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Supplementary Table S6. Multivariate analysis for investigating the comparative performance of the biomarker classifier and cytology for detecting recurrence. Of the 211 subjects that were included in the validation phase, voided urinary cytology (VUC) results were available for 96 urinary samples.

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. Clin Cancer Res; 22(16); 4077–86. ©2016 AACR.

Major interest lies in the evaluation of immune infiltrate in bladder cancer. CD8+ cytotoxic lymphocytes are key effectors of adaptive immune response.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.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.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+ /mmDifferences 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.

Fibroblast growth factor receptor 3 (FGFR3) is an actionable target in bladder cancer (BC). FGFR3 mutations are common in noninvasive BC and associated with favorable BC prognosis. Overexpression was reported in up to 40% of FGFR3 wild-type muscle-invasive BC. We analyzed FGFR3 mutations, FGFR3, and p53 protein expression and assessed their prognostic value in a cohort of 1000 chemotherapy-naive radical cystectomy specimens. FGFR3 mutations were found in 11%, FGFR3 overexpression was found in 28%, and p53 overexpression was found in 69% of tumors. Among FGFR3 mutant tumors, 73% had FGFR3 overexpression versus 22% among FGFR3 wild-type tumors. FGFR3 mutations were significantly associated with lower pT stage, tumor grade, absence of carcinoma in situ, pN0, low-level p53, and longer disease-specific survival (DSS). FGFR3 overexpression was associated only with lower pT stage and tumor grade. Moreover, FGFR3 overexpression was not associated with DSS in patients with FGFR3 wild-type tumors. In conclusion, FGFR3 mutations identified patients with favorable BC at cystectomy. Our results suggest that FGFR3 mutations have a driver role and are functionally distinct from FGFR3 overexpression. Hence, patients with FGFR3 mutations would be more likely to benefit from anti-FGFR3 therapy. Ideally, further research is needed to test this hypothesis. Patient summary: Oncogenic fibroblast growth factor receptor 3 (FGFR3) mutations are very common in bladder cancer. In this report, we found that these FGFR3 mutations were associated with favorable features and prognosis of bladder cancer compared with patients with FGFR3 overexpressed tumors only. As a consequence, patients with FGFR3 mutant tumors would be more likely to benefit from anti-FGFR3 therapy than patients with FGFR3 protein overexpression only.

Background and aims: The overall evidence on the association between gallbladder conditions (GBC: gallstones and cholecystectomy) and pancreatic cancer (PC) is inconsistent. To our knowledge, no previous investigations considered the role of tumour characteristics on this association. Thus, we aimed to assess the association between self-reported GBC and PC risk, by focussing on timing to PC diagnosis and tumour features (stage, location, and resection). Methods: Data derived from a European case-control study conducted between 2009 and 2014 including 1431 PC cases and 1090 controls. We used unconditional logistic regression models to estimate odds ratios (ORs) and corresponding 95% confidence intervals (CIs) adjusted for recognized confounders. Results: Overall, 298 (20.8%) cases and 127 (11.6%) controls reported to have had GBC, corresponding to an OR of 1.70 (95% CI 1.33-2.16). The ORs were 4.84 (95% CI 2.96-7.89) for GBC diagnosed = 3 years. The risk was slightly higher for stage I/II (OR = 1.71, 95% CI 1.15-2.55) vs. stage III/IV tumours (OR = 1.23, 95% CI 0.87-1.76); for tumours sited in the head of the pancreas (OR = 1.59, 95% CI 1.13-2.24) vs. tumours located at the body/tail (OR = 1.02, 95% CI 0.62-1.68); and for tumours surgically resected (OR = 1.69, 95% CI 1.14-2.51) vs. non-resected tumours (OR = 1.25, 95% CI 0.88-1.78). The corresponding ORs for GBC diagnosed >= 3 years prior PC were close to unity. Conclusion: Our study supports the association between GBC and PC. Given the time-risk pattern observed, however, this relationship may be non-causal and, partly or largely, due to diagnostic attention and/or reverse causation.

