PD-L1 expression as a predictive biomarker for cytokine-induced killer cell immunotherapy in patients with hepatocellular carcinoma

Figures & data

Figure 1. Kaplan–Meier curves of differences in survival among patients with hepatocellular carcinoma (HCC) who received adjuvant CIK cell treatment (CIK treatment group) or hepatectomy alone (surgery alone group). (A) Overall survival (OS) and (B) recurrence-free survival (RFS) curves. Significantly improved OS and PFS were observed in the CIK treatment group versus the surgery alone group.

Table 1. Demographics and clinical characteristics of patients in the CIK treatment and surgery alone groups.

Clinicopathological variables	Surgery alone group (n = 217)	CIK treatment group (n = 231)	p value
Age (y)			0.292
<50	118	137
≥50	99	94
Median (range)	48 (22–74)	47 (14–80)
Gender			0.068
Male	198	198
Female	19	33
Capsule			0.880
Complete	183	196
Incomplete	34	35
Tumor size (cm)			0.781
≤5	93	96
>5	124	135
TNM stage			0.539
I	122	136
II	31	25
III	64	70
Tumor number			0.142
Single	181	180
Multiple	36	51
Histological differentiation			0.653
Well	34	43
Moderate	133	133
Poor	50	55
Virus infection			0.090
HBV	195	198
HCV	1	1
HBV+HCV	3	1
Non	18	31
Child-Pugh classes			0.414
A	205	222
B	12	9
Alcoholic liver disease			0.924
Yes	9	10
No	208	221
NASH			0.700
Yes	8	7
No	209	224
AFP (ng/mL)			0.433
Median	173.3	254.5
Range	0.64–1,21,000	0-1,21,000
PD-L1			0.960
<1%	111	116
1–5%	54	57
≥5%	52	58

HBsAg, Hepatitis B surface antigen; AFP, α-fetoprotein; NASH, non-alcoholic steatohepatitis.

Figure 2. Immunohistochemical analysis of PD-L1 expression in primary hepatocellular carcinoma surgical specimens. Positive staining results are reported as the percentage of tumor cells showing membranous PD-L1 expression (frequency). (A and D) PD-L1 expression was scored as <1%, (B and E) PD-L1 expression was scored as 1–5%, (C and F) PD-L1 expression was scored as ≥5%. PD-L1 staining is indicated by the presence of brown chromogen. Blue represents the hematoxylin counterstain (A–C, 100× magnification; D–F, 400× magnification).

Figure 3. Correlation of PD-L1 expression by HCC cells with the survival benefit from adjuvant CIK cell immunotherapy. Differences in levels of PD-L1 expression between patients showing a long-term versus a minimal survival benefit are shown before (A) and after (B) the propensity score weighting method was used to balance all covariates. C and D depicts the Kaplan–Meier curves for overall survival (OS) and recurrence-free survival (RFS) in the CIK cell treatment group for patients with ≥5%, 1–5% and <1 % PD-L1 expression, respectively.

Table 2. Demographics and clinical characteristics of patients according to the survival benefit from CIK cell therapy before and after use of PS weighting.

Clinicopathological variables	Original			PS weight
	Minimal survival benefit (n = 115)	Long-term survival benefit (n = 116)	p value	Minimal survival benefit (n = 83)	Long-term survival benefit (n = 83)	p value
Age (y)			0.626			0.377
Median	46	48		46	43
Range	25–80	14–70		28–77	14–70
Gender			0.640			0.393
Male	99	98		68	72
Female	18	21		15	11
Capsule			0.039			0.822
Complete	94	107		71	72
Incomplete	23	12		12	11
Tumor size (cm)			0.008			0.347
≤5	38	59		33	39
>5	79	60		50	44
TNM stage			<0.001			0.791
I	54	84		51	55
II	12	15		10	8
III	51	20		22	20
Tumor number			0.006			0.431
Single	83	102		65	69
Multiple	34	17		18	14
Histological differentiation			0.104			0.684
Well	17	27		15	19
Moderate	67	70		50	45
Poor	33	22		18	19
HBsAg			0.332			0.659
Positive	97	104		70	72
Negative	20	15		13	11
AFP (ng/mL)			0.006			0.475
Median	460.3	100		293	255
Range	1–1,21,000	0–1,21,000		1–1,21,000	0–1,21,000

HBsAg, Hepatitis B surface antigen; AFP, α-fetoprotein.

Table 3. Univariate and multivariate analysis of overall survival (OS) in HCC patients who received adjuvant CIK cell immunotherapy.

