Establishment of diagnostic criteria for upper urinary tract urothelial carcinoma based on genome-wide DNA methylation analysis

ABSTRACT

The aim of this study was to develop a less invasive and accurate diagnostic system for upper urinary tract urothelial carcinoma (UTUC) based on genome-wide DNA methylation profiling. Genome-wide DNA methylation screening was performed using the Infinium HumanMethylation450 BeadChip, and DNA methylation quantification was verified using pyrosequencing. We analysed 26 samples of normal control urothelial tissue (C), an initial cohort of 62 samples (31 samples of non-cancerous urothelium [N] from UTUC patients and 31 samples of the corresponding UTUCs), a validation cohort of 82 samples (41 N and 41 UTUC samples), and 14 samples of urinary bladder urothelial carcinoma (BUC). In the initial cohort, we identified 2,448 CpG sites showing significant differences in DNA methylation levels between both C and UTUC and N and UTUC, but not showing differences between C and N. Among these CpG sites, 10 were located within CpG islands or their shores and shelves included in genomic domains where DNA methylation levels are stably controlled, allowing discrimination of UTUC even from BUC. Receiver operating characteristic curve analysis for discrimination of UTUC from N in these 10 CpG and neighbouring sites (37 diagnostic panels in total) yielded area under the curve values of 0.959–1.000, with a sensitivity and specificity of 86.6–100% and 93.5–100%, respectively. The diagnostic impact was successfully confirmed in the validation cohort. Our criteria were useful for diagnosis of UTUC, regardless of its clinicopathological features. Application of our criteria to voided urine samples will ultimately allow non-invasive DNA methylation diagnosis of UTUC.

KEYWORDS:

• Urothelial carcinoma
• upper urinary tract
• urinary bladder
• DNA methylation diagnostics
• genome-wide analysis

Abbreviations

AUC	=	Area under the curve
BUC	=	Urinary bladder urothelial carcinoma
C	=	Normal control urothelium
HPLC	=	High-performance liquid chromatography
N	=	Non-cancerous urothelium
PCA	=	Principal component analysis
ROC	=	Receiver operating characteristic
TCGA	=	The Cancer Genome Atlas
TSS	=	Transcription start site
UC	=	Urothelial carcinoma
UTUC	=	Upper urinary tract urothelial carcinoma

Author’s contributions

EA and YK conceived, designed, and supervised the study. TaY and YY developed the methodology. MF, EA, KT, AMM, HF, and TeY contributed to acquire the data. MF and YT contributed to analyze and interpret the data. MF, EA, and YK wrote, reviewed and/or revised the manuscript. All authors read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the author.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by the Japan Society for the Promotion of Science under Grant KAKENHI JP19K07444; the Japan Agency for Medical Research and Development under Grant JP19ae0101020.