The work was partially supported by Fondo de Investigaciones Sanitarias (FIS), Instituto de Salud Carlos III, Spain (PI061614, PI11/01542, PI0902102, PI12/01635, PI12/00815, PI15/01573, PI18/01347); Red Temática de Investigación Cooperativa en Cáncer, Spain (RD12/0036/0034, RD12/0036/ 0050, RD12/0036/0073); Spanish Ministerio de Ciencia, Innovación y Universidades (BFU2017-85926-P), López de Maturana, E., Rodríguez, J.A., Alonso, L., Lao, O., Molina-Montes, E., Martín-Antoniano, I.A., Gómez-Rubio, P., Lawlor, R., Carrato, A., Hidalgo, M., Iglesias, M., Molero, X., Löhr, M., Michalski, C., Perea, J., O’Rorke, M., Barberà, V.M., Tardón, A., Farré, A., Muñoz-Bellvís, L., Crnogorac-Jurcevic, T., Domínguez-Muñoz, E., Gress, T., Greenhalf, W., Sharp, L., Arnes, L., Cecchini, L., Balsells, J., Costello, E., Ilzarbe, L., Kleeff, J., Kong, B., Márquez, M., Mora, J., O’Driscoll, D., Scarpa, A., Ye, W., Yu, J., García-Closas, M., Kogevinas, M., Rothman, N., Silverman, D.T., Albanes, D., Arslan, A.A., Beane-Freeman, L., Bracci, P.M., Brennan, P., Bueno-de-Mesquita, B., Buring, J., Canzian, F., Du, M., Gallinger, S., Gaziano, J.M., Goodman, P.J., Gunter, M., LeMarchand, L., Li, D., Neale, R.E., Peters, U., Petersen, G.M., Risch, H.A., Sánchez, M.J., Shu, X.-O., Thornquist, M.D., Visvanathan, K., Zheng, W., Chanock, S.J., Easton, D., Wolpin, B.M., Stolzenberg-Solomon, R.Z., Klein, A.P., Amundadottir, L.T., Marti-Renom, M.A., Real, F.X., Malats, N., PanGenEU Investigators, SBC/EPICURO Investigators

The 5-year survival rate of patients with pancreatic ductal adenocarcinoma (PDAC) is around 5% due to the fact that the majority of patients present with advanced disease that is treatment resistant. Familial pancreatic cancer (FPC) is a rare disorder that is defined as a family with at least two affected first degree relatives, with an estimated incidence of 4%-10%. The genetic basis is unknown in the majority of families although around 10%-13% of families carry germline mutations in known genes associated with hereditary cancer and pancreatitis syndromes. Panel sequencing was performed of 35 genes associated with hereditary cancer in 43 PDAC cases from families with an apparent hereditary pancreatic cancer syndrome. Pathogenic variants were identified in 19% (5/26) of PDAC cases from pure FPC families in the genes MLH1, CDKN2A, POLQ and FANCM. Low frequency potentially pathogenic VUS were also identified in 35% (9/26) of PDAC cases from FPC families in the genes FANCC, MLH1, PMS2, CFTR, APC and MUTYH. Furthermore, an important proportion of PDAC cases harboured more than one pathogenic, likely pathogenic or potentially pathogenic VUS, highlighting the multigene phenotype of FPC. The genetic basis of familial or hereditary pancreatic cancer can be explained in 21% of families by previously described hereditary cancer genes. Low frequency variants in other DNA repair genes are also present in 35% of families which may contribute to the risk of pancreatic cancer development. This study was funded by the Instituto de Salud Carlos III (Plan Estatal de I + D + i 2013-2016): ISCIII (PI09/02221, PI12/01635, PI15/02101 and PI18/1034) and co-financed by the European Development Regional Fund ''A way to achieve Europe'' (ERDF), the Biomedical Research Network in Cancer: CIBERONC (CB16/12/00446), Red Temática de investigación cooperativa en cáncer: RTICC (RD12/0036/0073) and La Asociación Española contra el Cáncer: AECC (Grupos Coordinados Estables 2016). This study was funded by the Instituto de Salud Carlos III (Plan Estatal de I+D+i 2013-2016): ISCIII (PI09/02221, PI12/01635, PI15/02101 and PI18/1034) and co-financed by the European Development Regional Fund "A way to achieve Europe'' (ERDF), the Biomedical Research Network in Cancer: CIBERONC (CB16/12/00446), Red Tematica de investigacion cooperativa en cancer: RTICC (RD12/0036/0073) and La Asociacion Espanola contra el Cancer: AECC (Grupos Coordinados Estables 2016). Sí

Previous studies suggested an association between atopic conditions and specific cancers. The results on the association with urothelial bladder cancer (UBC) are scarce and inconsistent. To evaluate the association between asthma and risk of UBC, we considered 936 cases and 1022 controls from the Spanish Bladder Cancer/EPICURO Study (86% males, mean age 65.4 years), a multicenter and hospital based case-control study conducted during 1998-2001. Participants were asked whether they had asthma and detailed information about occupational exposures, smoking habits, dietary factors, medical conditions, and history of medication was collected through face-to-face questionnaires performed by trained interviewers. Since asthma and UBC might share risk factors, association between patients' characteristics and asthma was studied in UBC controls. Association between UBC and asthma was assessed using logistic regression unadjusted and adjusted for potential confounders. The complex interrelationships, direct, and mediating effect of asthma on UBC, were appraised using counterfactual mediation models. Asthma was associated with a reduced risk of UBC (odds ratio (OR)=0.54, 95% confidence interval (CI) 0.37, 0.79) after adjusting for a wide range of confounders. No mediating effect was identified. The reduced risk associated with asthma was restricted to patients with high-risk non-muscle invasive (OR=0.25, 95%CI 0.10, 0.62) and muscle invasive UBC (OR=0.32, 95%CI 0.15, 0.69). Our results support that asthma is associated with a decreased risk of UBC, especially among aggressive tumors. Further work on the relationship between asthma and other atopic conditions and cancer risk should shed light on the relationship between immune response mechanisms and bladder carcinogenesis. This article is protected by copyright. All rights reserved.

