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Alvarez-Mugica M.,Tumor Markers Group | Alvarez-Mugica M.,Hospital Valle Del Nalon | Fernandez-Gomez J.M.,Hospital Universitario Central Of Asturias | Cebrian V.,Tumor Markers Group | And 3 more authors.
European Urology | Year: 2013

Background: Bacillus Calmette-Guérin (BCG) is a standard treatment to reduce tumor recurrence and delay progression of high-risk non-muscle-invasive (NMI) bladder tumors. However, it is not clear yet which patients are more likely to respond to BCG. Objective: The aim was to evaluate the role of polyamine-modulated factor-1 (PMF-1) methylation in predicting clinical outcome of T1 high-grade (T1HG) bladder tumors treated with BCG. Design, setting, and participants: In a retrospective design, PMF-1 methylation was analyzed on tumor specimens belonging to 108 patients with T1HG NMI bladder cancer undergoing BCG treatment. Median follow-up was 77 mo (range: 5-235 mo). Outcome measurements and statistical analysis: PMF-1 methylation was assessed by methylation-specific polymerase chain reactions. Recurrence, progression into muscle-invasive tumors, and disease-specific survival rates were analyzed using competing risks regression analysis. Results and limitations: Among the 108 patients analyzed, 35 had recurring disease (32.4%), 21 progressed (19.4%), and 16 died of disease (14.8%); 71.3% of tumors had PMF-1 methylation. Univariate analyses using cumulative incidence curves revealed that an unmethylated PMF-1 was significantly associated with increased recurrence (p = 0.026), progression (p = 0.01), and shorter disease-specific survival (log-rank, p = 0.03). Multivariate analyses indicated that among sex, age, focality, tumor size, and concomitant carcinoma in situ, only PMF-1 methylation provided significant hazard ratios (HRs) for recurrence of (HR: 2.032; p = 0.042), and progression (HR: 2.910; p = 0.020). Limitations of the study include its retrospective design, lymphovascular invasion status not available, short maintenance BCG, and that a second transurethral resection was not performed. Conclusions: Epigenetic analyses revealed that the methylation status of PMF-1 was associated with the clinical outcome of patients with T1HG tumors undergoing BCG treatment. An unmethylated PMF-1 correlated to recurrence and progression in T1HG disease using univariate and multivariate analyses. Thus, assessing the methylation status of PMF-1 may serve to distinguish patients responding to BCG from those who may require more aggressive therapeutic approaches. © 2012 European Association of Urology. Published by Elsevier B.V. All rights reserved.


Maraver A.,Tumor Suppression Group | Fernandez-Marcos P.,Tumor Suppression Group | Herranz D.,Tumor Suppression Group | Herranz D.,Columbia University | And 11 more authors.
Cancer Cell | Year: 2012

Here, we have investigated the role of the Notch pathway in the generation and maintenance of Kras G12V-driven non-small cell lung carcinomas (NSCLCs). We demonstrate by genetic means that γ-secretase and RBPJ are essential for the formation of NSCLCs. Of importance, pharmacologic treatment of mice carrying autochthonous NSCLCs with a γ-secretase inhibitor (GSI) blocks cancer growth. Treated carcinomas present reduced HES1 levels and reduced phosphorylated ERK without changes in phosphorylated MEK. Mechanistically, we show that HES1 directly binds to and represses the promoter of DUSP1, encoding a dual phosphatase that is active against phospho-ERK. Accordingly, GSI treatment upregulates DUSP1 and decreases phospho-ERK. These data provide proof of the in vivo therapeutic potential of GSIs in primary NSCLCs. © 2012 Elsevier Inc.


