T-Lymphocyte Gene-Regulated CCL5 and Its Association with Extrahepatic Metastasis in Hepatocellular Carcinoma

Figures & data

Table 1 Patient Characteristics in Primary and Validation Cohort

Characteristic	Primary Cohort (N=315)	Validation Cohort (N=65)	p-value
Gender			0.863
Male	249 (79.0%)	52 (80.0%)
Female	66 (21.0%)	13 (20.0%)
BMI	24.4 ± 3.1	25.1 ± 3.2	0.073
Smoke			0.072
No	193 (61.3%)	32 (49.2%)
Yes	122 (38.7%)	35 (50.8%)
Alcohol consumption			0.110
No	274 (78.4%)	45 (69.2%)
YES	68 (21.6%)	20 (30.8%)
AGE	58.8 ± 10.4	59.5 ± 9.7	0.640
ECOG			0.168
0	306 (97.1%)	65 (100%)
1	9(2.9%)	0(0.0%)
Tumor size	2.8 ± 1.3	2.7 ± 1.2	0.850
Tumor number	1.7 ± 1.4	1.8 ± 1.5	0.520
Hepatitis			0.288
None	34 (10.8%)	13 (20.0%)	0.098
HBV	235 (74.6%)	45 (69.2%)
HCV	36 (11.4%)	4 (6.2%)
HBV&HCV	4 (1.3%)	0 (0.0%)
Others	6 (1.9%)	3 (4.6%)
Comorbidities			0.154
None	92 (29.2%)	21 (32.3%)
Hypertension	37 (11.7%)	13 (20.0%)
Diabetes	59 (18.7%)	13 (20.0%)
Others	127 (10.3%)	18 (27.7%)
AFP	255.2 ± 1620.7	753.3 ± 2968.4	0.538
WBC	4.7 ± 1.7	4.8 ± 1.9	0.384
Lymphocytes	0.3 ± 0.1	0.3 ± 0.1	0.188
Neutrophils	0.6 ± 0.1	0.6 ± 0.1	0.177
HB	134.5 ± 18.0	132.9 ± 17.3	0.840
Albumin	40.2 ± 4.3	39.8 ± 4.2	0.513
PLTs	125.7 ± 57.7	122.4 ± 49.7	0.854
Prothrombin	14.3 ± 2.2	14.0 ± 1.0	0.790
BCLC Staging			0.455
A	264 (83.8%)	52 (80.0%)
B	51 (16.2%)	13 (20.0%)
Child-Pugh Grade			0.060
A	312 (99.0%)	63 (96.9%)
B	1 (0.3%)	2 (3.1%)
C	2 (0.6%)	0 (0.0%)

Note: Values are mean±standard deviation or number (%).

Abbreviations: PLT, platelets; AFP, alpha fetoprotein; WBC, white blood cells; HB, hemoglobin.

Figure 1 Flowchart of data sampling.

Table 2 Univariate and Multivariate Analyses of Prognostic Factors Associated with EHM

Characteristic	Univariate Analysis			Multivariate Analysis
	HR	95% CI	p value	HR	95% CI	p value
WBC	1.0	0.9–1.1	0.856	–	–	–
Lymphocytes	1.3	0.3–5.8	0.715	–	–	–
Neutrophils	0.7	0.2–2.7	0.646	–	–	–
B Cells	1.0	1.0–1.0	0.119	–	–	–
CD4^+
CD4^low (≤25)	1.0
CD4^high (>25)	1.7	1.2–2.3	0.002*	1.4693	1.0390–2.0779	0.0295*
CD28^+	1.0	1.0–1.0	0.165	–	–	–
CD28^−	1.0	1.0–1.0	0.336	–	–	–
CD3^+	1.0	1.0–1.0	0.258	–	–	–
CD4^+/CD8^+	1.0	0.9–1.2	0.908	–	–	–
CD8^+
CD8^low (≤30)	1.0
CD8^high (>30)	0.6	0.4–0.8	<0.001**	0.6385	0.4612–0.8840	0.0069*
NK	1.0	1.0–1.0	0.095	–	–	–
NKT	1.0	0.9–1.0	0.179	–	–	–
CD45RA^+	1.0	1.0–1.0	0.851	–	–	–
CD45RO^+	1.0	1.0–1.0	0.991	–	–	–
Treg cells
Treg (≤2.9)	1.0
Treg (>2.9)	0.6	0.4–0.8	0.004*	0.7926	0.5445–1.1536	0.2249

Notes: The bold value indicated identifying significant variables through univariate or multivariate Cox regression analysis. *P < 0.05, **P < 0.001.

Figure 2 Kaplan-Meier analyses for MFS according to CD4⁺ cell ratio (A), CD8⁺ cell ratio (B), and combined (CD4⁺/CD8⁺) cell ratio (C) in the primary cohort.

Figure 3 CCL5 modulated the intratumoral infiltration level of CD8⁺ T cells. (A) The association between CCL5 expression and immune infiltration levels of multiple CD8⁺ T cell types estimated using the TCGA database. (B) Evaluation of immune cell infiltration in the TME of HCC patients according to CCL5 gene expression using CIBERSORT. *P < 0.05, **P < 0.001. (C) Violin plot showing the CCL5 expression levels in the 12 lymphocyte subtypes in tumors of HCC patients. (D) Quantitative analysis of the ratio of CD3, CD8, and CD68 positive cells in the tumor region. **P < 0.001. (E) Immunohistochemistry done to detect the expression pattern of CD34, CD3, CD8, and CD68 positive cells in HCC patients with different serum CCL5 levels (n = 3). Scale bar: 100μm.