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Cisplatin-based neoadjuvant chemotherapy (NAC) followed by radical cystectomy is recommended for patients with muscle-invasive bladder cancer (MIBC). It has been shown that somatic deleterious mutations in ERCC2, gain-of-function mutations in ERBB2, and alterations in ATM, RB1, and FANCC are correlated with pathological response to NAC in MIBC. The objective of this study was to validate these genomic biomarkers in pretreatment transurethral resection material from an independent retrospective cohort of 165 patients with MIBC who subsequently underwent NAC and radical surgery. Patients with ypT0/Tis/Ta/T1N0 disease after surgery were defined as responders. Somatic deleterious mutations in ERCC2 were found in nine of 68 (13%) evaluable responders and two of 95 (2%) evaluable nonresponders (p = 0.009; FDR = 0.03). No correlation was observed between response and alterations in ERBB2 or in ATM, RB1, or FANCC alone or in combination. In an exploratory analysis, no additional genomic alterations discriminated between responders and nonresponders to NAC. No further associations were identified between the aforementioned biomarkers and pathological complete response (ypT0N0) after surgery. In conclusion, we observed a positive association between deleterious mutations in ERCC2 and pathological response to NAC, but not overall survival or recurrence-free survival. Other previously reported genomic biomarkers were not validated. Patient summary: It is currently unknown which patients will respond to chemotherapy before definitive surgery for bladder cancer. Previous studies described several gene mutations in bladder cancer that correlated with chemotherapy response. This study confirmed that patients with bladder cancer with a mutation in the ERCC2 gene often respond to chemotherapy.

The view of prostate cancer (PCa) progression as a result of the interaction of epithelial cancer cells with the host's immune system is supported by the presence of tumor infiltrating lymphocytes (TILs). TILs fate and interaction with the tumor microenvironment is mediated by accessory molecules such as CD5 and CD6, two signal-transducing coreceptors involved in fine-tuning of T cell responses. While the nature of the CD5 ligand is still controversial, CD6 binds CD166/ALCAM, a cell adhesion molecule involved in progression and dissemination of epithelial cancers, including PCa. The purpose of the present study was to determine the role of CD5, CD6, and CD166/ALCAM gene variants in PCa.Functionally relevant CD5 (rs2241002 and rs2229177), CD6 (rs17824933, rs11230563, and rs12360861) and CD166/ALCAM (rs6437585, rs579565, rs1044243, and rs35271455) single nucleotide polymorphisms (SNPs) were genotyped in germline DNA samples from 376 PCa patients. Their association with PCa prognostic factors, namely biochemical recurrence (BCR) and International Society of Urological Pathology (ISUP) grade was analyzed by generalized linear models and survival analyses.Proportional hazards regression showed that the minor CD6 rs12360861The results show the impact on PCa aggressiveness and recurrence brought about by gene variants involved in modulation of lymphocyte activation (CD5, CD6) and immune-epithelial cell adhesion (CD166/ALCAM) in PCa aggressiveness and recurrence, thus supporting a role for host immune response in PCa pathophysiology.

Molecular characterization of radical prostatectomy specimens after systemic therapy may identify a gene expression profile for resistance to therapy. This study assessed tumor cells from patients with prostate cancer participating in a phase II neoadjuvant docetaxel and androgen deprivation trial to identify mediators of resistance. Transcriptional level of 93 genes from a docetaxel-resistant prostate cancer cell lines microarray study was analyzed by TaqMan low-density arrays in tumors from patients with high-risk localized prostate cancer (36 surgically treated, 28 with neoadjuvant docetaxel + androgen deprivation). Gene expression was compared between groups and correlated with clinical outcome. VIM, AR and RELA were validated by immunohistochemistry. CD44 and ZEB1 expression was tested by immunofluorescence in cells and tumor samples. Parental and docetaxel-resistant castration-resistant prostate cancer cell lines were tested for epithelial-to-mesenchymal transition (EMT) markers before and after docetaxel exposure. Reversion of EMT phenotype was investigated as a docetaxel resistance reversion strategy. Expression of 63 (67.7%) genes differed between groups (P < 0.05), including genes related to androgen receptor, NF-κB transcription factor, and EMT. Increased expression of EMT markers correlated with radiologic relapse. Docetaxel-resistant cells had increased EMT and stem-like cell markers expression. ZEB1 siRNA transfection reverted docetaxel resistance and reduced CD44 expression in DU-145R and PC-3R. Before docetaxel exposure, a selected CD44+ subpopulation of PC-3 cells exhibited EMT phenotype and intrinsic docetaxel resistance; ZEB1/CD44+ subpopulations were found in tumor cell lines and primary tumors; this correlated with aggressive clinical behavior. This study identifies genes potentially related to chemotherapy resistance and supports evidence of the EMT role in docetaxel resistance and adverse clinical behavior in early prostate cancer. Mol Cancer Ther; 13(5); 1270–84. ©2014 AACR.

PDF - 265K, Biochemical-relapse analysis according to gene expression levels of markers differentially expressed in treated vs non-treated patients.