Garcia-Baquero R.,Tumor Markers Group | Garcia-Baquero R.,Hospital Puerta Del Mar | Puerta P.,Tumor Markers Group | Beltran M.,Hospital Puerta Del Mar | And 4 more authors.
Journal of Urology | Year: 2013

Purpose: Changes in DNA methylation of tumor suppressor genes early in carcinogenesis represent potential indicators of cancer detection and disease evolution. We examined the diagnostic, stratification and prognostic biomarker roles in urine of the methylation of a novel panel of tumor suppressor genes in bladder cancer. Material and Methods: We evaluated the methylation of 18 tumor suppressor genes in 2 prospective, independent sets of urine samples (training set of 120 preparations and validation set of 128) from patients with bladder cancer (170) and controls (78) using methylation specific multiplex ligation-dependent probe amplification. Diagnostic performance was evaluated with ROC curves. Recurrence, progression and disease specific survival were analyzed using univariate and multivariate Cox models. Results: PRDM2, HLTF, ID4, DLC1, BNIP3, H2AFX, CACNA1G, TGIF and CACNA1A were methylated in bladder cancer. CCND2, SCGB3A1, BNIP3, ID4 and RUNX3 were the most frequently methylated tumor suppressor genes in each urine set. Methylation of several tumor suppressor genes correlated with clinicopathological variables, such as stage, tumor grade, focality or age. ROC analysis revealed significant diagnostic accuracy for RUNX3 and CACNA1A in the training set, and for RUNX3 and ID4 in the validation set. On univariate and multivariate analysis CACNA1A methylation correlated with recurrence in the training set, while in the validation set PRDM2 and BNIP3 were significantly associated with recurrence and disease specific survival, respectively. Conclusions: Tumor suppressor gene methylation allowed for histopathological and clinical stratification. Urine methylation has noninvasive usefulness not only for diagnostic assessment but also as independent bladder cancer prognosticators. © 2013 American Urological Association Education and Research, Inc.


Moya P.,Tumor Markers Group | Esteban S.,Tumor Markers Group | Fernandez-Suarez A.,Hospital Alto Guadalquivir andujar | Maestro M.,Hospital Clinico Universitario | And 2 more authors.
Tumor Biology | Year: 2013

KISS1 is a metastasis suppressor lost in several solid malignancies. We evaluated the clinical relevance of KiSS-1 methylation and its protein expression in colorectal cancer. The epigenetic silencing of KiSS-1 by hypermethylation was tested in colon cancer cells (n = 5) before and after azacytidine treatment. KiSS-1 methylation was evaluated by methylation-specific PCR in colorectal cancer cells, and normal, benign, and tumor tissues (n = 352) were grouped in a training set (n = 62) and two independent validation cohorts (n = 100 and n = 190). KiSS-1 protein expression was analyzed by immunohistochemistry on tissue arrays. KiSS-1 hypermethylation correlated with transcript and protein expression loss, being increased in vitro by azacytidine. Methylation rates were 53.1, 70.0, and 80.0 % in the training and validation sets, respectively. In the training set, KiSS-1 methylation rendered a diagnostic accuracy of 72.7 % (p = 0.002). Combination of KiSS-1 methylation and serum CEA (p = 0.001) increased the prognostic utility of CEA alone (p = 0.022). In the first validation set, KiSS-1 methylation correlated with tumor grade (p = 0.011), predicted recurrence (p = 0.009), metastasis (p = 0.004), disease-free (p = 0.034), and overall survival (p = 0.015). In the second validation cohort, KiSS-1 methylation predicted disease-specific survival (p = 0.030). In the training set, cytoplasmic KiSS-1 expression was significantly higher in nonneoplastic biopsies as compared to colorectal tumors (p < 0.0005). In the validation set, loss of cytoplasmic expression correlated with tumor stage (p = 0.007), grade (p = 0.035), recurrence (p = 0.017), and disease-specific survival (p = 0.022). KiSS-1 was revealed epigenetically modified in colorectal cancer. The diagnostic and prognostic utility of KiSS-1 methylation and expression patterns suggests their assessment for the clinical management of colorectal cancer patients. © 2012 International Society of Oncology and BioMarkers (ISOBM).


