Aberrant promoter methylation profiles and association with survival in patients with hepatocellular carcinoma

Figures & data

Table 1 Characteristics of patients with hepatocellular cell carcinoma from the TCGA data portal

Category	Cohort N (n=45)	Cohort T (n=323)	P-value
Age (years)			0.072
≤60	16	161
>60	29	162
Sex			0.239
Female	18	101
Male	27	222
Race			0.000
Asian	6	152
African	6	16
White	30	144
NA	3	11
Vital status			0.000
Alive	18	245
Dead	27	78
Tumor grade			0.113
G1	5	46
G2	22	152
G3	16	110
G4	0	13
NA	2	2
Tumor pathologic stage			0.523
T1	21	159
T2	16	88
T3	5	62
T4	3	13
TX	0	1
Node pathologic stage			0.355
N0	30	234
N1	1	4
NX	13	84
NA	1	1
Metastasis pathologic stage			0.727
M0	32	245
M1	1	4
MX	12	74
AJCC pathologic stage			0.052
Stage I	20	151
Stage II	12	80
Stage III	5	70
Stage IV	1	6
NA	7	16

Note: Cohort N represents adjacent non-tumor tissues, while Cohort T represents hepatocellular carcinoma tissues.

Abbreviations: AJCC, American Joint Committee on Cancer; NA, not applicable; TCGA, The Cancer Genome Atlas.

Figure 1 Volcano plot showing the distribution of genes from the promoter-level test assessed by methylation differences and adjusted P-values.

Note: All of gene promoters are with FDR <0.05, gene promoters in blue are differentially methylated >0.2 or < −0.2.
Abbreviation: FDR, false discovery rate.

Figure 2 Heatmap of differentially methylated genes within promoters between HCC cancer tissues and adjacent tissues.

Notes: Hierarchical clustering by 323 HCC cancer tissues (orange color bar) and 45 adjacent tissues (green color bar) with 2,489 significant genes within promoters (FDR <0.05, |delta beta| >0.2); red/blue gradient represents beta values of the methylations.
Abbreviations: FDR, false discovery rate; HCC, hepatocellular carcinoma.

Table 2 Univariate and multivariate analysis of prognostic variables for cancer-specific survival in patients with hepatocellular cell carcinoma

Variables	No. of patients	Univariate analysis		Multivariate analysis
		P-value	Hazard ratios (95% CI)	P-value	Hazard ratios (95% CI)
Age (years)
>60	162	0.063		–	–
≤60	161		1.558 (0.976–2.486)
Sex
Female	101	0.040
Male	222		1.605 (1.021–2.521)	0.344	0.781 (0.468–1.304)
Tumor stage
pT1 + pT2	247	0.205		–	–
pT3 + pT4 + pTX	76		1.414 (0.827–2.417)
Node stage
pN0	238	0.005
pNX	84		2.020 (1.238–3.297)	0.311	1.454 (0.704–3.000)
Metastasis stage
pM0 + pM1	249	0.002
pMX	74		2.186 (1.348–3.546)	0.264	1.533 (0.724–3.245)
Fuhrman grade
G1 + G2	198	0.086		–	–
G3 + G4	123		1.495 (0.944–2.366)
Pathological stage
I + II	234	0.502		–	–
III + IV	73		1.232 (0.671–2.261)
RNA5SP38 promoter	323	0.035	0.294 (0.094–0.917)	0.008	0.141 (0.033–0.605)
IL21 promoter	323	0.040	4.653 (1.073–20.173)	<0.001	32.706 (5.642–189.582)
SDC4P promoter	323	0.004	0.157 (0.045–0.549)	0.035	0.163 (0.030–0.883)
MIR4439 promoter	323	0.024	0.256 (0.079–0.836)	0.419	0.532 (0.115–2.461)

Abbreviations: CI, confidence of interval; IL21, interleukin-21; MIR4439, microRNA 4439; RNA5SP38, RNA 5S ribosomal pseudogene 38; SDC4P, syndecan 4 pseudogene.

Figure 3 Kaplan–Meier plot of overall survival.

Notes: (A) Overall survival for HCC with high and low methylation levels of the RNA5SP38 promoter; (B) overall survival for HCC with high and low methylation levels of the IL21 promoter; (C) overall survival for HCC with high and low methylation levels of the SDC4P promoter.
Abbreviations: HCC, hepatocellular carcinoma; IL21, interleukin-21; RNA5SP38, RNA 5S ribosomal pseudogene 38; SDC4P, syndecan 4 pseudogene.

Figure 4 The areas under the ROC curves for the three gene promoter methylations and their combination that differentiate between HCCs and adjacent tissues.

Notes: (A) The ROC curve for the RNA5SP38 promoter. (B) The ROC curve for the IL21 promoter. (C) The ROC curve for the SDC4P promoter. (D) The ROC curve for the combination of the three gene promoter methylations.
Abbreviations: AUC, area under the curve; HCC, hepatocellular carcinoma; IL21, interleukin-21; RNA5SP38, RNA 5S ribosomal pseudogene 38; ROC, receiver operating characteristic; SDC4P, syndecan 4 pseudogene.

Table 3 KEGG and PANTHER pathways enriched in differentially methylated genes within promoters

Category	Term	Count genes	P-value
KEGG	hsa04740: olfactory transduction	254	5.740E-188
KEGG	hsa05332: graft-versus-host disease	12	8.876E-04
KEGG	hsa05320: autoimmune thyroid disease	14	8.926E-04
KEGG	hsa04650: natural killer cell–mediated cytotoxicity	23	8.702E-03
KEGG	hsa04060: cytokine–cytokine receptor interaction	37	1.962E-02
KEGG	hsa04640: hematopoietic cell lineage	16	1.764E-02
KEGG	hsa04612: antigen processing and presentation	15	2.800E-02
KEGG	hsa05416: viral myocarditis	13	3.959E-02
PANTHER	P00031: inflammation mediated by chemokine and cytokine signaling pathway	29	4.300E-04

Abbreviation: KEGG, Kyoto Encyclopedia of Genes and Genomes.