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

Figures & data

Figure 1. DNA methylation profiling of tissue samples in the initial cohort. (a) Principal component analysis based on the results of the Infinium assay. The DNA methylation profile of non-cancerous urothelium (N, n = 31, blue) from patients with upper urinary tract urothelial carcinoma (UTUC) tended to differ from that of normal control urothelium (C, n = 26, black). Furthermore, the DNA methylation profile of the corresponding UTUC (n = 31, red) clearly differed from those of both C and N samples. (b) Unsupervised hierarchical clustering (Ward’s linkage using Euclidean distances). The distinct difference of the DNA methylation profile of UTUC (red) from that of C (black) or N (blue) samples is again confirmed. (c) Representative results of receiver operating characteristic curve analysis for candidate marker CpG sites identified using the procedures described in the Results section. The area under the curve values for Infinium probes cg01921432 and cg07418387 for discrimination of UTUC from C and those of the same probes for discrimination of UTUC from N are all 1

Table 1. DNA methylation levels obtained by Infinium assay for tissue samples of normal control urothelium (C), non-cancerous urothelium (N) from patients with upper urinary tract urothelial carcinoma (UTUC), and the corresponding UTUCs in the initial cohort and urinary bladder urothelial carcinoma (BUC)

		DNA methylation levels (mean ± SD)				UTUC vs C		UTUC vs N		N vs C	UTUC vs BUC
Probe ID^a	CpG type^b	C (n = 26)	N (n = 31)	UTUC (n = 31)	BUC (n = 14)	P	AUC	P	AUC	P	P
cg01227537	Island	0.116 ± 0.050	0.147 ± 0.080	0.424 ± 0.304	0.499 ± 0.198	4.51 × 10^−16	1.00	1.10 × 10^−16	1.00	1.00	2.75 × 10^−3
cg01921432	Island	0.151 ± 0.032	0.180 ± 0.053	0.455 ± 0.303	0.579 ± 0.161	1.57 × 10^−13	1.00	1.50 × 10^−13	1.00	1.00	7.63 × 10^−3
cg05492975	Island	0.859 ± 0.038	0.830 ± 0.044	0.538 ± 0.310	0.382 ± 0.137	1.27 × 10^−17	1.00	3.80 × 10^−17	1.00	1.00	5.37 × 10^−3
cg07197785	Island	0.230 ± 0.087	0.185 ± 0.083	0.479 ± 0.316	0.626 ± 0.183	2.85 × 10^−18	1.00	5.03 × 10^−20	1.00	1.00	1.51 × 10^−2
cg07418387	Shelf	0.779 ± 0.045	0.710 ± 0.072	0.440 ± 0.287	0.283 ± 0.127	4.43 × 10^−23	1.00	2.86 × 10^−23	1.00	1.00	7.91 × 10^−3
cg08070771	Shore	0.128 ± 0.041	0.189 ± 0.081	0.489 ± 0.314	0.487 ± 0.268	7.45 × 10^−23	1.00	1.55 × 10^−24	1.00	1.00	1.28 × 10^−3
cg08364561	Shore	0.092 ± 0.046	0.122 ± 0.075	0.444 ± 0.344	0.662 ± 0.143	8.12 × 10^−19	1.00	4.34 × 10^−20	1.00	1.00	3.33 × 10^−2
cg10216717	Shore	0.806 ± 0.034	0.782 ± 0.055	0.501 ± 0.303	0.338 ± 0.132	3.52 × 10^−16	1.00	9.28 × 10^−17	1.00	1.00	1.14 × 10^−2
cg10256242	Shore	0.864 ± 0.032	0.856 ± 0.046	0.558 ± 0.321	0.443 ± 0.150	7.27 × 10^−15	1.00	3.23 × 10^−15	1.00	1.00	9.87 × 10^−4
cg10874111	Shore	0.772 ± 0.046	0.803 ± 0.059	0.529 ± 0.296	0.415 ± 0.162	5.85 × 10^−15	1.00	2.10 × 10^−16	1.00	1.00	4.40 × 10^−3
cg14302471	Shore	0.783 ± 0.072	0.814 ± 0.075	0.501 ± 0.335	0.370 ± 0.210	2.06 × 10^−18	1.00	2.73 × 10^−19	1.00	1.00	9.18 × 10^−3
cg14851578	Shore	0.768 ± 0.055	0.781 ± 0.060	0.482 ± 0.319	0.335 ± 0.208	6.56 × 10^−18	1.00	2.21 × 10^−18	1.00	1.00	2.33 × 10^−2
cg15822765	Island	0.120 ± 0.038	0.185 ± 0.082	0.493 ± 0.330	0.692 ± 0.128	9.32 × 10^−19	1.00	2.81 × 10^−20	1.00	1.00	1.66 × 10^−2
cg16529477	Shore	0.128 ± 0.043	0.155 ± 0.056	0.428 ± 0.303	0.513 ± 0.195	3.85 × 10^−13	1.00	8.16 × 10^−13	1.00	1.00	5.42 × 10^−3
cg16705627	Island	0.198 ± 0.072	0.247 ± 0.104	0.519 ± 0.297	0.695 ± 0.112	2.63 × 10^−21	1.00	4.41 × 10^−20	1.00	1.00	1.79 × 10^−2
cg18077971	Shore	0.137 ± 0.060	0.165 ± 0.079	0.475 ± 0.335	0.583 ± 0.192	2.46 × 10^−18	1.00	1.62 × 10^−18	1.00	1.00	2.23 × 10^−3
cg24035245	Island	0.186 ± 0.070	0.225 ± 0.094	0.500 ± 0.296	0.653 ± 0.161	1.01 × 10^−22	1.00	5.05 × 10^−22	1.00	1.00	2.12 × 10^−2
cg26132774	Island	0.085 ± 0.040	0.104 ± 0.046	0.428 ± 0.348	0.646 ± 0.141	9.09 × 10^−16	1.00	1.94 × 10^−15	1.00	1.00	3.80 × 10^−2

