Detection of promoter hypermethylation of Wnt antagonist genes in fecal samples for diagnosis of early colorectal cancer

Table 1 Correlation of the methylation status of fecal SFRP2 and WIF-1 genes with patients’ clinicopathological parameters n (%)

Parameter	Case	WIF-1		SFRP2		WIF-1+SFRP2		Sensitivity	Specificity
	(n)	Methylation	Pvalue	Methylation	Pvalue	Methylation	Pvalue
CRC	48	27 (56.3)	0.0001	29 (60.4)	0.0001	39 (81.3)	0.0001	81.3%
Gender
Male	24	17 (70.8)	0.140	14 (58.3)	0.771
Female	24	12 (29.2)		13 (54.2)
Age (yr)
> 55	32	21 (65.6)	0.297	18 (56.3)	1.000
≤ 55	16	8 (50)		9 (56.3)
Tumor location
Left colon	32	21 (65.6)	0.267	18 (56.3)	1.000
Right colon	16	8 (50)		9 (56.3)
TNM stage
A-B	27	18 (66.7)	0.315	15 (55.6)	0.912
C-D	21	11 (52.4)		12 (57.1)
Lymph node metastasis
Positive	21	11 (52.4)	0.315	12 (57.1)	0.912
Negative	27	18 (66.7)		15 (55.6)
Distant metastasis
Positive	7	4 (57.1)	0.848	4 (57.1)	0.959
Negative	41	25 (61.0)		23 (56.1)
Adenoma	35	18 (51.4)	0.0001	16 (45.7)	0.0001	23 (65.7)	0.0001	65.7%
Advanced	15
Non-advanced	20
Hyperplastic polyp	32	4 (12.5)	0.0451	6 (18.7)	0.0551	8 (25.0)	0.0161	25.0%	96.7%
Normal control	30	0 (0)		1 (3.3)		1 (3.3)

¹Represents the P value vs normal subjects. Sensitivity represents the ability of the DNA methylation test to detect the presence of at least one hypermethylated gene. CRC: Colorectal cancer; SFRP: Secreted frizzled related protein; WIF-1: Wnt inhibitory factor-1.

Table 2 SFRP2 and WIF-1 gene primer sequences for methylation-specific polymerase chain reaction assays

Gene	Primer (5’-3’)	Product size (bp)	Annealing temperature (°C)
M: SFRP2	GGGTCGGAGTTTTTCGGAGTTGCGC	138	62
	CCGCTCTCTTCGCTAAATACGACTCG		(38)
U: SFRP2	TTTTGGGTTGGAGTTTTTTGGAGTTGTGT	145	52
	AACCCACTCTCTTCACTAAATACAACTCA		(38)
M: WIF-1	GGGCGTTTTATTGGGCGTAT	197	58
	AAACCAACAATCAACGAAC		(38)
U: WIF-1	GGGTGTTTTATTGGGTGTAT	198	55
	AAACCAACAATCAACAAAAC		(38)

SFRP: Secreted frizzled related protein; WIF-1: Wnt inhibitory factor-1.

Table 3 Hypermethylation status of SFRP2 and WIF-1 in fecal DNA samples n (%)

Group	Case	Hypermethylated genes
		SFRP2	WIF-1	SFRP2+WIF-1
CRC	48	27 (56.3)	29 (60.4)	39 (81.3)
Dukes A-B	27	15 (55.6)	18 (66.7)	22 (81.5)
Dukes A	7	5 (71.4)	4 (57.1)	6 (85.7)
Dukes B	20	10 (50.0)	14 (70.0)	16 (80.0)
Dukes C-D	21	12 (57.1)	11 (52.4)	17 (81.0)
Dukes C	14	8 (57.1)	7 (50.0)	10 (71.4)
Dukes D	7	4 (57.1)	4 (57.1)	7 (100)
Adenoma	35	18 (51.4)	16 (45.7)	23 (65.7)
Advanced	15	9 (60.0)	8 (53.3)	12 (80.0)
Non-advanced	20	8 (40.0)	7 (35.0)	11 (55.0)
Hyperplastic polyp	32	4 (12.5)	6 (18.7)	8 (25.0)
Normal control	30	0 (0)	1 (3.3)	1 (3.3)

CRC: Colorectal cancer; SFRP: Secreted frizzled related protein; WIF-1: Wnt inhibitory factor-1.

Table 4 Hypermethylated SFRP2 and WIF-1 genes and fecal occult blood test for the detection of colorectal tumors n (%)

Group	n	HypermethylatedSFRP2andWIF-1	Fecal occult blood test	Pvalue
CRC	48	39 (81.3)	27 (56.3)	0.008
Dukes A-B	27	22 (81.5)	14 (51.9)	0.021
Dukes A	7	6 (85.7)	2 (28.5)	0.031
Dukes B	20	16 (80.0)	12 (60.0)	0.168
Dukes C-D	21	17 (81.0)	13 (61.9)	0.170
Dukes C	14	10 (71.4)	8 (57.1)	0.430
Dukes D	7	7 (100)	5 (71.4)	0.127
Adenoma	35	23 (65.7)	9 (25.7)	< 0.001
Advanced	15	12 (80.0)	5 (33.3)	0.010
Non-advanced	20	11 (55.0)	4 (20.0)	0.022
Hyperplastic polyp	32	8 (25.0)	5 (15.6)	0.351
Normal control	30	1 (3.3)	1 (3.3)	1.000

CRC: Colorectal cancer; SFRP: Secreted frizzled related protein; WIF-1: Wnt inhibitory factor-1.