MT1M and MT1G promoter methylation as biomarkers for hepatocellular carcinoma

Table 1 Primers for polymerase chain reaction

Primers	Sequences	Annealing temp (°C)	Size (bp)
MT1M
U	5'-TTGAAAATGGTGGGGTGA-3'	54	163
	5'-AAACTATACACCAAATAATACACAATATCC-3'
M	5'-GACGTTCGCGACGTTAAG-3'	54	124
	5'-ACGCCGAATAATACGCAAT-3'
MT1G
U	5'-GGGGTTGTTTTGTGGTGTGTG-3'	59	135
	5'-AAACACCCCACCCCACCCTT-3'
M	5'-TTCGCGAGTCGGTGCGAAAG-3'	50	96
	5'-CCGCGATCCCGACCTAAACT-3'

Table 2 Clinicopathological data and serum metallothionein 1M and metallothionein 1G promoter methylation in hepatocellular carcinoma patients

Characteristics	n	MT1M			MT1G
		1M	2U	P value	1M	2U	P value
Total number	121	59	62		85	36
Gender
Male	100	50	50	0.552	70	30	0.896
Female	21	9	12		15	6
Age (yr)
≥ 55	67	32	35	0.807	46	21	0.670
< 55	54	27	27		39	15
HBV infection
Yes	100	50	50	0.552	69	31	0.512
No	21	9	12		16	5
Vascular invasion or metastasis
Yes	44	26	18	0.086	33	11	0.387
No	77	33	44		52	25
Histological differentiation
Poor	41	18	23	0.610	26	15	0.426
Moderate	50	27	23		38	12
Well	30	14	16		21	9
TNM stage
I-II	64	26	38	0.058	44	13	0.115
III-IV	57	33	24		41	23
Tumor multiplicity
Single	72	26	36	0.741	52	20	0.565
Multiple	49	23	26		33	16
Tumor size(cm)
≥ 5	59	38	21	0.001	38	21	0.170
< 5	62	21	41		47	15

¹Methylated; ²Unmethylated.

Table 3 Correlation of metallothionein 1M and metallothionein 1G promoter methylation with tumor size

Gene	Tumor size (cm)		rs	Pvalue
	MethylatedM (P25-P75)	UnmethylatedM (P25-P75)
MT1M	6.5 (4.0-9.0)	3.9 (2.2-6.4)	0.321	0.000
MT1G	4.4 (2.9-8.0)	5.0 (3.5-7.9)	-0.049	0.590

M: Median; P₂₅: First quartile; P₇₅: Third quartile; rs: Spearman correlation coefficient; MT1M: Metallothionein 1M; MT1G: Metallothionein 1G.

Table 4 Vascular invasion or metastasis and a combination of metallothionein 1M and metallothionein 1G promoter methylation

Characteristic	n	MT1M and MT1G
		1M	2U	P value
Vascular invasion or metastasis
Yes	44	18	26	0.018
No	77	16	61

¹Both MT1M and MT1G were methylated; ²One of MT1M and MT1G was methylated or neither of them methylated. MT1M: Metallothionein 1M; MT1G: Metallothionein 1G.

Table 5 Sensitivity and specificity of gene sets for hepatitis B virus (+) hepatocellular carcinoma detection in chronic hepatitis B group

No.	Marker	TP/FN	FP/TN	Sensitivity (%)TP/(TP + FN)	Specificity (%)TN/(TN + FP)
1	AFP	56/44	14/23	56.0	62.1
2	MT1M	50/50	2/35	50.0	94.6
3	MT1G	69/31	6/31	69.0	83.8
4	MT1M/MT1G	90/10	7/30	90.0	81.1

Sensitivity (%), TP/(TP + FN) and specificity (%), TN/(TN + FP) of each gene set were calculated and plotted. MT1M/MT1G, MT1M or MT1G promoter methylation. TP: True positive; FN: False negative; FP: False positive; TN: True negative.

Table 6 Sensitivity and specificity of gene sets for hepatocellular carcinoma detection in normal controls group

No.	Gene	TP/FN	FP/TN	Sensitivity (%)TP/(TP+FN)	Specificity (%)TN/(TN+FP)
1	MT1M	59/62	2/29	48.8	93.5
2	MT1G	85/36	4/27	70.2	87.1
3	MT1M/MT1G	110/11	5/26	90.9	83.9

Sensitivity (%), TP/(TP + FN) and specificity (%), TN/(TN + FP) of each gene set were calculated and plotted. MT1M/MT1G, MT1M or MT1G promoter methylation. TP: True positive; FN: False negative; FP: False positive; TN: True negative; MT1M: Metallothionein 1M; MT1G: Metallothionein 1G.