sequence still provides a solid model for research on carcinogenesis. The finding of local renin-angiotensin systems (RAS) has been crucial to understand the role of this peptidergic system in cancer and has opened new perspectives in the study of colorectal carcinogenetic processes. Methods: In this study we analyzed the immunohistochemical expression of three main RAS receptors (AT1, AT2 and MAS) in a large series of CRC samples (n=161), including uninvolved intestinal mucosa-adenoma-adenocarcinoma sequences from the same patients (n=50). Results: 1) AT1 and AT2 showed a biphasic expression pattern along the sequence. The expression significantly decreased in adenomas with respect to uninvolved mucosa but increased in CRCs. 2) AT2 expression was lower in advanced CRCs with high local invasion (pT4), high stage (IV), high nodal (N2) and vascular invasion. 3) MAS receptor was moderately expressed in the uninvolved mucosa and in adenomas. This expression increased very significantly in CRC tissues. Conclusions: These results suggest that: 1) RAS receptors are differentially regulated as the genetic and epigenetic alterations accumulate throughout the uninvolved mucosa-adenoma-CRC sequence. 2) Loss of AT2 expression could contribute to the aggressive behavior of advanced CRC cells.

The coexpression of fibroblast activation protein (FAP) and basal-type markers (CK 5/6 and CD44) predicts prognosis in high-grade invasive urothelial carcinoma of the bladder.

High-grade urothelial carcinoma (UC) of the bladder is a heterogeneous disease with dismal prognosis. Bladder tumors with basal phenotype are intrinsically aggressive, and morphological parameters that define disease staging remain main prognosticators. We intend to evaluate the role of cancer-associated fibroblasts (CAFs) in the prognosis of bladder cancer and its association with basal and luminal phenotypes. Clinical and pathological parameters, including the immunohistochemical expression of fibroblast activation protein (FAP) and markers of basal (CK5/6, CD44) and luminal (CK20, GATA3) phenotypes, have been investigated in a series of 121 patients with UC of the bladder treated by radical cystectomy with lymph node dissection, and their implication in long-term cancer-specific survival has been evaluated. A cytoplasmic immunostaining of FAP in CAFs implies worse disease-specific survival (hazard ratio [HR] = 1.68; P = .048). FAP expression is associated with tumor staging (P < .0001), with best discrimination at T2a/T2b level, and with negative expression of markers of luminal phenotype, such as CK20 (P < .0001) and GATA3 (P = .005). In the multivariate analysis, simultaneous expression of FAP, CK5/6, and CD44 is a strong prognosticator of disease-specific survival (HR = 2.3; P = .001), together with nodal invasion (HR = 3.47; P < .0001) and bladder infiltration up to deep muscle or beyond (HR = 2.47; P = .02). There is no association between positive FAP expression in primary tumor and nodal disease (P = .22). FAP expression in CAFs favors tumor invasion in high-grade invasive UC of the bladder with basal phenotype. This new immunohistochemical marker could be added to the routine immunohistochemical protocol to predict clinical behavior in these patients.