Acyl-CoA Binding Domain Containing 4 Polymorphism rs4986172 and Expression Can Serve as Overall Survival Biomarkers for Hepatitis B Virus-Related Hepatocellular Carcinoma Patients After Hepatectomy

Figures & data

Table 1 Survival Analysis of ACBD4 Gene in GSE14520 HBV-Related HCC Cohort

Variables	Patients (n=212)	No. of Event	MST (Months)	Crude HR (95% CI)	Crude P	Adjusted HR (95% CI)	Adjusted P^ɠ
RFS
Low ACBD4	106	62	30	1		1
High ACBD4	106	54	53	0.711(0.494–1.025)	0.068	0.818(0.563–1.187)	0.29
OS
Low ACBD4	106	51	54	1		1
High ACBD4	106	31	NA	0.493(0.315–0.771)	0.002	0.561(0.348–0.903)	0.017

Notes: ^ɠRFS adjusted for gender, BCLC and cirrhosis in multivariate Cox proportional risk regression model, while OS adjusted for tumor size, cirrhosis, BCLC and AFP.

Abbreviations: HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HR, hazard ratio; CI, confidence interval; MST, median survival time; RFS, recurrence-free survival; OS, overall survival; BCLC, Barcelona Clinic Liver Cancer; AFP, α-fetoprotein; NA, not available; ACBD4, Acyl-CoA binding domain containing 4.

Table 2 Joint Effect Survival Analysis of ACBD4 Expression and Clinical Parameters in GSE14520 HBV-Related HCC Patients

Group	Variables	Gene Expression	Patients (n=212)	No. of Event	MST (Months)	Crude HR (95% CI)	Crude P	Adjusted HR (95% CI)	Adjusted P^ɠ
	Tumor Size^&
a1	≤5cm	High ACBD4	71	18	NA	1		1
b2	>5cm	High ACBD4	35	13	NA	1.844(0.903–3.766)	0.093	1.146(0.532–2.466)	0.728
c3	≤5cm	Low ACBD4	66	28	NA	1.966(1.087–3.556)	0.025	1.819(0.975–3.393)	0.06
d4	>5cm	Low ACBD4	39	23	20	4.065(2.182–7.572)	<0.001	1.993(0.958–4.149)	0.065
	BCLC stage
11	0	High ACBD4	10	1	NA	1		1
22	A	High ACBD4	79	21	NA	3.257(0.438–24.215)	0.249	2.934(0.392–21.928)	0.294
33	B	High ACBD4	9	4	47	6.914(0.771–61.984)	0.084	5.312(0.573–49.232)	0.142
44	C	High ACBD4	8	5	13	12.393(1.444–106.328)	0.022	11.260(1.244–101.952)	0.031
55	0	Low ACBD4	10	1	NA	1.114(0.070–17.813)	0.939	0.991(0.062–15.971)	0.995
66	A	Low ACBD4	64	27	NA	5.887(0.800–43.341)	0.082	5.459(0.731–40.757)	0.098
77	B	Low ACBD4	13	8	30	11.845(1.474–95.191)	0.02	9.049(1.081–75.725)	0.042
88	C	Low ACBD4	19	15	10	25.657(3.370–195.363)	0.002	19.515(2.451–155.358)	0.005
	Serum AFP^φ
A1	≤300 ng/mL	High ACBD4	74	19	NA	1		1
B2	>300 ng/mL	High ACBD4	30	12	NA	1.691(0.821–3.485)	0.154	1.372(0.655–2.874)	0.402
C3	≤300 ng/mL	Low ACBD4	41	20	51	2.301(1.226–4.317)	0.009	2.125(1.124–4.019)	0.02
D4	>300 ng/mL	Low ACBD4	64	31	54	2.453(1.385–4.347)	0.002	1.992(1.106–3.590)	0.022
	Cirrhosis
aa	Yes	Low ACBD4	98	49	NA	1		1
bb	NO	Low ACBD4	8	2	NA	0.378(0.092–1.555)	0.178	0.538(0.128–2.260)	0.397
cc	Yes	High ACBD4	97	31	NA	0.513(0.327–0.806)	0.004	0.593(0.367–0.958)	0.033
dd	NO	High ACBD4	9	0	NA	1.000×10^−6(7.141×10^−289-2.661×10^276)	0.968	2.000×10^−6(2.938×10^−268-1.065×10^256)	0.966

Notes: ^ɠOS adjusted for tumor size, cirrhosis, BCLC and AFP. ^&Information of tumor size was unavailable in 1 patient; ^φInformation of serum AFP was unavailable in 3 patients.