Deciphering the underlying genetic basis behind pancreatic cancer (PC) and its associated multimorbidities will enhance our knowledge toward PC control. The study investigated the common genetic background of PC and different morbidities through a computational approach and further evaluated the less explored association between PC and autoimmune diseases (AIDs) through an epidemiological analysis. Gene‐disease associations (GDAs) of 26 morbidities of interest and PC were obtained using the DisGeNET public discovery platform. The association between AIDs and PC pointed by the computational analysis was confirmed through multivariable logistic regression models in the PanGen European case–control study population of 1,705 PC cases and 1,084 controls. Fifteen morbidities shared at least one gene with PC in the DisGeNET database. Based on common genes, several AIDs were genetically associated with PC pointing to a potential link between them. An epidemiologic analysis confirmed that having any of the nine AIDs studied was significantly associated with a reduced risk of PC (Odds Ratio (OR) = 0.74, 95% confidence interval (CI) 0.58–0.93) which decreased in subjects having ≥2 AIDs (OR = 0.39, 95%CI 0.21–0.73). In independent analyses, polymyalgia rheumatica, and rheumatoid arthritis were significantly associated with low PC risk (OR = 0.40, 95%CI 0.19–0.89, and OR = 0.73, 95%CI 0.53–1.00, respectively). Several inflammatory‐related morbidities shared a common genetic component with PC based on public databases. These molecular links could shed light into the molecular mechanisms underlying PC development and simultaneously generate novel hypotheses. In our study, we report sound findings pointing to an association between AIDs and a reduced risk of PC. Grant sponsor: Acción Especial de Genómica, Spain; Grant numbers: #GEN2001-4748-c05-03; Grant sponsor: Swedish ALF; Grant numbers: #SLL20130022; Grant sponsor: Cancer Focus Northern Ireland and Department for Employment and Learning; Grant sponsor: EU H2020 Programme 2014-2020; Grant numbers: 634143 MedBioinformatics676559 Elixir-Excelerate; Grant sponsor: EU-6FP Integrated Project; Grant numbers: #018771-MOLDIAG-PACA; Grant sponsor: EU-FP7-HEALTH; Grant numbers: #256974-EPC-TMNet#259737-ANCERALIA#602783- Cam-Pac; Grant sponsor: Italian Foundation for Cancer Research (FIRC); Grant sponsor: Italian Ministry of Health; Grant numbers: FIMPCUP_J33G13000210001; Grant sponsor: Red Temática de Investigación Cooperativa en Cáncer, Spain; Grant numbers: #RD12/0036/0050#RD12/0036/ 0073(#RD12/0036/0034; Grant sponsor: The work was partially supported by Fondo de Investigaciones Sanitarias (FIS), Instituto de Salud Carlos III-FEDER, Spain; Grant numbers: #PI0902102#PI11/01542#PI12/00815#PI12/01635#PI13/ 00082CP10/00524PI15/01573; Grant sponsor: World Cancer Research Fund; Grant numbers: WCR #15-0391.