PDF - 72K, Genes expression values by qRT-PCR in treated patients vs non-treated patients; TLDA: TaqMan Low Density Arrays; FDR: False Discovery Rate.

PDF - 475K, Radiological-progression analysis according to gene expression levels of markers differentially expressed in treated vs non-treated patients.

PDF - 35K, Legends for Supplementary Figures 1 and 2.

Supplementary Data from Gene Expression Signature in Urine for Diagnosing and Assessing Aggressiveness of Bladder Urothelial Carcinoma

Purpose: To develop an accurate and noninvasive method for bladder cancer diagnosis and prediction of disease aggressiveness based on the gene expression patterns of urine samples.Experimental Design: Gene expression patterns of 341 urine samples from bladder urothelial cell carcinoma (UCC) patients and 235 controls were analyzed via TaqMan Arrays. In a first phase of the study, three consecutive gene selection steps were done to identify a gene set expression signature to detect and stratify UCC in urine. Subsequently, those genes more informative for UCC diagnosis and prediction of tumor aggressiveness were combined to obtain a classification system of bladder cancer samples. In a second phase, the obtained gene set signature was evaluated in a routine clinical scenario analyzing only voided urine samples.Results: We have identified a 12+2 gene expression signature for UCC diagnosis and prediction of tumor aggressiveness on urine samples. Overall, this gene set panel had 98% sensitivity (SN) and 99% specificity (SP) in discriminating between UCC and control samples and 79% SN and 92% SP in predicting tumor aggressiveness. The translation of the model to the clinically applicable format corroborates that the 12+2 gene set panel described maintains a high accuracy for UCC diagnosis (SN = 89% and SP = 95%) and tumor aggressiveness prediction (SN = 79% and SP = 91%) in voided urine samples.Conclusions: The 12+2 gene expression signature described in urine is able to identify patients suffering from UCC and predict tumor aggressiveness. We show that a panel of molecular markers may improve the schedule for diagnosis and follow-up in UCC patients. Clin Cancer Res; 16(9); 2624–33. ©2010 AACR.

BACKGROUND: Various germline genetic variants are associated with the prognosis of non-muscle invasive bladder cancer (NMIBC). Germline variants in genes frequently somatically mutated in bladder cancer have not been studied thoroughly in relation to risk of recurrence or progression in NMIBC. OBJECTIVE: To identify germline DNA variants in bladder carcinogenesis-related genes associated with recurrence or progression in NMIBC. METHODS: We analysed associations between single-nucleotide polymorphisms (SNPs) and NMIBC recurrence and progression using data from the Nijmegen Bladder Cancer Study (NBCS, 1,443 patients). We included 5,053 SNPs within 46 genes known to have mutation, overexpression or amplification in bladder cancer. We included all recurrences in the statistical analysis and performed both single variant analysis and gene-based analysis. SNPs and genes that showed significant or suggestive association (false discovery rate P value

This chapter aims to introduce the moleculartechnologies that have been optimized for theanalysis of biomarkers in recent years and that are currentlybeing applied in clinical practice.The chapter is divided into three blocks, which dealwith the concepts and techniques of molecular biology related to DNA-, RNA- and proteins-based biomarkers.In addition, real case examples in which these moleculartechnologies are used in clinical routine are given.Este capítulo pretende introducir lastecnologías moleculares que se han ido optimizandopara el análisis de los biomarcadores en los últimosaños y que están siendo aplicadas actualmente en lapráctica clínica.El capítulo está dividido en tres bloques, en los quese tratan los conceptos y técnicas de biología molecularrelacionadas con los biomarcadores basados enDNA, RNA y proteínas. Además, se dan ejemplos de casosreales en los que se utilizan estas tecnologías molecularesen la rutina clínica.

Purpose Current clinical prognostic factors are not accurate enough to identify and monitor those muscle-invasive bladder cancer (MIBC) patients at high risk of progression after radical cystectomy (RC). Here, we determined genetic alterations in the tumor and circulating tumor cell (CTC) enumeration to find biomarkers useful for the management of MIBC after RC. Methods Thirty-nine MIBC patients undergoing RC were included. Tumoral tissue DNA was analyzed by next generation sequencing. CTCs were isolated from blood collected before RC and one, four and 12 months later. Results Sixteen (41%) patients progressed in a median time of 8.5 months and 11 (69%) of these patients harbored the TERT c.-124C > T mutation. All progressive patients harboring the TERT c.-124C > T mutation presented a significant increase in CTC number 12 months after RC compared to those without the mutation. Additionally, CTC number at 12 months was identified as an independent prognostic biomarker for tumor progression and cancer specific survival (CSS). Ten (63%) progressive patients showed an increment of CTC number with a median anticipation period of four months compared with imaging techniques. Conclusions The TERT c.-124C > T mutation could be considered a biomarker of aggressivity. CTC enumeration is a useful tool for identifying MIBC patients at high risk of progression and CSS after RC and for detecting tumor progression earlier than imaging techniques.

