Pancreatic cancer risk in relation to lifetime smoking patterns, tobacco type, and dose-response relationships.

BACKGROUND: Despite smoking being a well-established risk factor for pancreatic cancer (PC), there is a need to further characterize PC risk according to lifespan smoking patterns and other smoking features. Our aim was to deeply investigate them within a large European case-control study. METHODS: Tobacco smoking habits and other relevant information was obtained from 2,009 cases and 1,532 controls recruited in the PanGenEU study using standardized tools. Multivariate logistic regression analysis was performed to evaluate PC risk by smoking characteristics and interactions with other PC risk factors. Fractional polynomials and restricted cubic splines were used to test for non-linearity of the dose-response relationships and to analyse their shape. RESULTS: Relative to never-smokers, current smokers (OR=1.72, 95%CI: 1.39-2.12), those inhaling into the throat (OR=1.48, 95%CI: 1.11-1.99), chest (OR=1.33, 95%CI: 1.12-1.58), or using non-filtered cigarettes (OR=1.69, 95%CI: 1.10-2.61), were all at an increased PC risk. PC risk was highest in current black tobacco smokers (OR=2.09, 95%CI: 1.31-3.41), followed by blond tobacco smokers (OR=1.43, 95%CI: 1.01-2.04). Childhood exposure to tobacco smoke relative to parental smoking was also associated with increased PC risk (OR=1.24, 95%CI: 1.03-1.49). Dose-response relationships for smoking duration, intensity, cumulative dose, and smoking cessation were non-linear and showed different shapes by tobacco type. Effect modification by family history of PC and diabetes was likely. CONCLUSIONS: This study reveals differences in PC risk by tobacco type and other habit characteristics, as well as non-linear risk associations. IMPACT: This characterization of smoking-related PC risk profiles may help in defining PC high-risk populations

Framework for the Integration of Genomics, Epigenomics and Transcriptomics in Complex Diseases.

peer reviewedOBJECTIVES: Different types of '-omics' data are becoming available in the post-genome era; still a single -omics assessment provides limited insights to understand the biological mechanism of complex diseases. Genomics, epigenomics and transcriptomics data provide insight into the molecular dysregulation of neoplastic diseases, among them urothelial bladder cancer (UBC). Here, we propose a detailed analytical framework necessary to achieve an adequate integration of the three sets of -omics data to ultimately identify previously hidden genetic mechanisms in UBC. METHODS: We built a multi-staged framework to study possible pair-wise combinations and integrated the data in three-way relationships. SNP genotypes, CpG methylation levels and gene expression levels were determined for a total of 70 individuals with UBC and with fresh tumour tissue available. RESULTS: We suggest two main hypothesis-based scenarios for gene regulation based on the -omics integration analysis, where DNA methylation affects gene expression and genetic variants co-regulate gene expression and DNA methylation. We identified several three-way trans-association 'hotspots' that are found at the molecular level and that deserve further studies. CONCLUSIONS: The proposed integrative framework allowed us to identify relationships at the whole-genome level providing some new biological insights and highlighting the importance of integrating -omics data

CD8+ Cytotoxic Immune Infiltrate in Non-Muscle Invasive Bladder Cancer: A Standardized Methodology to Study Association with Clinico-Pathological Features and Prognosis

Background: Major interest lies in the evaluation of immune infiltrate in bladder cancer. CD8+ cytotoxic lymphocytes are key effectors of adaptive immune response. Objectives: The aims of the study were to set up a standardized methodology for CD8+ lymphocytes estimation in NMIBC and investigate how intra-tumoral heterogeneity influences CD8+ immune infiltrate. Methods: We considered 995 NMIBC included in the Spanish Bladder Cancer (SBC)/EPICURO Study. Duplicate 0.6mm TMA spots and paired full sections (FS) for 50 selected cases were double stained with anti-pan cytokeratin antibody and anti-CD8 antibody. Slides were digitalized and CD8+ cells were automatically counted after tissue recognition (tumor vs stroma). Spatial heterogeneity was assessed and a resampling strategy was applied to estimate the proper number of 0.6mm TMA spots providing an adequate CD8+ cell estimate. Association between CD8+ count and expression of urothelial differentiation markers was estimated. Cox regression models were performed to assess association between CD8+ cell count and risk of recurrence and progression. Results: Microscopic examination of full sections showed spatial heterogeneity for CD8+ infiltrates. Simulation analyses demonstrated that 5 TMA regions provided a correct sampling of tumor and stromal compartments in Ta while 2 and 6 TMA regions were necessary in T1, respectively. CD8+ cells infiltration was associated with stage, regardless of the histological compartment analyzed (median CD8+/mm(2) were 25/mm(2) and 129/mm(2) in tumor and stroma respectively in Ta and 111/mm(2) and 344/mm(2) in T1; p-value = 0.006). CD8+ infiltration in tumor compartment was significantly associated with low FGFR3 expression. CD8+/mm(2) count in the tumor compartment was not associated with prognosis. Conclusion: Differences identified between Ta and T1 tumours supported the hypothesis that rigorous efforts should be placed in proper study design. These results provide a new framework to investigate microenvironment complexity in bladder cancer