Background: Family history (FH) of pancreatic cancer (PC) has been associated with an increased risk of PC, but little is known regarding the role of inherited/environmental factors or that of FH of other comorbidities in PC risk. We aimed to address these issues using multiple methodological approaches. Methods: Case-control study including 1431 PC cases and 1090 controls and a reconstructed-cohort study (N = 16 747) made up of their first-degree relatives (FDR). Logistic regression was used to evaluate PC risk associated with FH of cancer, diabetes, allergies, asthma, cystic fibrosis and chronic pancreatitis by relative type and number of affected relatives, by smoking status and other potential effect modifiers, and by tumour stage and location. Familial aggregation of cancer was assessed within the cohort using Cox proportional hazard regression. Results: FH of PC was associated with an increased PC risk [odds ratio (OR) = 2.68; 95% confidence interval (CI): 2.27-4.06] when compared with cancer-free FH, the risk being greater when ≥ 2 FDRs suffered PC (OR = 3.88; 95% CI: 2.96-9.73) and among current smokers (OR = 3.16; 95% CI: 2.56-5.78, interaction FHPC*smoking P-value = 0.04). PC cumulative risk by age 75 was 2.2% among FDRs of cases and 0.7% in those of controls [hazard ratio (HR) = 2.42; 95% CI: 2.16-2.71]. PC risk was significantly associated with FH of cancer (OR = 1.30; 95% CI: 1.13-1.54) and diabetes (OR = 1.24; 95% CI: 1.01-1.52), but not with FH of other diseases. Conclusions: The concordant findings using both approaches strengthen the notion that FH of cancer, PC or diabetes confers a higher PC risk. Smoking notably increases PC risk associated with FH of PC. Further evaluation of these associations should be undertaken to guide PC prevention strategies. The work was partially supported by: Fondo de Investigaciones Sanitarias (FIS), Instituto de Salud Carlos III, Spain (#PI11/01542, #PI0902102, #PI12/01635, #PI12/00815, #PI15/01573); Red Temática de Investigación Cooperativa en Cáncer, Spain (#RD12/0036/0034, #RD12/0036/0050, #RD12/0036/0073); WCR (15-0391); European Cooperation in Science and Technology – COST Action #BM1204: EUPancreas EU-6FP Integrated Project (#018771-MOLDIAG-PACA), EU-FP7-HEALTH (#259737-CANCERALIA, #256974-EPC-TM-Net); Associazione Italiana Ricerca sul Cancro (12182); Cancer Focus Northern Ireland and Department for Employment and Learning; and ALF (#SLL20130022), Sweden.

Objectives: 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.

BACKGROUND: Progression of non-muscle-invasive bladder cancer (NMIBC) to muscle-invasive bladder cancer (MIBC) is life-threatening and cannot be accurately predicted using clinical and pathological risk factors. Biomarkers for stratifying patients to treatment and surveillance are greatly needed.OBJECTIVE: To validate a previously developed 12-gene progression score to predict progression to MIBC in a large, multicentre, prospective study.DESIGN, SETTING, AND PARTICIPANTS: We enrolled 1224 patients in ten European centres between 2008 and 2012. A total of 750 patients (851 tumours) fulfilled the inclusion and sample quality criteria for testing. Patients were followed for an average of 28 mo (range 0-76). A 12-gene real-time qualitative polymerase chain reaction assay was performed for all tumours and progression scores were calculated using a predefined formula and cut-off values.OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We measured progression to MIBC using Cox regression analysis and log-rank tests for comparing survival distributions.RESULTS AND LIMITATIONS: The progression score was significantly (pCONCLUSIONS: The 12-gene progression score had independent prognostic power beyond clinical and histopathological risk factors, and may help in stratifying NMIBC patients to optimise treatment and follow-up regimens.PATIENT SUMMARY: Clinical use of a 12-gene molecular test for disease aggressiveness may help in stratifying patients with non-muscle-invasive bladder cancer to optimal treatment regimens.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is usually diagnosed in late adulthood; therefore, many patients suffer or have suffered from other diseases. Identifying disease patterns associated with PDAC risk may enable a better characterization of high-risk patients. METHODS: Multimorbidity patterns (MPs) were assessed from 17 self-reported conditions using hierarchical clustering, principal component, and factor analyses in 1705 PDAC cases and 1084 controls from a European population. Their association with PDAC was evaluated using adjusted logistic regression models. Time since diagnosis of morbidities to PDAC diagnosis/recruitment was stratified into recent (

PURPOSE: Only 3% to 28% of patients referred to the urology clinic for hematuria are diagnosed with bladder cancer. Cystoscopy leads to high diagnostic costs and a high patient burden. Therefore, to improve the selection of patients for cystoscopy and reduce costs and over testing we aimed to validate a recently developed diagnostic urine assay.MATERIALS AND METHODS: Included in study were 200 patients from a total of 3 European countries who underwent cystoscopy for hematuria, including 97 with bladder cancer and 103 with nonmalignant findings. Voided urine samples were collected prior to cystoscopy. DNA was extracted and analyzed for mutations in FGFR3, TERT and HRAS, and methylation of OTX1, ONECUT2 and TWIST1. Logistic regression was used to analyze the association between predictor variables and bladder cancer.RESULTS: Combining the methylation and mutation markers with age led to an AUC of 0.96 (95% CI 0.92-0.99) with 93% sensitivity and 86% specificity, and an optimism corrected AUC of 0.95. The AUC was higher for T1 or greater tumors compared to Ta tumors (0.99 vs 0.93). The AUC was also higher for high grade tumors compared to low grade tumors (1.00 vs 0.93). Overall negative predictive value was 99% based on the 5% to 10% prevalence of bladder cancer in patients with hematuria. This would lead to a 77% reduction in diagnostic cystoscopy.CONCLUSIONS: Analyzing hematuria patients for the risk of bladder cancer using novel molecular markers may lead to a reduction in diagnostic cystoscopy. Combining methylation analysis (OTX1, ONECUT2 and TWIST1) with mutation analysis (FGFR3, TERT and HRAS) and patient age resulted in a validated accurate prediction model.

