T cell exhaustion markers in hepatitis C virus-related hepatocellular carcinoma: Expression patterns and prognostic significance in an Egyptian cohort

Table 1 Baseline demographic and clinical characteristics of study participants, mean ± SD/median (interquartile range)

Parameter	HCC (n = 50)	Cirrhosis (n = 50)	HCV (n = 50)	Controls (n = 50)	Test	P value
Age (years)	57.4 ± 8.2	55.9 ± 7.8	54.8 ± 8.0	54.2 ± 7.6	ANOVA	0.312
Sex (male/female)	34/16	32/18	31/19	30/20	χ2	0.894
ALT (U/L)	55.0 (48.0-63.0)	52.0 (46.0-61.0)	50.0 (44.0-58.0)	24.0 (20.0-28.0)	Kruskal-Wallis	< 0.001a
AST (U/L)	62.0 (54.0-70.0)	58.0 (50.0-66.0)	56.0 (49.0-62.0)	25.0 (21.0-29.0)	Kruskal-Wallis	< 0.001a
Albumin (g/dL)	3.2 ± 0.4	3.3 ± 0.4	3.5 ± 0.3	4.2 ± 0.3	ANOVA	< 0.001a
INR	1.42 ± 0.12	1.40 ± 0.11	1.35 ± 0.10	1.01 ± 0.05	ANOVA	< 0.001a

^aP < 0.001.

ANOVA adjusted for Child-Pugh class for hepatocellular carcinoma and cirrhosis groups to control for liver function severity. All participants were adults (age > 18 years), with age and sex matched across groups to control for demographic confounders. ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; INR: International normalized ratio; HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus.

Table 2 Immune checkpoint expression and T-cell profiles across study groups, mean ± SD/median (interquartile range)

Parameter	HCC (n = 50)	Cirrhosis (n = 50)	HCV (n = 50)	Control (n = 50)	8F/Kruskal-Wallis	P value
CD4/PD1	35.14 ± 0.43	20.87 ± 0.9	12.59 ± 0.84	12.0 ± 1.33	6700.7	< 0.001c
Post hoc7	1P < 0.001c, 2P < 0.001c, 3P = 0.001b, 4P < 0.001c, 5P < 0.001c, 6P < 0.001c
CD8/PD1	35.57 ± 0.58	23.89 ± 0.81	15.6 ± 0.84	15.0 ± 1.33	5854.15	< 0.001c
Post hoc7	1P < 0.001c, 2P < 0.001c, 3P = 0.007b, 4P < 0.001c, 5P < 0.001c, 6P < 0.001c
CD4/TIGIT	13.77 ± 0.37	8.76 ± 0.46	6.18 ± 0.79	5.6 ± 1.33	1011.91	< 0.001c
Post hoc7	1P < 0.001c, 2P > 0.999, 3P = 0.003b, 4P < 0.001c, 5P < 0.001c, 6P < 0.001c
CD8/TIGIT	17.66 ± 0.38	6.56 ± 0.46	7.6 ± 0.77	7.0 ± 1.32	2088.86	< 0.001c
Post hoc7	1P < 0.001c, 2P < 0.001c, 3P =0.002b, 4P < 0.001c, 5P < 0.001c, 6P =0.043a
CD4/TIM3	14.7 ± 0.34	6.34 ± 0.46	3.6 ± 0.77	3.0 ± 1.33	2169.06	< 0.001c
Post hoc7	1P < 0.001c, 2P > 0.999, 3P = 0.002b, 4P < 0.001c, 5P < 0.001c, 6P < 0.001c
CD8/TIM3	17.14 ± 0.44	8.56 ± 0.46	4.1 ± 0.77	3.5 ± 1.33	2868.84	< 0.001c
Post hoc7	1P < 0.001c, 2P < 00.001c, 3P = 0.002b, 4P < 0.001c, 5P < 0.001c, 6P < 0.001c
CD3	66.12 ± 0.68	67.84 ± 0.83	71.25 ± 0.88	71.15 ± 1.37	339.21	< 0.001c
Post hoc7	1P < 0.001c, 2P < 0.001c, 3P = 0.945, 4P < 0.001c, 5P < 0.001c, 6P < 0.001c
CD4	39.06 ± 0.75	43.95 ± 0.91	47.25 ± 0.88	47.15 ± 1.37	733.67	< 0.001c
Post hoc7	1P < 0.001c, 2P < 0.001c, 3P = 0.95, 4P < 0.001c, 5P < 0.001c, 6P < 0.001c
CD8	21.06 ± 0.69	23.98 ± 0.87	24.25 ± 0.88	24.15 ± 1.37	122.07	< 0.001c
Post hoc7	1P < 0.001c, 2P = 0.05, 3P = 0.947, 4P < 0.001c, 5P < 0.001c, 6P = 0.829
CD4/CD8	1.86 ± 0.03	1.83 ± 0.03	1.95 ± 0.03	1.96 ± 0.05	140.79	< 0.001c
Post hoc7	1P = 0.023a, 2P < 0.001c, 3P = 0.852, 4P < 0.001c, 5P < 0.001c, 6P < 0.001c
AFP (ng/mL)	13210 (11008.3-14956.8)	4 (2-6)	6.85 (6.08-7.5)	4.3 (3.08-5.53)	144.208	< 0.001c
Post hoc7	1P < 0.001c, 2P < 0.001c, 3P < 0.001c, 4P < 0.001c, 5P < 0.001c, 6P = 0.883