Non-invasive urine-based biomarkers can potentially improve current diagnostic and monitoring protocols for bladder cancer (BC). Here we assess the performance of earlier published biomarker panels for BC detection (BC-116) and monitoring of recurrence (BC-106) in combination with cytology, in two prospectively collected patient cohorts.Of the 602 patients screened for BC, 551 were found eligible. For the primary setting, 73 patients diagnosed with primary BC (n = 27) and benign urological disorders, including patients with macroscopic haematuria, cystitis and/or nephrolithiasis (n = 46) were included. In total, 478 patients under surveillance were additionally considered (83 BC recurrences; 395 negative for recurrence). Urine samples were analysed with capillary electrophoresis-mass spectrometry. The biomarker score was estimated via support vector machine-based software.Validation of BC-116 biomarker panel resulted in 89% sensitivity and 67% specificity (AUCBC-116 biomarker panel is a useful test for detecting primary BC. BC-106 classifier integrated with cytology showing95% negative predictive value, might be useful for decreasing the number of cystoscopies during surveillance.

Background: Non-muscle-invasive bladder cancer (NMIBC) is characterized by frequent recurrences and a risk of progression in stage and grade. Increased knowledge of underlying biological mechanisms is needed.Objective: To identify single nucleotide polymorphisms (SNPs) associated with recurrence-free (RFS) and progression-free (PFS) survival in NMIBC.Design, setting, and participants: We analyzed outcome data from 3400 newly diagnosed NMIBC patients from the Netherlands, the UK, Canada, and Spain. We generated genome-wide germline SNP data using Illumina OmniExpress and Infinium Global Screening Array in combination with genotype imputation.Outcome measurements and statistical analysis: Cohort-specific genome-wide association studies (GWASs) for RFS and PFS were performed using a Cox proportional hazard model. Results were combined in a fixed-effect inverse-variance weighted meta-analysis. Candidate genes for the identified SNP associations were prioritized using functional annotation, gene-based analysis, expression quantitative trait locus analysis, and transcription factor binding site databases. Tumor expression levels of prioritized genes were tested for association with RFS and PFS in an independent NMIBC cohort.Results and limitations: This meta-analysis revealed a genome-wide significant locus for RFS on chromosome 14 (lead SNP rs12885353, hazard ratio [HR] C vs T allele 1.55, 95% confidence interval [CI] 1.33-1.82, p = 4.0 x 10(-8)), containing genes G2E3 and SCFD1. Higher expression of SCFD1 was associated with increased RFS (HR 0.70, 95% CI 0.59-0.84, p(FDR) = 0.003). Twelve other loci were suggestively associated with RFS (p < 10(-5)), pointing toward 18 additional candidate genes. For PFS, ten loci showed suggestive evidence of association, indicating 36 candidate genes. Expression levels of ten of these genes were statistically significantly associated with PFS, of which four (IFT140, UBE2I, FAHD1, and NME3) showed directional consistency with our meta-analysis results and published literature.Conclusions: In this first prognostic GWAS in NMIBC, we identified several novel candidate loci and five genes that showed convincing associations with recurrence or progression.Patient summary: In this study, we searched for inherited DNA changes that affect the outcome of non-muscle-invasive bladder cancer (NMIBC). We identified several genes that are associated with disease recurrence and progression. The roles and mechanisms of these genes in NMIBC prognosis should be investigated in future studies. (C) 2021 The Authors. Published by Elsevier B.V. on behalf of European Association of Urology.

PurposeNon-invasive urine-based biomarkers for bladder cancer (BC) diagnosis and surveillance can potentially improve current diagnostic and monitoring protocols by guiding cystoscopy. Here, we aim to access the diagnostic performance of nomograms based on published biomarker panels for BC detection (BC-116) and monitoring of recurrence (BC-106) in combination with cytology, in two prospectively collected patient cohorts.Experimental Design602 recruited patients were screened for presence of BC, out of which 551 were found eligible for further analysis. For the primary setting, urine samples from 73 eligible patients were analyzed from those diagnosed with primary BC (n=27) and benign urological disorders (n=46). For the surveillance setting, 478 eligible patients were considered (83 BC recurrences; 395 negative for recurrence). Urine samples were analyzed with capillary electrophoresis coupled to mass spectrometry and the biomarker score was estimated via a support vector machine-based software.ResultsValidation of the BC-116 biomarker panel resulted in 89% sensitivity and 67% specificity (AUCBC-116=0.82), similar to the published estimates. The nomogram based on cytology and BC-116 resulted in good (AUCNom116=0.85) but not significantly better performance than the BC-116 alone (P=0.5672). BC-106 biomarker panel showed 89% sensitivity and 32% specificity for surveillance, while improved performance was achieved when a nomogram including BC-106 and cytology was evaluated (AUCNom106=0.82), significantly outperforming both cytology (AUCcyt=0.72;P=0.0022) and BC-106 alone (AUCBC-106=0.67;P=0.0012).ConclusionsBC-116 biomarker panel is a useful test for detecting primary BC. BC-106 classifier integrated with cytology and showing >95% negative predictive value, might be useful for decreasing the number of cystoscopies during surveillance.