Abbreviations: HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HR, hazard ratio; CI, confidence interval; MST, median survival time; RFS, recurrence-free survival; OS, overall survival; BCLC, Barcelona Clinic Liver Cancer; AFP, α-fetoprotein; NA, not available; ACBD4, Acyl-CoA binding domain containing 4.

Figure 1 Violin diagram of ACBD4 gene expression level distribution in normal human organ tissues from GTEx database.

Figure 2 The diagnostic and prognostic values of ACBD4 in GSE14520 HBV-related HCC cohort. (A) Expression distribution of ACBD4 in tumor and adjacent paracancerous tissues; (B) the ROC curve of ACBD4 gene in distinguished tumor and adjacent paracancerous tissues; (C) Kaplan-Meier curve of ACBD4 in HBV-related HCC recurrence-free survival time; (D) Kaplan-Meier curve of ACBD4 in HBV-related HCC overall survival time.

Figure 3 Stratified survival analysis results of ACBD4 gene in GSE14520 HBV-related HCC cohort.

Figure 4 Joint effect survival analysis of ACBD4 expression and clinical parameters in GSE14520 HBV-related HCC patients. (A) Joint effect survival analysis of ACBD4 and tumor size; (B) Joint effect survival analysis of ACBD4 and cirrhosis; (C) Joint effect survival analysis of ACBD4 and BCLC stage; (D) Joint effect survival analysis of ACBD4 and serum AFP.

Note: The detailed information on grouping is shown in Table 2.

Figure 5 The nomogram of ACBD4 and clinical parameters in the GSE14520 HBV-related HCC cohort.

Figure 6 Prognostic value of ACBD4 gene in TCGA HCC cohort. (A) Kaplan-Meier curve of ACBD4 in HCC recurrence-free survival time; (B) Kaplan-Meier curve of ACBD4 in HCC overall survival time; (C) Kaplan-Meier curve of ACBD4 in HCC progression-free survival time; (D) Kaplan-Meier curve of ACBD4 in HCC disease-specific survival time.

Table 3 Survival Analysis of ACBD4-rs4986172 in Guangxi HBV-Related HCC Patients

Genotype	Patients (n=474)	MST (Months)	Crude HR (95% CI)	Crude P	Adjusted HR (95% CI)	Adjusted P^¥
rs4986172
GG	106	35	1		1
AG	231	75	0.607(0.442–0.833)	0.002	0.617(0.438–0.870)	0.006
AA	137	73	0.665(0.467–0.947)	0.024	0.755(0.519–1.099)	0.142
AA+AG	368	74	0.628(0.468–0.841)	0.002	0.666(0.486–0.913)	0.012
rs4986172
AA	137	73	1		1
AG+GG	337	51	1.078(0.802–1.450)	0.619	0.962(0.705–1.312)	0.807

Note: ^¥Adjusted for AFP, BCLC, tumor number, tumor size, PVTT and radical resection.

Abbreviations: HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HR, hazard ratio; CI, confidence interval; MST, median survival time; ACBD4, Acyl-CoA binding domain containing 4.

Table 4 Correlation Analysis Between ACBD4-rs4986172 and Clinical Parameters in Guangxi HBV-Related HCC Patients