Thomas S.,University of Virginia | Overdevest J.B.,University of Virginia | Nitz M.D.,University of Virginia | Williams P.D.,University of Virginia | And 6 more authors.
Cancer Research | Year: 2011

In bladder cancer, increased caveolin-1 (Cav-1) expression and decreased Src expression and kinase activity correlate with tumor aggressiveness. Here, we investigate the clinical and functional significance, if any, of this reciprocal expression in bladder cancer metastasis. We evaluated the ability of tumor Cav-1 and Src RNA and protein expression to predict outcome following cystectomy in 257 patients enrolled in two independent clinical studies. In both, high Cav-1 and low Src levels were associated with metastasis development. We overexpressed or depleted Cav-1 and Src protein levels in UMUC-3 and RT4 human bladder cancer cells and evaluated the effect of this on actin stress fibers, migration using Transwells, and lung metastasis following tail vein inoculation. Cav-1 depletion or expression of active Src in metastatic UMUC-3 cells decreases actin stress fibers, cell migration, and metastasis, while Cav-1 overexpression or Src depletion increased the migration of nonmetastatic RT4 cells. Biochemical studies indicated that Cav-1 mediates these effects via its phosphorylated form (pY14), whereas Src effects are mediated through phosphorylation of p190RhoGAP and these pathways converge to reduce activity of RhoA, RhoC, and Rho effector ROCK1. Treatment with a ROCK inhibitor reduced UMUC-3 lung metastasis in vivo, phenocopying the effect of Cav-1 depletion or expression of active Src. Src suppresses whereas Cav-1 promotes metastasis of bladder cancer through a pharmacologically tractable common downstream signaling pathway. Clinical evaluation of personalized therapy to suppress metastasis development based on Cav-1 and Src profiles seems warranted. ©2010 AACR.


Puerta-Gil P.,Tumor Markers Group | Garca-Baquero R.,Hospital Puerta Del Mar | Jia A.Y.,Columbia University | Ocaa S.,Hospital Fundacion Of Alcorcon | And 5 more authors.
American Journal of Pathology | Year: 2012

Altered microRNA (miRNA) expression may occur early in bladder cancer and may play a role in carcinogenesis and tumor behavior. We evaluated whether alterations in miRNA expression could improve disease stratification and outcome prognosis in bladder tumors and noninvasive diagnosis in urinary samples. miR-143, miR-222, and miR-452 expression levels were analyzed by quantitative RT-PCR (RT-qPCR) in paired urinary and matching tumors and in two independent prospective series of tumors and urinary specimens. Differential expression of miR-143, miR-222, and miR-452 in urine were verified by in situ hybridization in matching tumors. Tumor miRNA expression by RT-qPCR correlated with tumor grade, size, and presence of carcinoma in situ for miR-222, recurrence (miR-222 and miR-143), progression (miR-222 and miR-143), disease-specific survival (miR-222), and overall survival (miR-222). Protein expression patterns of potential miRNA targets, including vascular endothelial growth factor, BCL2, v-erb-b2 erythroblastic leukemia viral oncogene (ERBB) homolog 3, and ERBB4, were evaluated by IHC in tissue arrays containing tumors for which miRNAs were assessed by RT-qPCR. Target expression correlated with expression of their predicted regulatory miRNAs, recurrence (ERBB3), progression (ERBB4), disease-specific survival (ERBB3 and ERBB4), and overall survival (ERBB3 and ERBB4). Furthermore, RT-qPCR of miR-452 (area under the curve, 0.848) and miR-222 (area under the curve, 0.718) in urine provided high accuracies for bladder cancer diagnosis. Thus, bladder tumors were characterized by changes in miRNA expression that could aid in tumor stratification and clinical outcome prognosis, and miRNAs were detected in urinary specimens for noninvasive diagnosis. © 2012 American Society for Investigative Pathology.


Karni-Schmidt O.,Columbia University | Castillo-Martin M.,Columbia University | HuaiShen T.,Columbia University | Gladoun N.,Columbia University | And 6 more authors.
American Journal of Pathology | Year: 2011

The TP63 gene, a member of the TP53 tumor suppressor gene family, can be expressed as at least six isoforms due to alternative promoter use and alternative splicing. The lack of p63 isoform-specific antibodies has limited the analysis of the biological significance of p63. We report a novel set of well-defined antibodies to examine p63 isoforms in mouse and human urothelium during embryogenesis and tumor progression, respectively. We provide evidence that basal and intermediate urothelial cells express p63 isoforms, with the TAp63 variant the first to be detected during development, whereas umbrella cells are characterized by a p63-negative phenotype. Notably, we report that p63-null mice develop a bladder with an abnormal urothelium, constituted by a single layer of cells that express uroplakin II and low molecular weight cytokeratins, consistent with an umbrella cell phenotype. Finally, analysis of 202 human bladder carcinomas revealed a new categorization of invasive tumors into basal-like (positive for ΔNp63 and high molecular weight cytokeratins and negative for low molecular weight cytokeratins) versus luminal-like (negative for ΔNp63 and high molecular weight cytokeratins and positive for low molecular weight cytokeratins) phenotypes, with ΔNp63 expression associated with an aggressive clinical course and poor prognosis. This study highlights the relevance of p63 isoforms in both urothelial development and bladder carcinoma progression, with ΔNp63 acting as an oncogene in certain invasive bladder tumors. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.


