Hepatocellular carcinoma (HCC) is an established indication for liver transplantation (LT). It is a major indication for living donor liver transplantation (LDLT). Because HCC recurrence after LT is associated with inferior outcomes, LT candidates are carefully selected through various selection criteria to minimize the risk of tumor recurrence [1-6].

Cancer immune suppression and immune escape are known to play essential roles in tumor progression. Activation of programmed death protein 1 (PD-1) pathway is one of the most critical mechanisms of tumor evasion, inhibiting T-cell proliferation, inducing T-cell exhaustion, and enhancing the activity of regulatory T cells [7]. Soluble PD-1 (sPD-1) can be detected in the peripheral blood of HCC patients. We have previous reported two contradictory results on the prognostic value of sPD-1 on posttransplant HCC recurrence [8,9]. So far, the influence of sPD-1 concentration on HCC prognosis remains debatable [10-12]. The objective of this study was to assess the prognostic influence of very high concentration of pretransplant serum sPD-1 in LT recipients diagnosed with HCC at LT.

This was a retrospective study of posttransplant outcomes of patients with HCC. This study aimed to assess the prognostic impact of pretransplant serum sPD-1 expression on tumor recurrence and patient survival.

The LT database of our institution was searched to identify adult patients with HCC who had undergone primary LT between January 2010 and December 2015. Among them, study patients were selected in the list of peripheral blood storage at the institutional Bio-Resource Center. Follow-up was conducted to determine tumor recurrence and patient survival until December 2021 or patient death through review of institutional medical records with assistance from the National Health Insurance Service in Korea. The study protocol was approved by the Institutional Review Board (IRB) of our institution (no. 2019-0599). The requirement for informed consent was waived by the IRB due to the retrospective nature of this study. This study was performed in accordance with ethical guidelines of the World Medical Association Declaration of Helsinki 2013.

Cutoff for Determination of Very High sPD-1 Concentration

In our precedent study with 229 patients [8], the mean concentration of pretransplant serum sPD-1 was 136.7±182.2 ng/mL and the distribution was left-skewed. Thus, we selected study patients who had serum sPD-1 greater than one standard deviation (sPD-1=318.9 ng/mL; upper 5% percentile).

Serum sPD-1 Assay

Concentrations of sPD-1 in sera were measured quantitatively using an enzyme-linked immunosorbent assay (ELISA) kit (Boster Biological Technology, Pleasanton, CA, USA). For sPD-1 detection, the plasma was diluted five times with a sample diluent provided in the kit. Testing samples, blank samples, and standard controls were used to analyze the quality of sPD-1. After standard controls or samples (50 μL each) were added to appropriate wells in triplicate, 100 μL of horseradish peroxidase–conjugated antibody was added to each well except for blank wells. Plates were covered with an adhesive strip and incubated for 1 hour at 37℃. After washing the microtiter plate five times, 50 μL of substrate A and 50 μL of substrate B were added to each well. Samples were gently mixed and incubated in the dark at 37℃ for 15 minutes. Finally, 50 μL of stop solution was added to each well. Plates were then analyzed at 450 nm using a Victor X3 Plate Reader (Bio-Rad Laboratories, Hercules, CA, USA). A standard curve was generated by plotting the average optical density at 450 nm on the vertical axis versus the corresponding concentration on the horizontal axis [8,9].

Calculation of ADV Score

ADV scores were calculated by multiplying α-fetoprotein (AFP) concentration (ng/mL), des-γ-carboxyprothrombin (DCP) (proteins induced by vitamin K antagonist or absence-II [PIVKA-II]) concentration (mAU/mL), and tumor volume (mL) expressed on a logarithmic scale (log10, simply log) [9]. Total tumor volume in patients with multiple tumors was calculated by multiplying the volume of the largest tumor by the number of tumors.

Statistical Analysis

Numerical data are presented as mean and standard deviation or median and range. Survival curves were generated using the Kaplan–Meier method and compared using a log-rank test. Cutoffs of sPD-1 concentration for predicting posttransplant HCC recurrence were determined using receiver operating characteristic curve analysis with optimal cutoff, sensitivity, and specificity determined using the Youden index. A p-value <0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS (version 22; IBM Corp., Armonk, NY, USA) and MedCalc version 20.010 (MedCalc Software Ltd., Ostend, Belgium).