Variables	Univariate analysis			Multivariate analysis
	HR	95% CI	p value	HR	95% CI	p value
Age (≤50 vs. >50)	1.188	0.843–1.676	0.326
Gender (Male vs. Female)	1.020	0.633–1.641	0.937
Capsule (Complete vs. Incomplete)	2.504	1.662–3.770	<0.001	1.189	0.754–1.876	0.456
Tumor size (cm) (≤5 vs. >5)	2.172	1.496–3.152	<0.001	1.151	0.755–1.755	0.514
TNM stage (I vs. II vs. III)	2.107	1.748–2.540	<0.001	1.952	1.492–2.553	<0.001^*
Tumor number (Single vs. Multiple)	2.690	1.856–3.898	<0.001	0.899	0.563–1.436	0.655
Histological differentiation (Well vs. Moderate vs. Poor)	1.590	1.220–2.072	0.001	1.276	0.945–1.722	0.111
HBsAg (Positive vs. Negative)	0.786	0.488–1.265	0.322
AFP (ng/mL) (<400 vs. ≥400)	1.747	1.239–2.462	0.001	1.313	0.969–1.778	0.079
PD-L1 (<5% vs. ≥5%)	0.527	0.336–0.827	0.005	0.526	0.329–0.839	0.007^*

HR, hazard ratio; CI, confidence interval; HBsAg, Hepatitis B surface antigen; AFP, α-fetoprotein.

* Statistically significant, p < 0 .05.

Table 4. Univariate and multivariate analysis of recurrence-free survival (RFS) in HCC patients who received adjuvant CIK cell immunotherapy.

Variables	Univariate analysis			Multivariate analysis
	HR	95% CI	p value	HR	95% CI	p value
Age (≤50 vs. >50)	1.183	0.872–1.605	0.279
Gender (Male vs. Female)	0.954	0.624–1.460	0.829
Capsule (Complete vs. Incomplete)	1.821	1.230–2.696	0.003	0.911	0.591–1.406	0.675
Tumor size (cm) (≤5 vs. >5)	1.988	1.439–2.747	<0.001	1.331	0.937–1.892	0.110
TNM stage (I vs. II vs. III)	1.818	1.536–2.152	<0.001	1.723	1.371–2.165	<0.001^*
Tumor number (Single vs. Multiple)	2.100	1.490–2.960	<0.001	0.904	0.589–1.388	0.645
Histological differentiation (Well vs. Moderate vs. Poor)	1.386	1.099–1.749	0.006	1.206	0.936–1.554	0.146
HBsAg (Positive vs. Negative)	0.807	0.527–1.234	0.807
AFP (ng/mL) (<400 vs. ≥400)	1.518	1.120–2.057	0.007	1.258	0.936–1.554	0.146
PD-L1 (<5% vs. ≥5%)	0.556	0.381–0.811	0.002	0.572	0.388–0.845	0.005^*

HR, hazard ratio; CI, confidence interval; HBsAg, Hepatitis B surface antigen; AFP, α-fetoprotein.

* Statistically significant, p <0 .05.

Figure 4. Kaplan–Meier curves for HCC patients who received hepatectomy alone based on expression of PD-L1 on tumor cells. (A) Overall survival (OS) and (B) recurrence-free survival (RFS) curves are shown. No correlation with PD-L1 expression was observed with the OS of RFS rates of patients with HCC; p values were calculated using the log-rank test.

Figure 5. Subgroup analysis to estimate the impact of PD-L1 expression on survival benefits of additional CIK treatment according to hepatitis B viral load. (A) OS and RFS curves for patients with high hepatitis B viral load. (B) OS and RFS curves for patients with low hepatitis B viral load.

Table 5. Correlation of PD-L1 expression and immunological status to HBV.

Variables	≥5 % PD-L1 expression (n = 110)	1–5% PD-L1 expression (n = 111)	<1% PD-L1 expression (n = 227)	p value
HBsAg
Positive	99	102	196	0.279
Negative	11	9	31
Anti-HBsAb				0.332
Positive	5	3	4
Negative	105	108	223
HBeAg				0.576
Positive	16	18	43
Negative	94	93	184
Anti-HBcAb				0.339
Positive	101	107	211
Negative	9	4	16
Anti-HBeAb				0.651
Positive	83	82	161
Negative	27	29	66
Viral load (copy/mL)				0.017 ^*
≥1,000	70	59	107
<1,000	40	52	120

* Statistically significant, p <0 .05.

Figure 6. Association between PD-L1 expression on tumor cells and infiltration of inflammatory cells in HCC microenvironment. (A) Gating routine for the identification of tumor cells and CD45⁺ cells is shown. Flow cytometric analysis was performed to measure PD-L1 expression on tumor cells(gate C) and CD4⁺, CD8⁺, CD56⁺, and CD68⁺ cell subsets gating on the CD45⁺fraction(gate B). Significantly higher percentages of CD4⁺ T cells and CD8⁺ T cells were observed in the HCC tumor tissues with high PD-L1 expression (case 1) compared to that with low PD-L1 expression (case 2). (B) A summary of correlation analysis was shown that PD-L1 positive tumor cells in tumor tissues significantly correlated with tumor-infiltrating CD4⁺ and CD8⁺ T cells, but not associated with CD56⁺ NK cell and CD68⁺ macrophage (n = 10).

Figure 7. PD-1 expression on the autologous CIK cells. (A) The percentages of PD-1 positive cells among the populations of CIK cells. (B) The percentages of CD4⁺PD-1⁺ CIK cells. (C) The percentages of CD8⁺PD-1⁺ CIK cells. (D) The percentages of CD3⁻CD56⁺ PD-1⁺ and CD3⁺CD56⁺ PD-1⁺ CIK cells. (E) PD-1 expression on each subgroup of CIK cells in five HCC patients. These data suggested CIK cells exhibited a low PD-1 expression.