AUC: area under the curve for receiver operating characteristic curve analysis. ^aProbe IDs for the Infinium HumanMethylation450 BeadChip (Illumina). ^bCpG islands, island shores (2000-bp region adjacent to a CpG island) and island shelves (2000-bp region adjacent to island shores) are identified based on the University of California, Santa Cruz (UCSC) genome browser (https://genome.ucsc.edu/).

Table 2. Marker CpG sites discriminating urinary tract urothelial carcinoma (UTUC) from normal control urothelium (C), non-cancerous urothelium from patients with UTUC (N), and urinary bladder urothelial carcinoma (BUC) located within stably regulated CpG islands or their shores and shelves, and for which DNA methylation levels based on Infinium assay were technically verified using pyrosequencing

Probe ID^a	Chromosome	Position^b	Gene symbol	Gene region^c
cg01921432	1	91,182,534	BARHL2	First exon
cg07197785	1	111,813,690	NA	NA
cg07418387	1	240,375,464	FMN2	Gene body
cg08364561	11	31,846,844	NA	NA
cg10256242	7	4,850,075	RADIL	Gene body
cg10874111	7	158,799,748	LOC154822	TSS1500
cg14302471	7	158,799,777	LOC154822	TSS1500
cg14851578	14	106,187,192	NA	NA
cg15822765	6	28,956,387	NA	NA
cg24035245	1	119,535,928	NA	NA

NA: not annotated (located within intergenic regions), TSS1500: from the transcription start site to 1500-bp upstream of it. ^aProbe IDs for the Infinium HumanMethylation450 BeadChip (Illumina). ^bNational Centre for Biotechnology Information database (Genome Built 37). ^cBased on the RefSeq database (http://www.ncbi.nlm.nih.gov/refseq/).