Variables	AA+AG (n=368)	GG (n=106)	OR (95% CI)	P
Age (years)
≤60	327	93	1
>60	41	13	1.115(0.573–2.168)	0.749
Gender
Male	324	95	1
Female	44	11	0.853(0.424–1.716)	0.655
Ethnicity
Han	227	71	1
Minority	141	35	0.794(0.503–1.252)	0.321
BMI
≤25	288	87	1
>25	80	19	0.786(0.451–1.369)	0.395
Smoking status
None	239	72	1
Ever	129	34	0.875(0.552–1.387)	0.57
Drinking status
None	219	66	1
Ever	149	40	0.891(0.571–1.389)	0.61
Child–Pugh^§
A	302	83	1
B	45	14	1.132(0.593–2.162)	0.707
Cirrhosis^£
No	46	11	1
Yes	321	95	1.238(0.617–2.484)	0.549
Radical resection^ƀ
Yes	203	61	1
No	157	43	0.911(0.586–1.419)	0.681
Pathological diagnosis^ƙ
Well differentiated	20	6	1
Moderately differentiated	284	8	0.939(0.365–2.417)	0.896
Poorly differentiated	9	3	1.111(0.226–5.468)	0.897
Tumor size
≤5 cm	155	36	1
>5 cm	213	70	1.415(0.900–2.224)	0.132
Tumor number
Single	269	81	1
Multiple	99	25	0.839(0.507–1.388)	0.494
BCLC
A	223	56	1
B	61	14	0.914(0.477–1.752)	0.786
C	84	36	1.707(1.047–2.781)	0.032
Portal vein tumor thrombus^ƕ
No	310	77	1
Yes	58	28	1.944(1.161–3.254)	0.012
Serum AFP^ƛ
AFP ≤400 (ng/mL)	196	47	1
AFP >400 (ng/mL)	147	50	1.418(0.903–2.229)	0.13

Notes: ^§Information of Child–Pugh was unavailable in 30 patients; ^£Information of cirrhosis was unavailable in 1 patient; ^ƀInformation of radical resection was unavailable in 10 patients; ^ƙInformation of pathological diagnosis was unavailable in 72 patients; ^ƕInformation of PVTT was unavailable in 1 patient; ^ƛInformation of AFP was unavailable in 34 patients.

Abbreviations: HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HR, hazard ratio; CI, confidence interval; MST, median survival time; ACBD4, Acyl-CoA binding domain containing 4; BMI, body mass index.

Table 5 Joint Effect Survival Analysis of ACBD4-rs4986172 and Clinical Parameters in Guangxi HBV-Related HCC Patients

Groups	Variables	Genotype	Patients (n=474)	MST (Months)	Crude HR (95% CI)	Crude P	Adjusted HR (95% CI)	Adjusted P^¥
	Radical resection^ƀ	rs4986172
1	Yes	GG	61	35	1		1
2	No	GG	43	27	1.552(0.939–2.563)	0.086	1.113(0.643–1.928)	0.702
3	Yes	AA+AG	203	76	0.681(0.452–1.027)	0.067	0.743(0.478–1.157)	0.189
4	No	AA+AG	157	63	0.862(0.569–1.306)	0.483	0.660(0.417–1.044)	0.076
	Tumor size
I	≤5 cm	GG	36	123	1		1
II	>5 cm	GG	70	24	2.887(1.566–5.320)	0.001	1.954(1.004–3.802)	0.049
III	≤5 cm	AA+AG	155	NA	0.850(0.460–1.569)	0.603	0.706(0.362–1.379)	0.308
IV	>5 cm	AA+AG	213	57	1.690(0.951–3.003)	0.074	1.280(0.683–2.399)	0.442
	Tumor number
a	Single	GG	81	40	1		1
b	Multiple	GG	25	24	1.793(1.045–3.078)	0.034	1.247(0.660–2.359)	0.497
c	Single	AA+AG	269	80	0.641(0.451–0.912)	0.013	0.655(0.447–0.962)	0.031
d	Multiple	AA+AG	99	65	0.969(0.650–1.446)	0.879	0.859(0.502–1.470)	0.579
	BCLC
A	A	GG	56	84	1		1
B	B	GG	14	27	1.527(0.684–3.409)	0.301	1.041(0.410–2.640)	0.933
C	C	GG	36	19	3.485(2.030–5.982)	<0.001	1.820(0.855–3.873)	0.12
D	A	AA+AG	223	101	0.686(0.432–1.090)	0.111	0.647(0.393–1.063)	0.085
E	B	AA+AG	61	73	1.311(0.773–2.221)	0.315	0.863(0.419–1.781)	0.691
F	C	AA+AG	84	27	2.228(1.371–3.619)	0.001	1.146(0.553–2.374)	0.714
	Portal vein tumor thrombus^ƕ
i	No	GG	77	42	1		1
ii	Yes	GG	28	14	4.187(2.513–6.975)	<0.001	2.223(1.093–4.522)	0.027
iii	No	AA+AG	310	80	0.758(0.524–1.097)	0.142	0.763(0.511–1.138)	0.185
iv	Yes	AA+AG	58	18	2.154(1.367–3.394)	0.001	1.148(0.598–2.203)	0.679
	Serum AFP^ƛ
AA	AFP ≤400 (ng/mL)	GG	47	35	1		1
BB	AFP >400 (ng/mL)	GG	50	27	0.924(0.550–1.551)	0.765	0.845(0.497–1.437)	0.534
CC	AFP ≤400 (ng/mL)	AA+AG	196	76	0.484(0.311–0.753)	0.001	0.562(0.358–0.882)	0.012
DD	AFP >400 (ng/mL)	AA+AG	147	73	0.676(0.431–1.059)	0.088	0.653(0.414–1.031)	0.067