¹P: Hepatocellular carcinoma vs cirrhosis.

²P: Hepatocellular carcinoma vs hepatitis C virus.

³P: Hepatitis C virus vs control.

⁴P: Hepatocellular carcinoma vs control.

⁵P: Cirrhosis vs control.

⁶P: Cirrhosis vs hepatitis C virus.

⁷Post hoc: Tukey’s test for ANOVA (CD4/programmed death 1, CD8/ programmed death 1, CD4/T cell immunoglobulin and ITIM domain, CD8/T cell immunoglobulin and ITIM domain, CD4/T cell immunoglobulin and mucin domain-containing protein 3, CD8/T cell immunoglobulin and mucin domain-containing protein 3, CD3, CD4, CD8, CD4/CD8).

⁸F: One-way ANOVA test; ANOVA adjusted for Child-Pugh class and age using ANCOVA for hepatocellular carcinoma and cirrhosis groups to control for liver function and demographic confounders; Kruskal-Wallis for alpha-fetoprotein used Dunn’s post hoc test.

^aP < 0.05.

^bP < 0.01.

^cP < 0.001.

PD-1: Programmed death 1; TIGIT: T-cell immunoglobulin and ITIM domain; TIM-3: T cell immunoglobulin and mucin domain-containing protein 3; AFP: Alpha-fetoprotein; HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus.

Table 3 Liver function scores in hepatocellular carcinoma and cirrhosis groups, mean ± SD/n (%)

Parameter	HCC (n = 50)	Cirrhosis (n = 50)	t/χ2	P value
MELD	2.5 ± 1.0	2.56 ± 1.09	-0.287	0.774
Child-Pugh class			1.795	0.18
A	9 (18)	13 (26)
B	9 (18)	12 (24)
C	32 (64)	25 (50)

χ² for trend test for Child-Pugh class. t-test for Model for End-Stage Liver Disease adjusted for age to control for demographic confounders. MELD: Model for End-Stage Liver Disease; HCC: Hepatocellular carcinoma.

Table 4 Tumor characteristics and outcomes in hepatocellular carcinoma patients, n (%)

Parameter	n = 50
Fate
Non-survivor	17 (34)
Survivor	33 (66)
Lesion size
< 3 cm	18 (36)
> 3 cm	32 (64)
BCLC stage
A	9 (18)
B	1 (2)
C	8 (16)
D	32 (64)

BCLC: Barcelona Clinic Liver Cancer.

Table 5 Correlations with disease severity in hepatocellular carcinoma patients

Parameter	MELD		Child-Pugh		AFP		BCLC stage
	r1	P value	r1	P value	r1	P value	r1	P value
CD4/PD-1 (%)	-0.730	< 0.001a	0.855	< 0.001a	0.750	< 0.001a	0.855	< 0.001a
CD8/PD-1 (%)	-0.738	< 0.001a	0.855	< 0.001a	0.752	< 0.001a	0.855	< 0.001a
CD4/TIGIT (%)	-0.764	< 0.001a	0.857	< 0.001a	0.758	< 0.001a	0.856	< 0.001a
CD8/TIGIT (%)	-0.764	< 0.001a	0.856	< 0.001a	0.756	< 0.001a	0.856	< 0.001a
CD4/TIM-3 (%)	-0.764	< 0.001a	0.858	< 0.001a	0.759	< 0.001a	0.858	< 0.001a
CD8/TIM-3 (%)	-0.764	< 0.001a	0.835	< 0.001a	0.745	< 0.001a	0.835	< 0.001a
CD3 (%)	-0.765	< 0.001a	0.837	< 0.001a	0.748	< 0.001a	0.837	< 0.001a
CD4 (%)	-0.756	< 0.001a	0.830	< 0.001a	0.746	< 0.001a	0.830	< 0.001a
CD8 (%)	-0.751	< 0.001a	0.835	< 0.001a	0.749	< 0.001a	0.835	< 0.001a
CD4/CD8 ratio	0.745	< 0.001a	-0.844	< 0.001a	-0.749	< 0.001a	-0.840	< 0.001a

