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Here we conducted an evidence-based study in developing and validating a urinary biomarker combination of gene methylation assays in patients with hematuria. A number of 99 urine samples were obtained and detected from Chinese patients with hematuria. The Cancer Genome Atlas cohort with methylation (HM450) beta-values and clinical data of 412 bladder cancer and 21 matching normal tissue was included as a validation series. A risk score formula was then developed and calculated by the targeted genes, weighted by their estimated regression coefficients from the multivariable binary logistic regression analyses, and evaluated by receiver operating characteristic (ROC) curves analysis. The combination assay of HOXA9 , ONECUT2 , PCDH17 , PENK , TWIST1 , VIM and ZNF154 was singled out according to the results of multivariate logistic regression analysis. The higher probability of DNA methylation of all the selected 7 genes was found in bladder cancer group than the control group. Remarkable higher DNA methylation beta-values of all the selected 7 genes were also displayed in bladder cancer tissues compared with their matching normal bladder tissues. And the AUC value of our risk score model were 0.894 and 0.851 in respective cohort, revealing highlighted predictive value of our risk score model on bladder cancer diagnosis. In conclusions, a urinary combined methylation assay of HOXA9 , ONECUT2 , PCDH17 , PENK , TWIST1 , VIM and ZNF154 displayed accurate prediction of bladder cancer in hematuria patients, which provided the guidance for the patients at early stage tumor and during the follow-up after operation. Of course, prospective study based on a hematuria cohort with a large sample size should be conducted to validate these findings in the future.

Identification of Cancer-Specific Methylation of Gene Combination for the Diagnosis of Bladder Cancer