Upregulation of circulating PD-L1/PD-1 is associated with poor post-cryoablation prognosis in patients with HBV-related hepatocellular carcinoma

Abstract

Background: The programmed cell death-1 receptor/programmed cell death-1 ligand (PD-1/PD-L1) pathway plays a crucial role in tumor evasion from host immunity. This study was designed to evaluate the association between circulating PD-L1/PD-1 and prognosis after cryoablation in patients with HBV-related hepatocellular carcinoma (HCC).

Methodology/principal findings: In the present study, 141 HBV-related HCC patients were enrolled and of those 109 patients received cryoablation. Circulating PD-L1/PD-1 expression was tested by flow cytometry, and 23 patients were simultaneously evaluated for intratumoral PD-L1 expression by immunohistochemical staining. Circulating PD-1/PD-L1 expression was associated with severity of diseases in patients with HCC, and the circulating PD-L1 expression was closely correlated with intratumoral PD-L1 expression. Of the clinical parameters, PD-1/PD-L1 expression was associated with tumor size, blood vessel invasion and BCLC staging. Moreover, PD-1/PD-L1 expression dropped after cryoablation while being elevated at the time of tumor recurrence. Patients with higher expression of circulating PD-L1, as well as circulating PD-1, had a significantly shorter overall survival and tumor-free survival than those with lower expression. Multivariate analysis confirmed that circulating PD-L1 could serve as an independent predictor of overall survival and tumor-recurrence survival in HCC patients after cryoablation.

Conclusions/significance: Upregulation of circulating PD-L1/PD-1 is associated with poor post-cryoablation prognosis in patients with HBV-related hepatocellular carcinoma.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Gating strategies and expression profiles of circulating PD-L1 and PD-1 in 141 patients with HBV-related HCC.

The gating strategies and representative results of circulating PD-L1 (A) and PD-1 (C) expression are shown. FSC, forward scatter; SSC, side scatter. In the histogram, the black line indicates PD-L1 or PD-1, whereas the grey line represents its corresponding isotype control. Numbers in the histograms show the frequencies of positive cells. The statistical results for circulating PD-L1 (B) and PD-1 (D) were organized according to the HCC stages (stage A, n = 46; stage B, n = 63; stage C, n = 32), respectively. The black bar indicates the mean value.

Figure 2. Circulating PD-L1 expression was closely correlated with their corresponding intratumoral PD-L1 expression.

(A)Representative immunohistochemical results of PD-L1 for different degrees are shown(red for PD-L1, ×400). (B) Statistical analysis demonstrated that circulating PD-L1 was significantly correlated with intratumoral PD-L1(r = 0.706, P

Figure 3. Dynamic circulating PD-1/PD-L1 variation after cryoablation in HCC patients.

(A,B) HCC patients with postoperative recurrent tumors (n = 11, PR, post-recurrence)showed that circulating PD-1/PD-L1 expression individually increased 4 weeks after cryoablation (PC, post-cryoablation). (C,D)Circulating PD-1/PD-L1 expression in 109 HCC patients increased significantly 1 week after cryoablation when compared with that before treatment, and dropped 4 weeks later. (E)A HCC patient(stageA, E1) receiving cryoablation showed tumor completely necrosis one week later(E2) and the necrotic tumor tissue shrank 33 months later with no tumor recurrence(E3). (F) A HCC patient(stageB, F1) receiving cryoablation showed tumor completely necrosis one week later(F2) and tumor recurred at the margin of necrotic tumor tissue 18 months later(F3, arrow indicates the recurred tumor).

Figure 4. Increased expression of circulating PD-L1/PD-1 predicts poor prognosis in HCC patients after cryoablation.

109 HCC patients were divided into 2 groups by the median value of circulating PD-1/PD-L1 at the time before treatment as low PD-1 (n = 23, 4.05±1.28% for stageA; n = 31, 9.26±1.63% for stage B) and high PD-1 (n = 23, 9.26±3.09% for stage A; n = 32, 18.05±3.78% for stage B), low PD-L1 (n = 23, 4.26+0.93% for stageA; n = 31, 7.17±2.19% for stage B) and high PD-L1 (n = 23, 8.51+2.48% for stage A; n = 32, 19.37±9.41% for stage B). (A,B)HCC patients in stage A with lower expression of circulating PD-1 have longer TFS and OS than those with higher expression(P = 0.03 and P = 0.002,respectively). (C,D) HCC Patients in stage B with lower expression of circulating PD-1 have longer TFS and OS than those with higher expression(P = 0.002 and P = 0.045,respectively). (E,F)HCC patients in stage A with lower expression of circulating PD-L1 have longer TFS and OS than those with higher expression(P = 0.004 and P = 0.004,respectively). (G,H)HCC patients in stage B with lower expression of circulating PD-L1 have longer TFS and OS than those with higher expression(P = 0.000 and P = 0.005,respectively).