Endotrophin, a pro-peptide of Type VI collagen, is a biomarker of survival in cirrhotic patients with hepatocellular carcinoma

Figures & data

Table 1. Patient demographics of the studied cohorts.

	Healthy controls	Noncirrhotic HBV	Cirrhosis	Cirrhosis with HCC	p-value four disease groups	p-value cirrhosis vs HCC
n	55	84	86	84
Age (years), mean ± SD	52.2 (7.7)	58.3 (8.6)	58.8 (10.0)	62.4(11.4)	< 0.0001	0.05
Gender (male), n (%)	51 (91.1)	73 (86.9)	75 (87.2)	74 (88.1)	0.89	0.86
BMI, mean ± SD	26.2 (2.9)	25.5 (4.0)	29.4 (5.6)	28.5 (6.5)	< 0.0001	0.12
Etiology					< 0.0001	0.26
HCV, n (%)	NA	0 (0)	43 (50.0)	40 (47.6)
HBV, n (%)	NA	84 (100)	23 (26.7)	13 (15.5)
EtOH, n (%)	NA	0 (0)	7 (8.1)	11 (13.1)
NASH, n (%)	NA	0 (0)	10 (11.6)	16 (19.0)
Other, n (%)	NA	0 (0)	3 (3.5)	0 (0)
Ethnicity					< 0.0001	< 0.0001
Caucasian, n (%)	44 (78.6)	9 (0.7)	49 (57.0)	54 (65.1)
Chinese, n (%)	7 (12.5)	55 (65.5)	12 (14.0)	11 (13.3)
Middle Eastern, n (%)	2 (3.6)	9 (10.7)	20 (23.3)	10 (12.0)
Indian, n (%)	3 (5.4)	7 (8.3)	3 (3.5)	3 (3.6)
African, n (%)	0 (0)	2 (2.4)	1 (1.2)	3 (3.6)
Polynesian, n (%)	0 (0)	2 (2.4)	1 (1.2)	2 (2.4)
Diabetics, n (y/n)	0/0	74/10	56/29	50/34	0.0001	0.40
Bilirubin, mean ± SD	11.4 (5.0)	13.4 (8.2)	21.2 (14.5)	21.8 (23.5)	< 0.0001	0.16
Albumin, mean ± SD	43.5 (2.3)	43.7 (2.9)	40.5 (5.3)	36.7 (6.6)	< 0.0001	0.0001
ALT, mean ± SD	31.2 (15.7)	40.7 (36.45)	65.3 (61.5)	85.7 (87.7)	< 0.0001	0.08
AST, mean ± SD	28.6 (7.0)	39.9 (12.8)	75.0 (58.2)	109.8 (98.8)	< 0.0001	0.001
PLT, mean ± SD	239.2 (56.6)	227.5 (50.9)	131.6 (66.0)	126.2 (64.1)	< 0.0001	0.61
PRO-C6 (ng/ml)	4.7 (1.4)	5.3 (2.4)	9.2 (11.4)	10.6 (5.0)	< 0.0001	0.0007
C6M (ng/ml)	6.6 (1.1)	7.7 (3.3)	8.4 (4.8)	9.6 (5.4)	0.001	0.046
AFP (IU/ml), mean ± SD	NA	2.6 (1.0)	6.5 (12.3)	2965.7 (13361.4)	< 0.0001	< 0.0001
Hyperlipidemia (Y/N)	NA	NA	NA	18/66	NA	NA
BCLC staging, 0/A/B/C/D	NA	NA	NA	4/31/32/13/3	NA	NA
Child-Pugh score, A/B/C/n/a	NA	NA	78/6/2/0	54/14/7/9	NA	0.0002
Size of largest lesion, mean ± SD	NA	NA	NA	4.6 (4.0)	NA	NA
Number of lesions, mean ± SD	NA	NA	NA	1.9 (1.5)	NA	NA
Metastasis, Y/N	NA	NA	NA	5/78	NA	NA
Portal vein invasion, Y/N	NA	NA	NA	13/70	NA	NA

Bold p-values indicate statistical significance.

Results are expressed as mean (standard deviation) or frequency (percentage). p-values were calculated using Kruskal–Wallis test with Dunn’s multiple comparisons or a chi-square test.

AFP: Alpha-fetoprotein; ALT: Alanine transaminase; AST: Aspartate transaminase; BCLC: Barcelona Clinic Liver Cancer; EtOH: Alcoholic liver disease; HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus; NA: Not applicable; NASH: Nonalcoholic steatohepatitis; PLT: Platelet count.