Objective Studies indicate an inverse association between ductal adenocarcinoma of the pancreas (PDAC) and nasal allergies. However, controversial findings are reported for the association with asthma. Understanding PDAC risk factors will help us to implement appropriate strategies to prevent, treat and diagnose this cancer. This study assessed and characterised the association between PDAC and asthma and corroborated existing reports regarding the association between allergies and PDAC risk. Design Information about asthma and allergies was collated from 1297 PDAC cases and 1024 controls included in the PanGenEU case-control study. Associations between PDAC and atopic diseases were studied using multilevel logistic regression analysis. Meta-analyses of association studies on these diseases and PDAC risk were performed applying random-effects model. Results Asthma was associated with lower risk of PDAC (OR 0.64, 95% CI 0.47 to 0.88), particularly long-standing asthma (>=17 years, OR 0.39, 95% CI 0.24 to 0.65). Meta-analysis of 10 case-control studies sustained our results (metaOR 0.73, 95% CI 0.59 to 0.89). Nasal allergies and related symptoms were associated with lower risk of PDAC (OR 0.66, 95% CI 0.52 to 0.83 and OR 0.59, 95% CI 0.46 to 0.77, respectively). These results were supported by a meta-analysis of nasal allergy studies (metaOR 0.6, 95% CI 0.5 to 0.72). Skin allergies were not associated with PDAC risk. Conclusions This study shows a consistent inverse association between PDAC and asthma and nasal allergies, supporting the notion that atopic diseases are associated with reduced cancer risk. These results point to the involvement of immune and/or inflammatory factors that may either foster or restrain pancreas carcinogenesis warranting further research to understand the molecular mechanisms driving this association.

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. Hotspot mutations in the promoter of the gene coding for telomerase reverse transcriptase (TERT) have been described and proposed to activate gene expression.

Francisco Real and colleagues report exome sequencing in urothelial bladder tumors. They show that STAG2, a subunit of the cohesin complex, is recurrently mutated and provide evidence that STAG2 loss does not lead to increases in aneuploidy. Urothelial bladder cancer (UBC) is heterogeneous at the clinical, pathological and genetic levels. Tumor invasiveness (T) and grade (G) are the main factors associated with outcome and determine patient management1. A discovery exome sequencing screen (n = 17), followed by a prevalence screen (n = 60), identified new genes mutated in this tumor coding for proteins involved in chromatin modification (MLL2, ASXL2 and BPTF), cell division (STAG2, SMC1A and SMC1B) and DNA repair (ATM, ERCC2 and FANCA). STAG2, a subunit of cohesin, was significantly and commonly mutated or lost in UBC, mainly in tumors of low stage or grade, and its loss was associated with improved outcome. Loss of expression was often observed in chromosomally stable tumors, and STAG2 knockdown in bladder cancer cells did not increase aneuploidy. STAG2 reintroduction in non-expressing cells led to reduced colony formation. Our findings indicate that STAG2 is a new UBC tumor suppressor acting through mechanisms that are different from its role in preventing aneuploidy.

Background: Increasing evidence points to the role of tumor immunologic environment on urothelial bladder cancer prognosis. This effect might be partly dependent on the host genetic context. We evaluated the association of SNPs in inflammation-related genes with non–muscle-invasive bladder cancer (NMIBC) risk-of-recurrence and risk-of-progression. Methods: We considered 822 NMIBC included in the SBC/EPICURO Study followed-up >10 years. We selected 1,679 SNPs belonging to 251 inflammatory genes. The association of SNPs with risk-of-recurrence and risk-of-progression was assessed using Cox regression single-marker (SMM) and multimarker methods (MMM) Bayes A and Bayesian LASSO. Discriminative abilities of the models were calculated using the c index and validated with bootstrap cross-validation procedures. Results: While no SNP was found to be associated with risk-of-recurrence using SMM, three SNPs in TNIP1, CD5, and JAK3 showed very strong association with posterior probabilities >90% using MMM. Regarding risk-of-progression, one SNP in CD3G was significantly associated using SMM (HR, 2.69; P = 1.55 × 10−5) and two SNPs in MASP1 and AIRE, showed a posterior probability ≥80% with MMM. Validated discriminative abilities of the models without and with the SNPs were 58.4% versus 60.5% and 72.1% versus 72.8% for risk-of-recurrence and risk-of-progression, respectively. Conclusions: Using innovative analytic approaches, we demonstrated that SNPs in inflammatory-related genes were associated with NMIBC prognosis and that they improve the discriminative ability of prognostic clinical models for NMIBC. Impact: This study provides proof of concept for the joint effect of genetic variants in improving the discriminative ability of clinical prognostic models. The approach may be extended to other diseases. Cancer Epidemiol Biomarkers Prev; 25(7); 1144–50. ©2016 AACR.