Development and Validation of Urine-based Peptide Biomarker Panels for Detecting Bladder Cancer in a Multi-center Study

Purpose: Urothelial bladder cancer presents high recurrence rates, mandating continuous monitoring via invasive cystoscopy. The development of noninvasive tests for disease diagnosis and surveillance remains an unmet clinical need. In this study, validation of two urine-based biomarker panels for detecting primary and recurrent urothelial bladder cancer was conducted. Experimental Design: Two studies (total n = 1,357) were performed for detecting primary (n = 721) and relapsed urothelial bladder cancer (n = 636). Cystoscopy was applied for detecting urothelial bladder cancer, while patients negative for recurrence had follow-up for at least one year to exclude presence of an undetected tumor at the time of sampling. Capillary electrophoresis coupled to mass spectrometry (CE-MS) was employed for the identification of urinary peptide biomarkers. The candidate urine-based peptide biomarker panels were derived from nested cross-sectional studies in primary (n = 451) and recurrent (n = 425) urothelial bladder cancer. Results: Two biomarker panels were developed on the basis of 116 and 106 peptide biomarkers using support vector machine algorithms. Validation of the urine-based biomarker panels in independent validation sets, resulted in AUC values of 0.87 and 0.75 for detecting primary (n = 270) and recurrent urothelial bladder cancer (n = 211), respectively. At the optimal threshold, the classifier for detecting primary urothelial bladder cancer exhibited 91% sensitivity and 68% specificity, while the classifier for recurrence demonstrated 87% sensitivity and 51% specificity. Particularly for patients undergoing surveillance, improved performance was achieved when combining the urine-based panel with cytology (AUC = 0.87). Conclusions: The developed urine-based peptide biomarker panel for detecting primary urothelial bladder cancer exhibits good performance. Combination of the urine-based panel and cytology resulted in improved performance for detecting disease recurrence. (C) 2016 AACR

Development and validation of urine-based peptide biomarker panels for detecting bladder cancer in a multi-center study

Purpose: Urothelial bladder cancer (UBC) presents high recurrence rates, mandating continuous monitoring via invasive cystoscopy. The development of non-invasive tests for disease diagnosis and surveillance remains an unmet clinical need. In this study, validation of two urine-based biomarker panels for detecting primary and recurrent UBC was conducted. Experimental Design: Two studies (total n=1357) were performed for detecting primary (n=721) and relapsed UBC (n=636). Cystoscopy was applied for detecting UBC, while patients negative for recurrence had follow-up for at least one year to exclude presence of an undetected tumor at the time of sampling. Capillary electrophoresis coupled to mass spectrometry (CE-MS) was employed for the identification of urinary peptide biomarkers. The candidate urine-based peptide biomarker panels were derived from nested cross-sectional studies in primary (n=451) and recurrent (n=425) UBC. Results: Two biomarker panels were developed based on 116 and 106 peptide biomarkers using support vector machine algorithms. Validation of the urine-based biomarker panels in independent validation sets, resulted in AUC values of 0.87 and 0.75 for detecting primary (n=270) and recurrent UBC (n=211), respectively. At the optimal threshold, the classifier for detecting primary UBC exhibited 91% sensitivity and 68% specificity, while the classifier for recurrence demonstrated 87% sensitivity and 51% specificity. Particularly for patients undergoing surveillance, improved performance was achieved when combining the urine-based panel with cytology (AUC of 0.87). Conclusions: The developed urine-based peptide biomarker panel for detecting primary UBC exhibits good performance. Combination of the urine-based panel and cytology resulted in improved performance for detecting disease recurrence

