Methylation of TIMP3 in esophageal squamous cell carcinoma

Table 1 Characteristics of patients with ESCC

Variable	n
Male	45
Female	19
Age, median (range)	57 (42-76)
Males	57 (42-70)
Females	62 (49-76)
Tumor volume (cm3), median (range)	57 (4-300)
Histological differentiation
Well/moderate	57
Poor	4
Not recorded	3
Tumor stage
T1N0M0	3
T2N0M0	9
T2N1M0	4
T3N0M0	29
T3N1M0	10
T3N1M1	1
T4N0M0	2
T4N0M1	1
T4N1M0	2
Not recorded	3

Table 2 Primer sequences for methylation analysis and quantitative real-time RT-PCR

	Forward primer	Reverse primer	Length (bp)
TIMP3 R1	TTTGGGTTAGAGATATTTAGTGGTT	RAATCRTTCAAATCCTTATAAAAAATAATA	175
TIMP3 R2	GGYGGTATTATTTTTTATAAGGATTTG	AAACCCCRCCTCRAACTATTAAA	158
TIMP3 R3	TTTGAGGGGGYGGGTTTTAATAGTT	AACRACCTCCCRACGAAAAAACAAA	174
TIMP3 qRT-PCR	CCAGGACGCCTTCTGCAAC	CCTCCTTTACCAGCTTCTTCCC	71

Table 3 TIMP3 methylation and expression in methylated ESCC samples

Patient	M/F	Age	Tumor			Methylation			Expression
			Volume (cm3)	Differentiation	Stage	R1	R2	R3
28	F	65	60	Moderate	T3N1M1	++	-	-	Strong heterogeneous
41	M	65	30	Moderate	T3N0M0	++	-	++	Strong heterogeneous
44	M	66	18	Moderate	T3N0M0	++	-	++	Strong heterogeneous
75	F	72	27	Moderate	T3N1M0	++	+	++	Focal loss