¹r: Pearson correlation coefficient for normally distributed variables (CD4/programmed death 1, CD8/ programmed death 1, CD4/T cell immunoglobulin and ITIM domain, CD8/T cell immunoglobulin and ITIM domain, CD4/T cell immunoglobulin and mucin domain-containing protein 3, CD8/T cell immunoglobulin and mucin domain-containing protein 3, CD3, CD4, CD8, CD4/CD8 ratio, Model for End-Stage Liver Disease); Spearman correlation coefficient for non-normally distributed variables (alpha-fetoprotein, Child-Pugh class, Barcelona Clinic Liver Cancer stage). Correlations adjusted for age and sex using partial correlation to control for demographic confounders.

^aP < 0.001.

Etiology controlled by study design (hepatitis C virus genotype 4 for hepatocellular carcinoma patients, confirmed by quantitative hepatitis C virus polymerase chain reaction targeting 5’ untranslated region). PD-1: Programmed death 1; TIGIT: T-cell immunoglobulin and ITIM domain; TIM-3: T cell immunoglobulin and mucin domain-containing protein 3; MELD: Model for End-Stage Liver Disease; AFP: Alpha-fetoprotein; BCLC: Barcelona Clinic Liver Cancer.

Table 6 Correlations with disease severity in cirrhosis patients

Parameter	MELD		Child-Pugh		AFP
	r1	P value	r1	P value	r1	P value
CD4/PD-1 (%)	0.926	< 0.001a	0.557	< 0.001a	0.142	0.324
CD8/PD-1 (%)	0.910	< 0.001a	0.566	< 0.001a	0.150	0.296
CD4/TIGIT (%)	0.919	< 0.001a	0.565	< 0.001a	0.138	0.339
CD8/TIGIT (%)	0.927	< 0.001a	0.565	< 0.001a	0.142	0.324
CD4/TIM-3 (%)	0.917	< 0.001a	0.565	< 0.001a	0.140	0.331
CD8/TIM-3 (%)	0.883	< 0.001a	0.565	< 0.001a	0.144	0.315
CD3 (%)	0.932	< 0.001a	0.563	< 0.001a	0.146	0.307
CD4 (%)	0.916	< 0.001a	0.562	< 0.001a	0.141	0.329
CD8 (%)	0.914	< 0.001a	0.562	< 0.001a	0.142	0.324
CD4/CD8 ratio	-0.901	< 0.001a	-0.562	< 0.001a	-0.145	0.309

¹r: Pearson correlation coefficient for normally distributed variables (CD4/programmed death 1, CD8/ programmed death 1, CD4/T cell immunoglobulin and ITIM domain, CD8/T cell immunoglobulin and ITIM domain, CD4/T cell immunoglobulin and mucin domain-containing protein 3, CD8/T cell immunoglobulin and mucin domain-containing protein 3, CD3, CD4, CD8, CD4/CD8 ratio, Model for End-Stage Liver Disease); Spearman correlation coefficient for non-normally distributed variables (alpha-fetoprotein, Child-Pugh class). Correlations adjusted for age and sex using partial correlation to control for demographic confounders.

^aP < 0.001.

Etiology controlled by study design (hepatitis C virus genotype 4 for hepatocellular carcinoma patients, confirmed by quantitative hepatitis C virus polymerase chain reaction targeting 5’ untranslated region). PD-1: Programmed death 1; TIGIT: T-cell immunoglobulin and ITIM domain; TIM-3: T cell immunoglobulin and mucin domain-containing protein 3; MELD: Model for End-Stage Liver Disease; AFP: Alpha-fetoprotein.