Non-muscle-invasive bladder cancer (NMIBC) is a heterogeneous disease with widely different outcomes. We performed a comprehensive transcriptional analysis of 460 early-stage urothelial carcinomas and showed that NMIBC can be subgrouped into three major classes with basal- and luminal-like characteristics and different clinical outcomes. Large differences in biological processes such as the cell cycle, epithelial-mesenchymal transition, and differentiation were observed. Analysis of transcript variants revealed frequent mutations in genes encoding proteins involved in chromatin organization and cytoskeletal functions. Furthermore, mutations in well-known cancer driver genes (e.g., TP53 and ERBB2) were primarily found in high-risk tumors, together with APOBEC-related mutational signatures. The identification of subclasses in NMIBC may offer better prognostication and treatment selection based on subclass assignment.

Purpose To describe the organisation of the registry and the preliminary results in terms of characteristics of high-risk pancreatic ductal adenocarcinoma (PDAC) families recruited to date and findings of the screening programme. To compare early onset sporadic cases (⩽50 years), sporadic cases (>50 years) and cases with family history of cancer, for PDAC possible risk factors. Methods/patients Families with hereditary cancer syndromes predisposing to PDAC were recruited from two main sources: Spanish hospitals participating in PanGenEU, a pan-European multicentre case–control study, and their genetic counseling unit. Individuals at high-risk of PDAC were enrolled into a screening programme, consisting of Endoscopic ultrasound, computerised tomography, magnetic resonance imaging. Genetic testing of candidate genes was offered according to each patient’s risk. Results Among 577 consecutive PDAC cases, recruited via PanGenEU, 36 (6%) had ⩾2 first-degree relative with PDAC: Familial pancreatic cancer (FPC). So far PanGen-Fam has recruited 42 high-risk PDAC families; 25 (60%) had FPC. Five index cases with cancer were positive for BRCA2 and one for BRCA1 germline mutations. In the second year of prospective PDAC screening, one neuroendocrine tumour and a high-grade dysplasia lesion suspicious of carcinoma were diagnosed among 41 high-risk individuals. Furthermore EUS detected chronic-pancreatitis-like parenchymal changes in 15 patients. Concluding statement The identification and recruitment of PDAC high-risk families into the PanGen-Fam registry provides an opportunity to detect early onset cancer and precursor pancreatic cancer lesions at a potentially curative stage and to increase the knowledge of the natural history of the disease.

Introduction: Germline variants in TP63 have been consistently associated with several tumors, including bladder cancer, indicating the importance of TP53 pathway in cancer genetic susceptibility. However, variants in other related genes, including TP53 rs1042522 (Arg72Pro), still present controversial results. We carried out an in depth assessment of associations between common germline variants in the TP53 pathway and bladder cancer risk. Material and Methods: We investigated 184 tagSNPs from 18 genes in 1,058 cases and 1,138 controls from the Spanish Bladder Cancer/EPICURO Study. Cases were newly-diagnosed bladder cancer patients during 1998–2001. Hospital controls were age-gender, and area matched to cases. SNPs were genotyped in blood DNA using Illumina Golden Gate and TaqMan assays. Cases were subphenotyped according to stage/grade and tumor p53 expression. We applied classical tests to assess individual SNP associations and the Least Absolute Shrinkage and Selection Operator (LASSO)-penalized logistic regression analysis to assess multiple SNPs simultaneously. Results: Based on classical analyses, SNPs in BAK1 (1), IGF1R (5), P53AIP1 (1), PMAIP1 (2), SERINPB5 (3), TP63 (3), and TP73 (1) showed significant associations at p-value#0.05. However, no evidence of association, either with overall risk or with specific disease subtypes, was observed after correction for multiple testing (p-value$0.8). LASSO selected the SNP rs6567355 in SERPINB5 with 83% of reproducibility. This SNP provided an OR = 1.21, 95%CI 1.05–1.38, p-value = 0.006, and a corrected p-value = 0.5 when controlling for over-estimation. Discussion: We found no strong evidence that common variants in the TP53 pathway are associated with bladder cancer susceptibility. Our study suggests that it is unlikely that TP53 Arg72Pro is implicated in the UCB in white Europeans. SERPINB5 and TP63 variation deserve further exploration in extended studies., This work was supported by the Fondo de Investigacion Sanitaria, Spain (grant numbers 00/0745, PI051436, PI061614, G03/174); Red Tematica de Investigacion Cooperativa en Cancer (grant number RD06/0020-RTICC), Spain; Marato TV3 (grant number 050830); European Commission (grant numbers EU-FP7-HEALTH-F2-2008-201663-UROMOL; US National Institutes of Health (grant number USA-NIH-RO1-CA089715); and the Intramural Research Program of the Division of Cancer Epidemiology and Genetics, National Cancer Institute at the National Institutes of Health, USA; Consolider ONCOBIO (Ministerio de Economia y Competitividad, Madrid, Spain). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The incidence and rate of recurrence of bladder cancer is high, particularly in developed countries, however current methods for diagnosis are limited to detecting high-grade tumours using often invasive methods. A panel of biomarkers to characterise tumours of different grades that could also distinguish between patients exhibiting the disease with first incidence or recurrence could be useful for bladder cancer diagnostics. In this study, potential metabolic biomarkers have been discovered through mass spectrometry based metabolomics of urine. Pre-treatment urine samples were collected from 48 patients diagnosed of urothelial bladder cancer. Patients were followed-up through the hospital pathological charts to identify whether and when the disease recurred or progressed. Subsequently, they were classified according to whether or not they suffered a tumour recurrence (recurrent or stable) as well as their risk group according to tumour grade and stage. Identified metabolites have been analysed in terms of disease characteristics (tumour stage and recurrence) and have provided an insight into bladder cancer progression. Using both liquid chromatography and capillary electrophoresis-mass spectrometry, a total of 27 metabolite features were highlighted as significantly different between patient groups. Some, for example histidine, phenylalanine, tyrosine and tryptophan have been previously linked with bladder cancer, however until now their connection with bladder cancer progression has not been previously reported. The candidate biomarkers revealed in this study could be useful in the clinic for diagnosis of bladder cancer and, through characterising the stage of the disease, could also be useful in prognostics.