Sanchez-Carbayo M.,Tumor Markers Group
Tumor Biology | Year: 2012

A compelling body of evidences sustains the importance of epigenetic mechanisms in the development and progression of cancer. Assessing the epigenetic component of bladder tumors is strongly improving our understanding of their biology and clinical behavior. In terms of DNA methylation, cancer cells show genome-wide hypomethylation and site-specific CpG island promoter hypermethylation. In the context of other epigenetic alterations, this review will focus on the hypermethylation of CpG islands in promoter regions, as the most widely described epigenetic modification in bladder cancer. CpG islands hypermethylation is believed to be critical in the transcriptional silencing and regulation of tumor suppressor and crucial cancer genes involved in the major molecular pathways controlling bladder cancer development and progression. In particular, several biological pathways of frequently methylated genes include cell cycle, DNA repair, apoptosis, and invasion, among others. Furthermore, translational aspects of bladder cancer methylomes described to date will be discussed towards their potential application as bladder cancer biomarkers. Several tissue methylation signatures and individual candidates have been evidenced, that could potentially stratify tumors histopathologically, and discriminate patients in terms of their clinical outcome. Tumor methylation profiles could also be detected in urinary specimens showing a promising role as non-invasive markers for cancer diagnosis towards an early detection and potentially for the surveillance of bladder cancer patients in a near future. However, the epigenomic exploration of bladder cancer has only just begun. Genome-scale DNA methylation profiling studies will further highlight the relevance of the epigenetic component to gain knowledge of bladder cancer biology and identify those profiles and candidates better correlating with clinical behavior. © International Society of Oncology and BioMarkers (ISOBM) 2011.


In the context of other proteomic technologies, targeted antibody arrays are strongly contributing for protein profiling and functional proteomics analyses in serum specimens. Protein-protein interactions, post-translational modifications, and interaction between protein and DNA or RNA can all shift the activity of a protein from what would have been predicted by its level of transcription. Functional proteomics studies the interaction of proteins within their cellular environment to determine how a given protein accomplishes its specific cellular task. Accordingly, the promise of protein profiling and functional proteomics is that by chronicling the function of aberrant or over-expressed proteins, it will be possible to characterize the mechanism of the disease-sustaining proteins. The further understanding of the disease networks will eventually lead to targeted cancer therapy and specific biomarkers for diagnosis, prognosis or therapeutic response prediction based on disease specific proteins. This review describes how such strategies reported to date in serum specimens may assist in characterizing tumor biology, and for the diagnosis, surveillance, prognosis, and potentially for predictive and therapeutic purposes for patients affected with solid and hematological neoplasias. © 2010 International Society of Oncology and BioMarkers (ISOBM).


Sanchez-Carbayo M.,Tumor Markers Group
Methods in Molecular Biology | Year: 2011

The cancer biomarkers field is being enriched by molecular profiling obtained by high-throughput approaches. Targeted antibody arrays are strongly contributing to the identification of protein cancer -biomarker candidates and functional proteomic analyses. Due to their versatility, novel technological and experimental design implementations are broadening the applications of antibody arrays. However, the cancer biomarker candidates delivered to date using this technology are still in their early developmental phase, requiring validation with high number of specimens focusing on specific clinical endpoints. Innovative strategies multiplexing protein measurements of protein extracts of cultured cells, tissue and body fluids using antibody arrays combined with appropriate validation approaches are enabling the -discovery of cancer-associated biomarkers. This review describes these strategies and cancer biomarker candidates reported to date that may assist in the diagnosis, surveillance, prognosis, and potentially for predictive and therapeutic purposes for patients affected with solid and hematological neoplasias. © Springer Science+Business Media, LLC 2011.

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