Development and Validation of Urine-based Peptide Biomarker Panels for Detecting Bladder Cancer in a Multi-center Study

Purpose: Urothelial bladder cancer (UBC) presents high recurrence rates, mandating continuous monitoring via invasive cystoscopy. The development of non-invasive tests for disease diagnosis and surveillance remains an unmet clinical need. In this study, validation of two urine-based biomarker panels for detecting primary and recurrent UBC was conducted. Experimental Design: Two studies (total n=1357) were performed for detecting primary (n=721) and relapsed UBC (n=636). Cystoscopy was applied for detecting UBC, while patients negative for recurrence had follow-up for at least one year to exclude presence of an undetected tumor at the time of sampling. Capillary electrophoresis coupled to mass spectrometry (CE-MS) was employed for the identification of urinary peptide biomarkers. The candidate urine-based peptide biomarker panels were derived from nested cross-sectional studies in primary (n=451) and recurrent (n=425) UBC. Results: Two biomarker panels were developed based on 116 and 106 peptide biomarkers using support vector machine algorithms. Validation of the urine-based biomarker panels in independent validation sets, resulted in AUC values of 0.87 and 0.75 for detecting primary (n=270) and recurrent UBC (n=211), respectively. At the optimal threshold, the classifier for detecting primary UBC exhibited 91% sensitivity and 68% specificity, while the classifier for recurrence demonstrated 87% sensitivity and 51% specificity. Particularly for patients undergoing surveillance, improved performance was achieved when combining the urine-based panel with cytology (AUC of 0.87). Conclusions: The developed urine-based peptide biomarker panel for detecting primary UBC exhibits good performance. Combination of the urine-based panel and cytology resulted in improved performance for detecting disease recurrence

Telomerase Reverse Transcriptase Promoter Mutations in Bladder Cancer: High Frequency Across Stages, Detection in Urine, and Lack of Association with Outcome

Background: Hotspot mutations in the promoter of the gene coding for telomerase reverse transcriptase (TERT) have been described and proposed to activate gene expression. Objectives: To investigate TERT mutation frequency, spectrum, association with expression and clinical outcome, and potential for detection of recurrences in urine in patients with urothelial bladder cancer (UBC). Design, setting, and participants: A set of 111 UBCs of different stages was used to assess TERT promoter mutations by Sanger sequencing and TERT messenger RNA (mRNA) expression by reverse transcription-quantitative polymerase chain reaction. The two most frequent mutations were investigated, using a SNaPshot assay, in an independent set of 184 non-muscle-invasive and 173 muscle-invasive UBC (median follow-up: 53 mo and 21 mo, respectively). Voided urine from patients with suspicion of incident UBC (n = 174), or under surveillance after diagnosis of non-muscle-invasive UBC (n = 194), was tested using a SNaPshot assay. Outcome measurements and statistical analysis: Association of mutation status with age, sex, tobacco, stage, grade, fibroblast growth factor receptor 3 (FGFR3) mutation, progression-free survival, disease-specific survival, and overall survival. Results and limitations: In the two series, 78 of 111 (70%) and 283 of 357 (79%) tumors harbored TERT mutations, C228T being the most frequent substitution (83% for both series). TERT mutations were not associated with clinical or pathologic parameters, but were more frequent among FGFR3 mutant tumors (p = 0.0002). There was no association between TERT mutations and mRNA expression (p = 0.3). Mutations were not associated with clinical outcome. In urine, TERT mutations had 90% specificity in subjects with hematuria but no bladder tumor, and 73% in recurrence-free UBC patients. The sensitivity was 62% in incident and 42% in recurrent UBC. A limitation of the study is its retrospective nature. Conclusions: Somatic TERT promoter mutations are an early, highly prevalent genetic event in UBC and are not associated with TERT mRNA levels or disease outcomes. A SNaPshot assay in urine may help to detect UBC recurrences. (C) 2013 European Association of Urology. Published by Elsevier B. V. All rights reserved