242 Background: The prognosis of patients diagnosed with Pancreatic Cancer (PC) is dismal with a 5-year survival rate of around 5%. Familial Pancreatic Cancer (FPC) is an autosomal dominant rare syndrome defined as families with two or more first-degree relatives with pancreatic cancer that do not fulfill the criteria of any other inherited tumor syndrome. The Spanish familial pancreatic cancer registry, Pan-Gen-FAM was established in 2009 in order to identify and manage families at high risk of developing PC. Methods: Information on the family history of cancer is collected in order to determine the phenotype of individual families and patients are offered genetic testing of known FPC associated genes pertinent to their familial syndrome. An individualized clinical screening program is devised for the early detection of a pancreatic tumor consisting of periodic monitoring by imaging techniques (EUS and MRI) and the evaluation of minimally-invasive tumor biomarkers approaches including Circulating Tumor Cells (CTC) and circulating free DNA in blood. Results: To date the registry includes 125 individuals representing some 41 families. Of 17 families tested so far BRCA2 germline mutations were detected in 5 families and a BRCA1 mutation in 1 family. 43 individuals underwent clinical screening. More pancreatic abnormalities were found by EUS (41%) than by MRI (31%). The most frequent abnormal findings were parenchymal changes associated with chronic pancreatitis. Eight cystic lesions were identified. One lesion was identified as a well differentiated neuroendocrine tumor and another was shown to have benign cytology and whilst carcinoma was found in a third patient, this patient underwent a subsequent partial pancreatectomy. The remaining patients with cystic lesions are undergoing close clinical observation. Seventy three patients underwent CTC determinations and all patients tested negative. Conclusions: Periodic screening of high risk individuals by EUS and RMI can detect small tumors and premalignant lesions. We hypothesize that these persons will have a higher probability of long-term survival than sporadic cases.

1529 Background: The incidence of pancreatic cancer (PC) is low, thus screening is recommended for individuals considered to be at high risk. These include members of families with ≥ 2 relatives with PC (FPC), Peutz Jegher syndrome, hereditary breast/ovarian cancer (HBOC), familial atypical multiple mole melanoma and Lynch syndrome. Previous studies have shown that non-invasive precursor lesions, such as intraductal papillary mucinous neoplasm (IPMN) and pancreatic intraepithelial neoplasia (PanIN) are more common and of a higher invasive grade in patients with a family history of PC. Methods: Baseline screening consisted of endoscopic ultrasound (EUS) and computed tomography (CT) and magnetic resonance imaging (MRI). FNA-EUS was performed when mass lesions or cysts ≥1 cm in size were encountered. The possibility of surgical intervention or ongoing clinical observation was discussed by a multidisciplinary team in the case of abnormal findings. Furthermore Circulating Tumor Cells (CTC) in peripheral blood as a diagnostic marker was assessed. Thirty-five individuals from FPC and HBOC families were screened between October 2011and December 2012. Results: The most frequent abnormal findings by EUS were parenchymal changes found in chronic pancreatitis (hyperechoic foci, echogenic strands, lobularity, cysts). Overall 13 (41%) patients showed at least 1 of these changes; of note, 7 patients were from the same FPC family. Four patients were diagnosed with cystic lesions by EUS; two lesions were confirmed by MRI but not by CT, one lesion was confirmed by both MRI and CT and was compatible with a mucinous tumor < 1cm in diameter and one lesion was detected only by EUS. One patient with cystic lesion >1 cm in size underwent EUS-FNA and the cytology showed benign celularity. One patient has a solid lesion, and it was a neuroendocrine tumor. The remaining patients with cystic lesions were proposed for ongoing close clinical observation. CTC have not been detected in 54 patients tested. Conclusions: A high prevalence of pancreatic abnormalities was found in patients from FPC families. Preliminary data suggest that EUS is the most sensitive method to detect small tumors and premalignant lesions.