Table 7 Immune profiles and mortality in hepatocellular carcinoma patients, mean ± SD/median (interquartile range)

Parameter	Survivors (n = 33)	Non-survivors (n = 17)	t/Z1	P value
CD4/PD-1 (%)	34.86 ± 0.25	35.63 ± 0.21	-10.52	< 0.001a
CD8/PD-1 (%)	36.20 ± 0.27	37.28 ± 0.26	-13.63	< 0.001a
CD4/TIGIT (%)	13.54 ± 0.21	14.21 ± 0.17	-11.32	< 0.001a
CD8/TIGIT (%)	17.43 ± 0.21	18.11 ± 0.17	-11.56	< 0.001a
CD4/TIM-3 (%)	14.50 ± 0.17	15.11 ± 0.17	-12.12	< 0.001a
CD8/TIM-3 (%)	16.86 ± 0.17	17.67 ± 0.27	-12.66	< 0.001a
CD3 (%)	65.72 ± 0.41	66.90 ± 0.32	-10.46	< 0.001a
CD4 (%)	38.60 ± 0.43	39.94 ± 0.36	-10.95	< 0.001a
CD8 (%)	20.64 ± 0.36	21.88 ± 0.35	-11.65	< 0.001a
CD4/CD8 ratio	1.87 ± 0.01	1.83 ± 0.01	11.82	< 0.001a
AFP (ng/mL)	12004 (10341-13654.5)	14030 (13672.5-14406)	-3.85	< 0.001a

¹t: Independent samples t-test, adjusted for age and Child-Pugh class using ANCOVA to control for demographic and liver function confounders. Z: Mann-Whitney U test for alpha-fetoprotein.

^aP < 0.001.

Etiology controlled by study design (hepatitis C virus genotype 4 for hepatocellular carcinoma patients, confirmed by quantitative hepatitis C virus polymerase chain reaction targeting 5’ untranslated region). PD-1: Programmed death 1; TIGIT: T-cell immunoglobulin and ITIM domain; TIM-3: T cell immunoglobulin and mucin domain-containing protein 3; AFP: Alpha-fetoprotein.

Table 8 Prognostic modeling and receiver operating characteristic curve performance for hepatocellular carcinoma mortality

Variable	Random forest AUC (95%CI)	Logistic regression OR (95%CI)	P value	ROC AUC (95%CI)	Cut-off	Sensitivity (%)	Specificity (%)
CD4/PD-1 (%)	0.92 (0.85-0.98)	2.3 (1.4-3.8)	< 0.001a	0.92 (0.85-0.98)	> 35%	88	85
CD8/TIM3 (%)	0.90 (0.83-0.97)	2.5 (1.5-4.2)	< 0.001a	0.90 (0.83-0.97)	> 17%	85	82
AFP (ng/mL)		3.2 (1.8-5.7)	< 0.001a	0.89 (0.81-0.96)	> 13000	90	80
AFP + CD8/TIM3	0.95 (0.90-0.99)		< 0.001a	0.95 (0.90-0.99)	> 13000, > 17%	92	90
BCLC stage (D vs A-C)		3.8 (2.0-7.2)	< 0.001a
Clustering: High-expression		50% mortality (n = 30)	< 0.001a		Elevated (PD-1, TIGIT, TIM-3)
Clustering: Low-expression		10% mortality (n = 20)	< 0.001a		Low (PD-1, TIGIT, TIM-3)

^aP < 0.001.

Random forest and logistic regression analyses, with receiver operating characteristic cut-offs determined by Youden’s index. Clustering based on K-means stratification using programmed death 1, T cell immunoglobulin and ITIM domain, and T cell immunoglobulin and mucin domain-containing protein 3 expression levels (standardized z-scores). PD-1: Programmed death 1; TIGIT: T-cell immunoglobulin and ITIM domain; TIM-3: T cell immunoglobulin and mucin domain-containing protein 3; AFP: Alpha-fetoprotein; BCLC: Barcelona Clinic Liver Cancer; AUC: Area under the curve; OR: Odds ratio; CI: Confidence interval; ROC: Receiver operating characteristic.

Table 9 Cox regression analysis for hepatocellular carcinoma mortality

Variable	HR (95%CI)	P value
CD8/TIM3 (high)	2.8 (1.5-5.2)	0.001a
BCLC stage D	3.1 (1.7-5.6)	< 0.001b
Age	1.2 (0.9-1.6)	0.15
MELD score	1.1 (0.8-1.5)	0.22

^aP < 0.01.

^bP < 0.001.