Patient Demographics

Twelve patients were included in this study. Their mean age was 51.8±5.1 years. There were 11 males (91.7%). All patients had hepatitis B virus-associated liver cirrhosis. Median AFP and PIVKA-II concentrations were 58.3 ng/mL (range, 1.2–5,135.7 ng/mL) and 44 mAU/mL (range, 12–19,400 mAU/mL), respectively. Mean model for end-stage liver disease score was 13.5±8.8. All patients received LDLT with right liver grafts. The mean graft-to-recipient weight ratio was 1.14%±0.22%. Explant liver pathology showed single tumor in 3 (25.0%) patients, two tumors in 5 (41.7%), and multiple tumors in 4 (33.3%). The number of patients was 6 (50.0%) who met the Milan criteria, 7 (58.3%) who met the University of California San Francisco criteria, and 8 (66.7%) who met the Asan Medical Center criteria.

Tumor Recurrence and Patient Survival

During the mean follow-up period of 71.0±49.7 months, HCC recurred in 9 (75.0%) patients. The cumulative tumor recurrence rate was 14.7% at 1 year, 75.0% at 3 years, and 75.0% at 5 years (Fig. 1).

Figure 1. Posttransplant tumor recurrence curve.

Eight (66.7%) patients died of HCC recurrence. The overall patient survival rate was 83.3% at 1 year, 66.7% at 3 years, 50.0% at 5 years, and 33.3% at 10 years (Fig. 2).

Figure 2. Posttransplant overall patient survival curve.

Post-recurrence patient survival rate was 71.4% at 6 months, 42.9% at 1 year, 28.6% at 3 years, 14.3% at 5 years, and 0% at 8 years (Fig. 3).

Figure 3. Patient survival curve after tumor recurrence.

Expression of Pretransplant Serum sPD-1 and HCC Recurrence

The distribution of serum sPD-1 concentration is depicted in Fig. 4. The median value was 365.7 ng/mL.

Figure 4. Distribution of pretransplant serum soluble PD-1 concentration. Bars indicate 25–75 percentiles. PD-1, programmed death protein 1.

Receiver operating characteristic curve analysis of serum sPD-1 concentration for tumor recurrence showed that the area under the curve was 0.556 (p=0.824; Fig. 5). The Youden index J was 0.333 (95% confidence interval: 0.222–0.333) at a sPD-1 cutoff of 444.8 ng/mL, which showed a sensitivity of 66.7% and a specificity of 66.7%. Application of this sPD-1 cutoff of 444.8 ng/mL did not result in any statistical difference in tumor recurrence (p=0.756; Fig. 6A) or patient survival (p=0.486; Fig. 6B).

Figure 5. Tumor recurrence (A) and patient survival (B) curves according to pretransplant serum soluble PD-1 concentration. PD-1, programmed death protein 1.

Figure 6. Tumor recurrence (A) and patient survival (B) curves according to the Milan criteria.

Prognostic Impact of High Serum sPD-1 on Milan Criteria and ADV Score

Tumor status within and beyond the Milan criteria did not show a statistically significant prognostic impact on tumor recurrence (p=0.377; Fig. 7A) or patient survival (p=0.514; Fig. 7B). ADV score with a cutoff of 5 log did not show a statistically significant prognostic impact on tumor recurrence (p=0.575; Fig. 8A) or patient survival (p=0.927; Fig. 8B) either.

Figure 7. Receiver operating characteristic curve analysis of posttransplant hepatocellular carcinoma recurrence. AUC, area under the curve.

Figure 8. Tumor recurrence (A) and patient survival (B) curves according to ADV score. ADV, multiplication of α-fetoprotein, des-γ-carboxyprothrombin and tumor.

