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Open Access Highly Accessed Technical advance

Diagnostic markers of urothelial cancer based on DNA methylation analysis

Yoshitomo Chihara12*, Yae Kanai3, Hiroyuki Fujimoto4, Kokichi Sugano5, Kiyotaka Kawashima6, Gangning Liang7, Peter A Jones7, Kiyohide Fujimoto1, Hiroki Kuniyasu2 and Yoshihiko Hirao1

Author Affiliations

1 Department of Molecular Pathology, Nara Medical University, 840, Shijyo-cho, Kashihara, Japan

2 Department of Urology, Nara Medical University, 840, Shijyo-cho, Kashihara, Japan

3 Division of Molecular Pathology, National Cancer Center Research Institute, 5-1-1, Tsukiji Chuo-ku, Tokyo, Japan

4 Department of Urology, National Cancer Center Hospital, 5-1-1, Tsukiji, Chuo-ku, Tokyo, Japan

5 Oncogene Research Unit/Cancer Prevention Unit, Tochigi Cancer Center Research Institute, 4-9-13, Yonan, Utsunomiya, Japan

6 Department of Urology, Tochigi Cancer Center Hospital, 4-9-13, Yonan, Utsunomiya, Japan

7 Department of Urology, Norris Comprehensive Cancer Center, University of Southern California, 1441 Eastlake Ave, Los Angeles, CA, 90033, USA

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BMC Cancer 2013, 13:275  doi:10.1186/1471-2407-13-275

Published: 4 June 2013

Abstract

Background

Early detection and risk assessment are crucial for treating urothelial cancer (UC), which is characterized by a high recurrence rate, and necessitates frequent and invasive monitoring. We aimed to establish diagnostic markers for UC based on DNA methylation.

Methods

In this multi-center study, three independent sample sets were prepared. First, DNA methylation levels at CpG loci were measured in the training sets (tumor samples from 91 UC patients, corresponding normal-appearing tissue from these patients, and 12 normal tissues from age-matched bladder cancer-free patients) using the Illumina Golden Gate methylation assay to identify differentially methylated loci. Next, these methylated loci were validated by quantitative DNA methylation by pyrosequencing, using another cohort of tissue samples (Tissue validation set). Lastly, methylation of these markers was analyzed in the independent urine samples (Urine validation set). ROC analysis was performed to evaluate the diagnostic accuracy of these 12 selected markers.

Results

Of the 1303 CpG sites, 158 were hyper ethylated and 356 were hypo ethylated in tumor tissues compared to normal tissues. In the panel analysis, 12 loci showed remarkable alterations between tumor and normal samples, with 94.3% sensitivity and 97.8% specificity. Similarly, corresponding normal tissue could be distinguished from normal tissues with 76.0% sensitivity and 100% specificity. Furthermore, the diagnostic accuracy for UC of these markers determined in urine samples was high, with 100% sensitivity and 100% specificity.

Conclusion

Based on these preliminary findings, diagnostic markers based on differential DNA methylation at specific loci can be useful for non-invasive and reliable detection of UC and epigenetic field defect.

Keywords:
Urothelial cancer; DNA methylation; Pyrosequencing; ROC; Piagnostic accuracy