DNA Methylation Consistency Implicates the Primary Tumor Cell Origin of Recurrent Hepatocellular Carcinoma

Figures & data

Figure 1.  The methylation patterns of the 24 genes chosen for analysis from primary and recurrent tumor tissues and the Hep-11 and Hep-12 cell lines.

(A) The methylation patterns between the primary and recurrent tumors of Cases 1 and 2, who were HBV negative. (B) The methylation patterns between the primary and recurrent tumors of Cases 3 to 9, who were HBV infection related. (C) The methylation pattern between Hep-11 and Hep-12 cell lines. The Spearman’s rank correlation coefficient test was used to analyze the correlations, p < 0.05 was considered significant.

P: Primary; R: Recurrent.

Figure 2.  Representative results of the HBV DNA integration patterns in the host cellular genome of the primary and recurrent tumors.

(A) The HBV DNA integration in Case 8. (B) The HBV DNA integration in Case 9. Left panel shows the Alu-PCR results detected by agarose gel electrophoresis separation. Lanes 1–4 represent different PCR results using different Alu-PCR primers. Lane 1: HBV X gene primer as forward and Alu (+) primer as reversed; Lane 2: HBV preC/C gene primer as forward and Alu (+) primer as reversed; Lane 3: HBV X gene primer as forward and Alu (−) primer as reversed; Lane 4: HBV preC/C gene primer as forward and Alu (−) primer as reversed. The right panel shows the sequencing results of the circled bands in the left panel. The arrows indicates the location of HBV–host genome junctions.

P: Primary hepatocellular carcinoma tumor; R: Recurrent hepatocellular carcinoma tumor.

Figure 3.  The mRNA expression levels of hepatocellular carcinoma cancer stem cell biomarkers in primary and recurrent hepatocellular carcinoma tissues.

(A) The mRNA expression levels of the 22 cancer stem cell biomarkers in primary and recurrent hepatocellular carcinoma tissues normalized by that in normal liver tissues. The housekeeping gene, CTBP, was also detected as a quality control. (B) The normalized mRNA expression levels of 22 cancer stem cell biomarkers in recurrent tumors compared with that in the primary.

*indicates that the gene’s expression level of either the primary tumor or the recurrent was significantly higher than that of normal liver tissues. The two-tailed unpaired t-test was used to analyze the difference of expression levels in different groups.

p < 0.05 was considered significant.

Table 1.  Clinicopathological parameters of the nine patients with primary and recurrent hepatocellular carcinoma.

Case No.	Sex/age	HBV	Interval^† (months)		Nodules (n)	Location	Size (cm)	BCLC stage	TNM stage	AFP (μg/l)
1	F/68	−	24.7	P	1	S4	6	A	II	>1210
				R	1	S3	6	A	II	1210
2	M/39	−	48.2	P	1	S5 & S6	6	A	II	160
				R	1	S8	8	A	II	1210
3	M/34	+	8.9	P	1	S6 & 7	6	A	II	2
				R	1	S6	6.5	A	II	2
4	M/52	+	13.1	P	1	S4 & S5 & S8	3	C	II	21
				R	1	S4 & S5 & S8	7	A	II	36
5	M/71	+	4.9	P	1	Right-lobe	10	A	II	202
				R	1	S6	3	A	II	55
6	M/41	+	5.0	P	1	Left-lobe	8	A	II	>1210
				R	1	Left-lobe	8	A	II	1000
7	M/43	+	3.3	P	1	S2 & S3	20	C	II	>1210
				R	Multiple^‡	Upper the greater omentum and mesentery	2	C	II	348
8	M/76	+	23.4	P	2^‡	S6,S7	10	C	III	>1210
				R	1	S7	4	C	II	1210
9	M/55	+	10.3	P	2^‡	S5,S6	3	A	III	21
				R	1	S7	5	A	II	17

† Interval between two operations.

‡ Only the largest nodule was available in our tissue bank.

AFP: α-fetoprotein; BCLC: Barcelona Clinic Liver Cancer; F: Female; M: Male; P: Primary hepatocellular carcinoma; R: Recurrent hepatocellular carcinoma S: Liver segment; TNM: Tumor, Lymph Node and Metastasis.

Table 2.  The HBV DNA integration status in host cellular genome of six patients with primary and recurrent hepatocellular carcinoma.