Figure 2. DNA methylation levels obtained by pyrosequencing of candidate marker CpG sites in tissue samples in the initial cohort. Normal control urothelium (C, n = 26), non-cancerous urothelium (N, n = 31) obtained from patients with upper urinary tract urothelial carcinoma (UTUC), and the corresponding UTUC (n = 30) were analysed. Infinium probe IDs and P values in Welch’s t-test are shown in each panel. DNA methylation levels for each of the Infinium probes cg01921432, cg07197785, cg08364561, cg15822765, and cg24035245 in UTUC samples are significantly higher than those in both C and N samples. The DNA methylation levels for each of the Infinium probes cg07418387, cg10256242, cg10874111, cg14302471, and cg14851578 in UTUC samples are significantly lower than those in both C and N samples

Table 3. DNA methylation diagnosis of upper urinary tract urothelial carcinoma (UTUC)

					Initial cohort		Validation cohort
Probe ID^a	Position^b	DNA methylation status^c	AUC	Cut-off value (%)	Sensitivity (%)^d	Specificity (%)^e	Sensitivity (%)^d	Specificity (%)^e
cg01921432	1	UTUC > N	0.997	22.54	96.6	96.7	82.9	100
	2		0.999	25.12	100	96.7	82.9	100
	All		0.999	21.70	100	96.7	85.3	100
cg07197785	1	UTUC > N	1.000	22.47	100	100	95.0	100
	2		1.000	23.42	100	100	92.5	100
	3		1.000	23.40	100	100	90.0	100
	All		1.000	23.41	100	100	95.0	100
cg07418387	1	UTUC < N	1.000	43.45	100	100	95.1	100
cg08364561	1	UTUC > N	0.996	43.89	96.6	100	90.2	100
	2		0.988	24.47	93.3	93.5	85.3	87.8
	3		0.959	22.81	96.6	93.5	92.6	87.8
	4		0.988	32.69	96.6	93.5	92.6	97.5
	All		0.997	39.00	96.6	96.7	87.8	100
cg10256242	1	UTUC < N	0.971	44.73	86.6	93.5	73.1	97.5
	2		0.998	72.48	96.6	100	92.6	100
	3		1.000	82.15	100	100	92.6	100
	All		1.000	70.43	100	100	90.2	97.5
cg10874111	1	UTUC < N	1.000	72.45	100	100	95.1	100
	2		1.000	72.36	100	100	97.5	100
	All		1.000	72.40	100	100	97.5	100
cg14302471	1	UTUC < N	1.000	77.05	100	100	97.5	100
	2		1.000	69.66	100	100	97.5	100
	3		0.998	66.37	96.6	100	95.1	100
	All		1.000	72.48	100	100	97.5	100
cg14851578	1	UTUC < N	1.000	39.49	100	100	95.1	100
	2		1.000	44.24	100	100	85.3	95.1
	All		1.000	39.81	100	100	90.2	100
cg15822765	1	UTUC > N	0.980	38.58	93.3	100	95.1	100
	2		0.970	35.56	93.3	96.7	95.1	100
	3		0.987	28.82	96.6	96.7	97.5	100
	4		0.994	28.19	96.6	96.7	95.1	100
	All		0.986	32.01	96.6	96.7	95.1	100
cg24035245	1	UTUC > N	0.992	37.54	96.6	100	90.2	100
	2		0.998	40.97	96.6	100	87.8	100
	3		1.000	39.70	100	100	90.2	100
	4		1.000	32.76	100	100	90.2	100
	All		1.000	35.17	100	100	90.2	100

AUC: area under the curve value of receiver operating characteristic curve analysis, N: non-cancerous urothelium from patients with UTUC. ^aProbe IDs for the Infinium HumanMethylation450 BeadChip (Illumina). ^bInfinium probe CpG sites and their neighbour CpG sites evaluated by pyrosequencing are numbered as shown in Table S1. Exact Infinium probe CpG sites are underlined. ‘All’ means the average for all CpG sites (exact Infinium probe CpG sites and their neighbour CpG sites). ^c‘UTUC < N’, when the DNA methylation level of the sample was lower than the cut-off value, the sample was diagnosed as UTUC; ‘UTUC > N’, when the DNA methylation level of the sample was higher than the cut-off value, the sample was diagnosed as UTUC. ^dSensitivity is defined as the ratio of the number of tissue samples diagnosed as UTUC based on the criteria relative to the exact number of UTUC samples. ^eSpecificity is defined as the ratio of the number of tissue samples not diagnosed as UTUC based on the criteria employed, relative to the exact number of N samples.