Figure 1. Levels of biomarkers in healthy controls (n = 86), hepatitis B virus noncirrhotics (n = 86), cirrhotics (n = 86) and hepatocellular carcinoma (n = 86) patients.

(A) Plasma levels of formation of collagen Type VI (PRO-C6, endotrophin); (B) plasma levels of degradation fragments of collagen Type VI (C6M); (C) alpha-fetoprotein (AFP) levels; (D) PRO-C6 levels in HCC patients divided into AFP negative (AFP<20) or AFP positive (AFP≥20). Statistical difference was calculated using a Mann–Whitney t-test. Statistical differences were analyzed using a Kruskal–Wallis test adjusted for Dunn’s multiple comparisons test. Data are presented as Tukey boxplots. Significance levels: *p < 0.05; **p = <0.01; ***p < 0.001; ****p < 0.0001.

AFP: Alphafeto-protein; HCC: Hepatocellular carcinoma.

Table 2. Discriminative performance of PRO-C6 and AFP biomarkers for the diagnosis of hepatocellular carcinoma in cirrhotic patients.

HCC vs cirrhotics in all cirrhotic patients	Cut-off value (ng/ml)	Sensitivity	Specificity	AUROC	p-value
PRO-C6 (n = 84 HCC; n = 86 cirrhotics)	6.31	79.8	50.0	0.65	0.0004
AFP (n = 84 HCC; n = 86 cirrhotics)	6.00	65.1	80.2	0.78	< 0.0001
HCC vs cirrhotics in AFP negative or positive cirrhotic patients (20 IU/ml ≥AFP<20 IU/ml)
PRO-C6 in AFP negative (n = 48 HCC; n = 80 cirrhotics)	7.49	66.7	73.2	0.69	< 0.0001
PRO-C6 in AFP positive (n = 36 HCC; n = 6 cirrhotics)	13.1	44.4	83.3	0.50	0.99

Bold p-values indicate statistical significance.

AFP: Alphafeto-protein; HCC: Hepatocellular carcinoma.

Figure 2. Kaplan–Meier curves for the univariate analysis of each marker in relation to overall survival and progression-free survival.

The median is used as cut-off (low vs high) for PRO-C6 (low n = 42; high n = 41) and C6M (low n = 21; high n = 24), and 20 IU/ml cut-off for AFP (negative n = 49/positive n = 37). (A, C & E) represents relation to overall survival and (B, D & F) represents relation to progression-free survival. Significance levels: ns = nonsignificant, *p < 0.05; ***p < 0.001; ****p < 0.0001.

AFP: Alphafeto-protein.

Table 3. Association between biomarker levels, clinical covariates and outcome for hepatocellular carcinoma patients.