Case No.	Integration No.	Chromosomal location	Chromosome location (UCSC)		Genbank	Related gene name	Integration site	Orientation	HBV break
3-P^†	1	8q13.2	68002219	68003916	NC_000008.11	PREX2	Intron 1	+	1815
3-R^†	1	8q13.2	68002219	68003916	NC_000008.11	PREX2	Intron 1	+	1815
4-P	1	4p14	40572076	40572142	NC_000004.12	RBM47	5’UTR	−	1821
4-R	1	4p14	40572076	40572142	NC_000004.12	RBM47	5’UTR	−	1821
6-P	1	20p12.3	5639689	5640156	NC_018931.2			−	2224
6-R	1	20p12.3	5639689	5640155	NC_018931.2			−	2230
	2	20q11.23	35833665	35833819	NC_018931.2	PHF20	Intron 1	+	1612
7-P	1	4q12	52251977	52252646	NC_000004.12			−	1621
7-R	1	4q12	52251977	52252646	NC_000004.12			−	1621
8-P	1	19q13.12	35722065	35721735	NC_000019.10	MLL4	Exon 3	+	1808
8-R	1	19q13.12	35722065	35721735	NC_000019.10	MLL4	Exon 3	+	1808
9-P	1	2q31.1	170846582	170846765	NC_000002.12	GAD1	Intron 9	+	1820
	2	19p13.11	17041317	17041506	NC_000002.12			−	1486
	3	2q11.2	97243605	97244162	NC_000002.1	ANKRD36	Intron 69	+	1786
	4	8q24.21	128187021	128187133	NC_018919.2			−	1825
9-R	1	16p11.2	28596750	28598781	NC_018927.2	SULT1A2	5’UTR	−	1832
	2	18q11.2	22003616	22003724	NC_000018.10			+	1742

Orientation: + means the inserted HBV fragment’s direction was the same with genome sequence; - means the inserted HBV fragment’s direction was the opposite to the genome sequence.

P:Primary hepatocellular carcinoma tumor; R:Recurrent hepatocellular carcinoma tumor; UTR:Untranslational region.

Table 3.  Correlation of every gene’s methylation between the eight paired primary and recurrent tumors.

No.	Gene ID	R	P
1	AKAP12	0.28571	0.4927
2	APC	0.7619	0.028
3	BASP1	0.976	<0.0001
4	BNC1	0.667	0.071
5	CDKN1C	0.762	0.028
6	CDKN2A	0.09524	0.8225
7	CFTR	0.71429	0.0465
8	CRMP1	0.69048	0.058
9	GSTP1	0.47619	0.2329
10	IRAK3	0.71429	0.0465
11	KL	0.69048	0.058
12	MIR196B	0.61905	0.1017
13	NPY	0.78571	0.0208
14	PRDM2	0.7619	0.028
15	PYDC1	0.64286	0.0856
16	RASSF1A	0.7381	0.0366
17	RASSF2A	0.7381	0.0366
18	RUNX3	0.78571	0.0208
19	SPINT2	0.88095	0.0039
20	TAL1	0.80952	0.0149
21	TLX3	0.97619	<0.0001
22	TMEFF2	0.78571	0.0208
23	UCHL1	0.88095	0.0039
24	WNK2	0.61905	0.1017

p-value of <0.05 (two sided) was considered as significant and written in bold text.

P: Primary hepatocellular carcinoma tumor; R: Recurrent hepatocellular carcinoma tumor.

Table 4.  The correlation between the paired primary and recurrent cell lines and tumor tissues by using the 15 constant methylated genes.

Sample ID	R	P
1-P&R	0.864	<0.0001
2-P&R	0.882	<0.0001
3-P&R	0.864	<0.0001
4-P&R	0.639	0.0103
5-P&R	0.843	<0.0001
6-P&R	0.811	0.0002
7-P&R	0.886	<0.0001
8-P&R	0.871	<0.0001
9-P&R	0.366	0.179
2-R&3-P	0.297	0.283
Hep11&12	0.799	0.0004

p-value of <0.05 (two sided) was considered as significant and written in bold text.

P: Primary hepatocellular carcinoma tumor; R: Recurrent hepatocellular carcinoma tumor.