Predicting the posttransplant prognosis in patients undergoing LT for HCC can be challenging as tumor burden prior to LT varies greatly. In addition, tumor biology is heterogeneous. It is influenced by continued immunosuppression. To reduce the risk of posttransplant HCC recurrence, many selection criteria have been proposed to date [1-6,13,14]. These criteria are usually based on tumor size and number, although some also include serologic tumor markers such as AFP and PIVKA-II. However, to accurately predict the risk of posttransplant HCC recurrence, it is essential to include other prognostic biomarkers that can provide additional information regarding tumor biology. We have proposed ADV score as a quantitative prediction model for posttransplant HCC recurrence [15,16].

However, the predictive power of currently available selection criteria is often limited because the majority of these criteria do not include biologic components of HCC. In our previous studies [8,9] and the present study, serum sPD-1 concentration served as the biologic component of tumor.

Cancer immune suppression and immune escape may play critical roles in the progression of malignant tumors. Activation of the PD-1 pathway is involved in tumor evasion, inhibiting T-cell proliferation, inducing T-cell exhaustion, and enhancing the activity of regulatory T cells [7]. During the process of HCC tumorigenesis, the immune surveillance mechanism is impaired involving PD-1/programmed death-ligand 1 (PD-L1) signaling pathways [17]. Expression of PD-1 in CD8+ T cells is higher in patients with HCC than in patients without HCC [18], with aggressive tumor progression being related to high frequencies of both circulating and tumor-infiltrating PD-1+ CD8+ T cells [19]. High PD-1 expression in HCC tissue has been associated with aggressive tumor features and the number of CD8+ T cells in peripheral blood [20]. PD-1 and PD-L1 can mediate immunosuppression within the tumor microenvironment, suggesting that levels of PD-1/PD-L1 expression may act as biomarkers for disease progression and patient survival [21]. Immunoregulatory pathways include costimulatory molecules present in both membrane-bound and soluble forms. Proteolytic cleavage of the membrane-bound form of a costimulatory protein in tissues can release the soluble form into the blood stream. Soluble forms of PD-1/PD-L1 are produced through this process.

A reliable prognostic cutoff of serum sPD-1 for HCC has not been reported yet. Our previous study has found that an sPD-1 cutoff of 300 ng/mL has a marginal impact on posttransplant tumor recurrence, whereas a cutoff of 93.6 ng/mL is not prognostic [8]. By contrast, our other precedent study has revealed that a serum sPD-1 cutoff of 177.1 ng/mL has a significant prognostic impact on posttransplant tumor recurrence [9]. These two studies differed from the present study in that the present study excluded patients previously treated for HCCs, had a slightly longer follow-up period, and included an additional number of patients with untreated HCCs. These studies suggest that HCC biology associated with sPD-1 expression is greatly influenced by responses to treatment. However, the real-world prognostic effect of very high serum sPD-1 was not fully evaluated in these studies.

Results of the present study demonstrated that very high expression of serum sPD-1 such as >318.9 ng/mL was closely associated with high tumor recurrence and inferior patient survival after LT. Its prognostic effect was greater than that of the Milan criteria. Patients meeting the Milan criteria do not guarantee acceptably favorable prognosis if their serum sPD-1 concentrations exceed 318.9 ng/mL. ADV score is a quantitative prognostic parameter reflecting the tumor biology of HCC [9,15,16]. However, a pretransplant ADV score cutoff <5 log could not be a favorable predictor of posttransplant HCC recurrence any more if serum sPD-1 concentration exceeds 318.9 ng/mL. Therefore, very high pretransplant serum sPD-1 over 318.9 ng/mL appears to be an independent prognostic factor. Thus, it is reasonable to consider other available clinical parameters to decide whether to perform LT or not.

The present study had several limitations. It was a retrospective, single-center study with a small sample size. In addition, all HCCs developed in hepatitis B virus-infected livers.

In conclusion, the results of the present study reveal that very high pretransplant serum sPD-1 over 318.9 ng/mL appears to be an independent risk factor which can surpass prognostic influences of the Milan criteria and ADV score. Therefore, eligibility of LT should be carefully evaluated in patients showing high serum sPD-1. Further high-volume, multicenter studies are needed to validate the role of serum sPD-1 in LT recipients.

There was no funding related to this study.

All authors have no conflicts of interest to declare.

Conceptualization: CSP, SH. Data curation: All. Methodology: All. Visualization: SH. Writing - original draft: All. Writing - review & editing: CSP, SH.