Variable		Progression-free survival			Overall survival
Univariate analysis		HR	95% CI	p-value	HR	95% CI	p-value
Age	Continuous	1.01	0.99–1.03	0.332	1.01	0.99–1.04	0.280
Gender (male)	Continuous	0.78	0.36–1.71	0.533	1.04	0.41–2.65	0.932
BMI	Continuous	1.01	0.97–1.05	0.672	0.99	0.94–1.04	0.634
Child-Pugh score	B/C vs A	2.39	1.37–4.19	0.002	5.06	2.58–9.93	< 0.0001
Number of lesions	Continuous	1.18	1.08–1.29	0.0003	1.41	1.22–1.63	< 0.0001
PRO-C6	High vs low (Median)	1.91	1.16–3.15	0.01	2.44	1.35–4.42	0.003
C6M	High vs low (Median)	1.05	0.58–1.92	0.87	2.37	1.12–5.03	0.02
AFP	High vs low (Median)	1.62	1.00–2.64	0.05	2.4	1.28–4.50	0.006
PRO-C6	High vs low (Q4 vs Q1-Q3)	2.32	1.22–4.42	0.011	3.18	1.49–6.82	0.003
C6M	High vs low (Q4 vs Q1-Q3)	1.51	0.71–3.20	0.28	2.89	1.12–7.46	0.03
AFP	High vs low (Q4 vs Q1-Q3)	2.77	1.40–2.51	0.004	3.4	1.50–7.75	0.004
AFP	High vs low (≥20 vs <20 IU/ml)	1.87	1.12–3.14	0.017	3.00	1.52–5.90	0.0015
Multivariate analysis		HR	95% CI	p-value	HR	95% CI	p-value
Adjusted for age, sex and BMI
PRO-C6	High vs low (Q4 vs Q1-Q3)	1.63	0.89–3.00	0.12	2.4	1.21–4.7	0.01
C6M	High vs low (Q4 vs Q1-Q3)	1.40	0.66–3.00	0.38	3.3	1.34–8.10	0.01
AFP	High vs low (≥20 vs <20 IU/ml)	1.58	0.97–2.60	0.07	2.51	1.36–4.64	0.003
High PRO-C6 and AFP (14.5%)	High PRO-C6 and high AFP vs low/high or high/low PRO-C6/AFP	1.94	0.78–4.83	0.16	6.94	1.19–40.86	0.03
High C6M and AFP (12.5%)	High C6M and high AFP vs low/high or high/low C6M/AFP	2.56	0.74–8.93	0.14	4.82	1.42–16.38	0.01
Adjusted for Child-Pugh score and number of lesions
PRO-C6	High vs low (Q4 vs Q1-Q3)	1.38	0.78–2.45	0.27	2.17	1.06–4.44	0.03
C6M	High vs low (Q4 vs Q1-Q3)	1.18	0.56–2.51	0.66	2.90	1.24–6.72	0.01
AFP	High vs low (≥20 vs <20 IU/ml)	1.81	1.06–3.09	0.03	3.68	1.80–7.51	0.0003
High PRO-C6 and AFP (14.5%)	High PRO-C6 (Q4) and high AFP vs low/high or high/low PRO-C6/AFP	1.99	0.96–4.11	0.07	5.67	2.30–13.94	0.0002
High C6M and AFP (12.5%)	High C6M (Q4) and high AFP vs low/high or high/low C6M/AFP	3.13	1.16–8.21	0.02	5.55	1.65–18.61	0.006
Adjusted for AFP
PRO-C6	High vs low (Q4 vs Q1-Q3)	1.78	1.05–3.02	0.03	2.40	1.29–4.47	0.006
C6M	High vs low (Q4 vs Q1-Q3)	1.48	0.76–2.90	0.25	2.22	1.01–4.90	0.05
Adjusted for Age, BMI, sex, Child-Pugh score, number of lesions.
High PRO-C6 (25.3%)	Q4	1.16	0.58–2.31	0.67	1.55	0.67–3.55	0.31
High C6M (25%)	Q4	1.25	0.57–2.73	0.57	3.38	1.32–8.68	0.01
High AFP (42.4/100%)	≥20 IU/ml	1.55	0.87–2.77	0.14	3.74	1.76–7.97	0.0006
High PRO-C6 and AFP (14.5/100%)	High (Q4) PRO-C6 and high AFP vs low/low, low/high or high/low PRO-C6/AFP	1.51	0.55–4.11	0.42	14.40	2.53–188.14	0.0001
High C6M and AFP (12.5/100%)	High (Q4) C6M and high AFP vs low/low, low/high or high/low C6M/AFP	3.90	1.00–15.37	0.0496	9.41	2.42–36.70	0.0012

Bold p-values indicate statistically significant HR.

Hazard ratios were calculated by univariate and multivariate Cox proportional-hazards analysis. By univariate analysis, Pro-C6 and C6M were analyzed divided into above or below the median, or quartiles with the lower levels (Q1-Q3) used as a reference to calculate the HR for patients in the upper quartile (Q4). The covariates were analyzed on a continuous scale and Child-Pugh score and AFP were further analyzed on a binominal scale. By multivariable analysis, PRO-C6, C6M and AFP were adjusted as indicated in the text.

AFP: Alpha-fetoprotein; HR: Hazard ratio.

Figure 3. Overall survival analysis.

(A) Hazard ratio for the prediction of overall survival by each indicated multivariate model, all corrected for sex, age, BMI, Child-Pugh and number of lesions. High PRO-C6 (Q4) and high C6M (Q4); high AFP (≥20 IU/ml) (HRs from Table 3). (B) Kaplan–Meier curves for the multivariate analysis of overall survival for high PRO-C6 (Q4) + high AFP (≥20 IU/ml). The 95% CI is seen for each group in brackets. Significance levels: ns = nonsignificant, *p < 0.05; ***p < 0.001; ****p < 0.0001.

AFP: Alpha-fetoprotein; HR: Hazard ratio.