Notes: ^¥Adjusted for AFP, BCLC, tumor number, tumor size, PVTT and radical resection. ^ƀInformation of radical resection was unavailable in 10 patients; ^ƕInformation of PVTT was unavailable in 1 patient; ^ƛInformation of AFP was unavailable in 34 patients.

Abbreviations: HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HR, hazard ratio; CI, confidence interval; MST, median survival time; ACBD4, Acyl-CoA binding domain containing 4.

Figure 7 Prognostic value of ACBD4-rs4986172 in Guangxi HBV-related HCC cohort. (A) Kaplan-Meier curve of AA, AG and GG; (B) Kaplan-Meier curve of AA+AG and GG; (C) Kaplan-Meier curve of AA and AG+GG.

Figure 8 Stratified survival analysis results of ACBD4-rs4986172 in Guangxi HBV-related HCC cohort.

Figure 9 Joint effect survival analysis of ACBD4-rs4986172 and clinical parameters in Guangxi HBV-related HCC patients. (A) Joint effect survival analysis of rs4986172 and serum AFP; (B) Joint effect survival analysis of rs4986172 and BCLC stage; (C) Joint effect survival analysis of rs4986172 and PVTT; (D) Joint effect survival analysis of rs4986172 and radical resection. (E) Joint effect survival analysis of rs4986172 and tumor size; (F) Joint effect survival analysis of rs4986172 and tumor number.

Note: The detailed information on grouping is shown in Table 5.

Figure 10 The nomogram of ACBD4-rs4986172 and clinical parameters in the Guangxi HBV-related HCC cohort. (A) AA, AG and GG genotypes in HBV-related HCC; (B) AA+AG and GG genotypes in HBV-related HCC.

Figure 11 Genome-wide co-expression network of ACBD4 gene in HBV-related HCC tumor tissues of patients in GSE14520 cohort.

Figure 12 Volcano plot of DEGs between low- and high-ACBD4 expression groups.

Figure 13 GSEA results between low- and high-ACBD4 expression groups by using c2 reference gene set. (A) nuclear receptor transcription pathway; (B) mTOR signaling up; (C) liver cancer recurrence down; (D) liver cancer poor survival down; (E) PPAR signaling pathway; (F) metabolism cytochrome P450; (G) oxidation by cytochrome P450; (H) bile acid and bile salt metabolism; (I) stress pathway; (J) fatty acid metabolism; (K) tumor invasiveness up; (L) metastasis up.

Figure 14 GSEA results between low- and high-ACBD4 expression groups by using c5 reference gene set. (A) fatty acid metabolic process; (B) polysaccharide catabolic process; (C) bile acid metabolic process; (D) alcohol catabolic process; (E) lipid accumulation in hepatocytes; (F) positive regulation of G protein coupled receptor signaling pathway; (G) fatty acid metabolic process; (H) lipoprotein metabolic process; (I) steroid metabolic process; (J) bile acid and bile salt transport; (K) drug metabolic process; (L) abnormality of hepatobiliary system physiology.

Figure 15 CMap result of ACBD4 in HCC. (A) Chemical construction of scopoletin; (B) Chemical construction of alfaxalone; (C) Chemical construction of bephenium hydroxynaphthoate; (D) Chemical construction of apramycin; (E) Chemical construction of 4,5-dianilinophthalimide; (F) Chemical construction of DL-thiorphan; (G) Chemical construction of aminohippuric acid; (H) Chemical construction of quinidine; (I) Summary table of CMap results.

Figure 16 Drug-gene interaction network of compounds identified by CMap.

Figure 17 Score of ACBD4 gene in HBV-HCC immune microenvironment.