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Background Accurate detection of pelvic lymph-node metastases in prostate cancer (PCa) is critical to individualize treatment strategies.We investigated the value of the hypermethylation of collapsin response mediator protein 4 (CRMP4) promoter in biopsies as a predictor for lymph-node metastases.Methods For the training cohort, two identified CpG sites on CRMP4 promoter(upstream of transcription starting site-848,-841 (UTSSs)) were used to quantitatively determined for methylation using pyrosequencing in 80 samples from matched cases with PCa including 40 lymph-node positive and 40 negative.The predictive cutoff value was accordingly established and sequentially validated in the internal testing group of 339 patients with PCa (from Southern China), and the external independent group of 328 cases (from Germany and across China).The sensitivity and specificity for lymph-node metastasis detection in PCa patients were evaluated by receiver operating characteristic analyses.We also investigated the biochemical recurrence-free and clinical progression-free survival in 142 PCa patients in which the identified region was herperrnethylated or not.Findings Analysis of the training group showed that hypermethylation on CRMP4 UTSSs was significantly associated with lymph-node metastases, in which 39 (97.5%) of 40 samples from negative lymph-node tumours were not hypermethylated, but 37 (92.5%) of 40 samples from positive lymph-node tumours were hypermethylated (≥ 15% methylated;p<0.001).Analysis of the internal testing set of patients demonstrated the cutoff value with the sensitivity of 92.3% (79.3-97.9) and specificity of 92.7% (80.2-99.1) to identify lymph-nodes.Successful validation was achieved in the external independent set of patients with the sensitivity of 92.2% (81.1-97.8) and specificity of 91.3% (87.4-94.4).UTSSs had positive predictive values of 75.0% (69.3-83.5) and 66.2% (54.0-77.0) in the internal and external group, respectively, and negative predictive values of 98.1% (82.4-99.6) and 98.4% (96.1-99.6) in the internal and external group, respectively.CRMP4 methylation alone as a continuous variable showed a larger area under the curve of compared with for Kattan nomogram variables (0.975 vs 0.705;p<0.001 in the internal testing set, and 0.971 vs 0.629;p<0.001 in the independent validation set).CRMP4 methylation is an independent predictor (odds ratio, 8.35;95%CI:5.64-12.35;p<0.001 in the internal testing;odds ratio, 12.46;95%CI: 5.82-26.70;p<0.0001 in the independent validation set) of lymph-node metastases in a multivariate analysis model.3-year clinical progression-free survival was 99.2% (94.2-100.0) for 119 patients with nonhypermethylated CRMP4 tumours and 95.5% (71.9-99.4) for 23 with hypermethylated tumours (p=0.0002) Interpretation In patients with PCa, the hypermethylation of UTSSs on CRMP4 promoter could efficaciously discriminate between patients with positive lymph-nodes and those negative.Furthermore, this region is easy to amplify, and the assay to establish hypermethylation can be performed on most tissues in most clinical laboratories.