Plasma DNA methylation of Wnt antagonists predicts recurrence of esophageal squamous cell carcinoma

Table 1 Primers for methylation-specific polymerase chain reaction

Gene		Primer sequence (5'–3')
WIF-1
U	F	GGGTGTTTTATTGGGTGTATTGT
	R	AAAAAAACTAACACAAACAAAATACAAAC
M	F	CGTTTTATTGGGCGTATCGT
	R	ACTAACGCGAACGAAATACGA
RUNX-3
U	F	TTATGAGGGGTGGTTGTATGTGGG
	R	AAAACAACCAACACAAACACCTCC
M	F	TTACGAGGGGCGGTCGTACGCGGG
	R	AAAACGACCGACGCGAACGCCTCC
SFRP-1
U	F	GAGTTAGTGTTGTGTGTTTGTTGTTTTGT
	R	CCCAACATTACCCAACTCCACAACCA
M	F	GTGTCGCGCGTTCGTCGTTTCGC
	R	AACGTTACCCGACTCCGCGACCG
DKK-3
U	F	TTAGGGGTGGGTGGTGGGGT
	R	CTACATCTCCACTCTACACCCA
M	F	GGGGCGGGCGGCGGGGC
	R	ACATCTCCGCTCTACGCCCG

M: Methylated sequence; U: Unmethylated sequence; F: Forward primer; R: Reverse primer.

Table 2 Characteristics of patients

Variables	Recurrence		P value
	No	Yes
Age (yr)			0.804
< 63	31	10
≥ 63	29	11
Gender			0.569
Female	16	4
Male	44	17
Smoking			0.614
No	31	9
Yes	29	12
Drinking			0.799
No	26	8
Yes	34	13
Operation			0.614
No	38	12
Yes	22	9
Chemo-/radio-therapy			0.751
No	11	5
Yes	49	16
Stage			< 0.001
I	25	3
II	22	3
III	13	15

Table 3 Correlation between methylation and recurrence in esophageal squamous cell carcinoma patients

Gene	n	Recurrence		Crude OR (95% CI)	Pvalue	Adjusted OR1 (95% CI)	P value1
		No	Yes
SFRP-1
Negative	57	49	8	1.00	< 0.001	1.00	< 0.001
Positive	24	11	13	7.24 (2.42-21.68)		10.8 (2.54-46)
WIF-1
Negative	52	42	10	1.00	0.069	1.00	0.107
Positive	29	18	11	2.57 (0.93-7.11)		2.61 (0.8-8.47)
DKK-3
Negative	51	44	7	1.00	0.001	1.00	0.003
Positive	30	16	14	5.50 (1.88-16.08)		6.07 (1.73-21.28)
RUNX-3
Negative	52	45	7	1.00	< 0.001	1.00	0.001
Positive	29	15	14	6.00 (2.04-17.65)		7.81 (2.30-47)

¹Adjusted for age, gender, smoking, drinking, stage, surgical operation and chemo-/radio-therapy. CI: Confidence intervals; OR: Odds ratios.

Table 4 Combined analysis of methylation and recurrence in esophageal squamous cell carcinoma patients

No. of M gene	n	Recurrence		Crude OR (95% CI)	P value	Adjusted OR1 (95% CI)	P value1
		No	Yes
0	32	29	3	Ref.		Ref.
1	25	21	4	1.84 (0.37-9.11)	0.454	1.71 (0.27-10.73)	0.570
2-3	24	10	14	13.53 (3.21-57.07)	< 0.001	15.69 (2.97-83.00)	0.001
Locus Trend				4.10 (1.92-8.75)	< 0.001	4.10 (1.73-9.72)	0.001

¹Adjusted for age, gender, smoking, drinking, stage, surgical operation and chemo-/radio-therapy. CI: Confidence intervals; OR: Odds ratios.