CpG island methylator phenotype in plasma is associated with hepatocellular carcinoma prognosis

Table 1 Primer sets for nested methylation-specific polymerase chain reaction

Genes			Primer sequence (5'-3')
APC	U	F	GTGTTTTATTGTGGAGTGTGGGTT
		R	CCAATCAACAAACTCCCAACAA
	M	F	TATTGCGGAGTGCGGGTC
		R	TCGACGAACTCCCGACGA
WIF-1	U	F	GGGTGTTTTATTGGGTGTATTGT
		R	AAAAAAACTAACACAAACAAAATACAAAC
	M	F	CGTTTTATTGGGCGTATCGT
		R	ACTAACGCGAACGAAATACGA
RUNX3	U	F	TTATGAGGGGTGGTTGTATGTGGG
		R	AAAACAACCAACACAAACACCTCC
	M	F	TTACGAGGGGCGGTCGTACGCGGG
		R	AAAACGACCGACGCGAACGCCTCC
DLC-1	U	F	AAACCCAACAAAAAAACCCAACTAACA
		R	TTTTTTAAAGATTGAAATGAGGGAGTG
	M	F	CCCAACGAAAAAACCCGACTAACG
		R	TTTAAAGATCGAAACGAGCGAGCG
SFRP-1	U	F	GAGTTAGTGTTGTGTGTTTGTTGTTTTGT
		R	CCCAACATTACCCAACTCCACAACCA
	M	F	GTGTCGCGCGTTCGTCGTTTCGC
		R	AACGTTACCCGACTCCGCGACCG
DKK	U	F	TTAGGGGTGGGTGGTGGGGT
		R	CTACATCTCCACTCTACACCCA
	M	F	GGGGCGGGCGGCGGGGC
		R	ACATCTCCGCTCTACGCCCG
E-cad	U	F	TAATTTTAGGTTAGAGGGTTATTGT
		R	CCACCCCAATACTAAATCACAACA
	M	F	TTAGGTTAGAGGGTTATCGCGT
		R	TAACTAAAAATTCACCTACCGAC

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

Table 2 Frequencies of gene methylation in hepatocellular carcinomas, corresponding non-neoplastic liver tissues and normal liver tissues

Genes	No. of methylated samples
	Tumor (n = 108)	Plasma (n = 108)	Non-neoplastic tissues (n = 102)	NormalTissues (n = 15)	Healthy plasma (n = 60)	χ2
APC	481	26	5	0	0	43.47
WIF- 1	531	35	6	0	0	48.44
DLC-1	381	23	4	0	0	32.05
DKK	391	25	5	0	0	30.85
SFRP-1	481	26	5	0	0	43.47
E-cad-1	371	18	3	0	0	33.37
RUNX3	521	42	4	0	0	52.47

¹χ² test comparing the frequencies of gene methylation between tumor and non-neoplastic samples, P < 0.05.

Table 3 Relationship between CpG island methylator phenotype and clinicopathological characteristics in hepatocellular carcinomas

Characteristics	CIMP+		CIMP-		χ21	χ22
	Tumor	Plasma	Tumor	Plasma
Age (yr)
< 52	27	25	21	23	0.5584	1.002
≥ 52	38	37	22	23
Gender
Female	9a	8a	13	15	4.28	6.115
Male	56	54	30	31
HBsAg
Negative	9a	7a	15	17	6.625	10.06
Positive	56	55	28	29
Anti-HCV
Negative	52	52	37	37	0.655	0.2151
Positive	13	10	6	9
AFP (μg/L)
< 20	2a	3a	40	41	88.1	77.72
20-400	29	30	3	5
> 400	34	29	0	0
Cirrhosis
Yes	53	50	32	36	0.7827	0.0925
No	12	12	11	10
Node number
Single	44	37	31	32	0.2362	1.119
Multiple	21	25	12	14
Tumor number
Single	41	37	28	31	0.0467	0.6738
Multiple	24	25	15	15
Tumor size
< 6	30	27	24	22	0.966	0.195
≥ 6	35	35	19	24
Edmondson-Steiner
I-II	38	35	24	21	0.0742	1.234
III-IV	27	27	19	25
TNM stage
I	31a	34a	20	17	5.325	4.872
II	16	12	4	8
III	18	16	19	21

¹Indicates χ² test comparing the clinicopathological characteristics between CIMP+ and CIMP- in tumor tissues; ²Indicates the comparison in the plasma.

^aP < 0.05. CIMP: CpG island methylator phenotype; HBsAg: Hepatitis B surface antigen; Anti-HCV: Hepatitis C virus antibody; AFP: Alpha-fetoprotein; TNM: Tumor-node-metastasis.

Table 4 Relationship between CpG island methylator phenotype and prognosis of hepatocellular carcinomas after a one-year follow-up (n = 98)

Prognosis	CIMP+		CIMP-		χ21	χ22
	Tumor	Plasma	Tumor	Plasma
Metastasis
Yes	22a	19a	2	5	11.032	5.762
No	40	38	34	36
Recurrence
Yes	18a	17a	3	4	5.796	4.257
No	44	40	33	37
Survival
Yes	26a	23a	24	27	5.574	4.621
No	36	32	12	16

¹Indicates χ² test comparing the prognosis between CIMP+ and CIMP- in tumor tissues; ²Indicates the comparison in the plasma.

^aP < 0.05. CIMP: CpG island methylator phenotype.