IM-3: T cell immunoglobulin and mucin domain-containing protein 3; BCLC: Barcelona Clinic Liver Cancer; MELD: Model for End-Stage Liver Disease; HR: Hazard ratio; CI: Confidence interval.

Table 10 Mediation analysis of CD4/programmed death 1 in alpha-fetoprotein-mortality relationship

Effect	1β (95%CI)	P value
Direct effect (AFP to mortality)	0.30 (0.15-0.45)	< 0.001a
Indirect effect (AFP to CD4/PD-1 to mortality)	0.45 (0.25-0.65)	< 0.001a
Total effect	0.75 (0.55-0.95)	< 0.001a

¹β: Standardized coefficient.

^aP < 0.001.

AFP: Alpha-fetoprotein; PD-1: Programmed death 1; CI: Confidence interval.

Table 11 Immune profiles by Barcelona Clinic Liver Cancer Stage in hepatocellular carcinoma patients, mean ± SD/median (interquartile range)/n (%)

Parameter	Early stage (BCLC A/B, n = 10)	Advanced stage (BCLC C/D, n = 40)	t/Z/χ2	P value
CD4/PD-1 (%)	33.50 ± 0.50	36.00 ± 0.40	t = -15.00	< 0.001b
CD8/TIM-3 (%)	15.50 ± 0.50	17.80 ± 0.40	t =-14.50	< 0.001b
AFP (ng/mL)	10000 (8000-12000)	14000 (12000-15000)	Z =-3.50	< 0.001b
Mortality	1 (10)	16 (40)	χ2 = 4.20	0.040a

^aP < 0.05.

^bP < 0.001.

Etiology controlled by study design (hepatitis C virus-positive hepatocellular carcinoma patients only). PD-1: Programmed death 1; TIM-3: T cell immunoglobulin and mucin domain-containing protein 3; AFP: Alpha-fetoprotein; BCLC: Barcelona Clinic Liver Cancer.

Table 12 Univariate and multivariate Cox proportional hazards regression for overall survival in hepatocellular carcinoma patients

Variable	Univariate HR (95%CI)	P value	Multivariate HR (95%CI)	P value
CD4/PD-1 (%)	2.5 (1.6-3.9)	< 0.001c	2.1 (1.3-3.4)	0.002b
CD8/PD-1 (%)	2.3 (1.5-3.5)	< 0.001c
CD4/TIGIT (%)	1.8 (1.1-2.9)	0.015a
CD8/TIGIT (%)	1.9 (1.2-3.0)	0.008b
CD4/TIM3 (%)	1.7 (1.0-2.8)	0.045a
CD8/TIM3 (%)	2.7 (1.7-4.3)	< 0.001c	2.4 (1.4-4.1)	0.001b
CD3 (%)	1.5 (0.9-2.5)	0.12
CD4 (%)	1.4 (0.8-2.4)	0.23
CD8 (%)	1.6 (0.9-2.8)	0.09
CD4/CD8 ratio	0.4 (0.2-0.7)	0.002b
AFP (ng/mL)	3.0 (1.9-4.7)	< 0.001c	2.6 (1.5-4.5)	< 0.001c
MELD score	1.1 (0.8-1.5)	0.53
Child-Pugh class (C vs A/B)	1.8 (1.0-3.2)	0.048a
BCLC stage (D vs A-C)	3.5 (2.2-5.6)	< 0.001c	3.0 (1.7-5.3)	< 0.001c

^aP < 0.05.

^bP < 0.01.

^cP < 0.001.

Multivariate model adjusted for all univariate variables (CD4/programmed death 1, CD8/ programmed death 1, CD4/T cell immunoglobulin and ITIM domain, CD8/T cell immunoglobulin and ITIM domain, CD4/T cell immunoglobulin and mucin domain-containing protein 3, CD8/T cell immunoglobulin and mucin domain-containing protein 3, CD3, CD4, CD8, CD4/CD8 ratio, alpha-fetoprotein, Model for End-Stage Liver Disease score, Child-Pugh class, Barcelona Clinic Liver Cancer stage). Child-Pugh class was not retained due to non-significant independent contribution. Etiology controlled by study design (hepatitis C virus-positive hepatocellular carcinoma patients only). PD-1: Programmed death 1; TIGIT: T-cell immunoglobulin and ITIM domain; TIM-3: T cell immunoglobulin and mucin domain-containing protein 3; AFP: Alpha-fetoprotein; MELD: Model for End-Stage Liver Disease; HR: Hazard ratio; CI: Confidence interval.