e12033 Background: Inherited predisposition to Pancreatic Cancer (PC) corresponds 10% of all cases and includes members of families affected with hereditary cancer syndromes as Familial Pancreatic Cancer (FPC), Peutz-Jeghers, familial melanoma, hereditary breast and ovarian cancer, hereditary pancreatitis. An inherited predisposition in early onset PC (≤ 50 years) has also been suggested. We report preliminary data on PanFAM patients and screening of high risk individuals. Methods: PamFAM is a part of the European PANGEN PC case/control study of hereditary PC, co-ordinated by the Ramón y Cajal (RC) hospital and the Spanish National Cancer Research Center, with 16 participating hospitals. All families with clinical evidence of an inherited PC syndrome were recruited and multi-generational pedigrees were constructed. Cancer diagnoses were confirmed, when possible, by review of medical records. Blood samples and epidemiological data were collected for all participating family members. A screening program for early detection of PC, based on endoscopic ultrasound (EUS), CT and circulating tumour cells (CTCs) was offered to high risk individuals. Results: Of 505 Spanish PCs collected by PANGEN, 31 (~6%) were FPC cases; 18 (58%) revealed only PC and the remaining showed clustering with other tumor types, gastric cancer was the most common (13%). Among FPC families, 3 had 3 cases of PC and the remaining had 2 cases. The mean age of diagnosis was 67 years (range 47-85), 20 male and 11 female. Four FPCs were previously diagnosed with cancer (Hodgkin lymphoma, breast and prostate cancer) and 3 with acute pancreatitis. 37 PCs with no family history of cancer were diagnosed at the age of 50 years or earlier (mean 45, range 30-50), 18 male and 19 female. Other 27 eligible families were recruited by RyC hospital, 8 (30%) with FPC and 3 (11%) with PC ≤ 50 years. A cohort of 61 high risk individuals participes in the screening study: 3 had abnormal EUS, 1 a benign pancreatic node and 1 a renal angiolipoma; one young man had 2 CTCs. Conclusions: PanFAM is the first registry in Spain collecting hereditary PC cases and it represents an important resource to identify underlying gene defects and to the development of screening methods precursor lesions detection in high risk individuals.

192 Background: 5-10% of pancreatic cancer (PC) cases show familial aggregation. 20% of these correspond to syndromes: Peutz Jeghers, hereditary pancreatitis, familial multiple melanoma, breast and ovarian cancer (HBOC) and others. Familial pancreatic cancer (FPC) is defined as: ≥2 first degree relatives with PC and with no other known syndromes. FPC seems to have autosomal dominant inheritance, but the genetic cause is unknown. Methods: The objectives are: 1) To develop the first FPC Spanish Registry, connecting different groups interested in this disease 2) To study inheritance, phenotypic and molecular characteristics of the FPC, and families with early PC 3) To establish a strategy for early detection of PC in high-risk individuals and to implement it 4) To characterize preneoplastic lesions and diagnosed PC by monitoring high-risk individuals. 16 Spanish hospitals are participating. This study has two components: 1) Cohort to identify families with FPC and hereditary PC. Sources for the families are the PanGen-ES Study, a case-control study of PC which identifies families through a questionnaire on family history of cancer, and Genetic Counseling Units, 2) Cohort of high-risk families. The latter will be followed up by endoscopic ultrasound (EUS) and CT ± abdominal magnetic resonance imaging. In addition, circulating tumor cells (CTC) in peripheral blood will be determined. Results: The assessment of family history of the 421 cases included in the PanGen Study has identified 32 (7.6%) families with FPC and 52 patients with PC ≤ 50 years (12.4%). In addition, the 190 families presenting PC aggregation with other neoplasms are being further evaluated. At this time we have obtained clinical data and blood samples to carry out molecular studies of 23 individuals: 17 belonging to 3 families with FPC, and 6 members of 2 families with an HBOC with some cases of pancreatic cancer. 18 relatives at risk began a follow-up with EUS and CT, with no detection of any suspicious pancreatic lesion; also CTC have not been detected. Conclusions: This initiative will permit to know more about FPC and will serve to evaluate protocols and PC markers in screening the high-risk population, and promote connections with other international groups.