Myeloid-derived suppressor cells in the patients with liver resection for hepatitis B virus-related hepatocellular carcinoma

Liver resection remains the popular treatment for hepatocellular carcinoma (HCC). The aim of this study was to explore the alteration of immune cells in HCC patients with liver resections. Nineteen patients were included and their peripheral blood samples were taken before and after liver resections for immune-cell analysis. The clinical characteristics showed that the median diameter of the resected tumors was 7.5 cm with a range from 1.4 to 16.5 cm. The analysis of immune cells showed that the percentage of CD4+ T-cells were not altered by liver resection, but the percentage of CD8+ T-cell was decreased from 31.7 ± 12.4% to 20.2 ± 10.4% at one week after liver resection (p = 0.006). For immunosuppressor cells, regulatory T-cells were not altered, but myeloid-derived suppressor cells (MDSC) were decreased from 7.75 ± 8.16% to 1.51 ± 1.32% at one month after liver resection (p = 0.022) in 10 of 19 patients with high frequency of MDSC. Furthermore, it was also found that MDSC population was linearly correlated to tumor volume. In conclusion, CD8+ T-cells and MDSC were altered by liver resection. The percentage of CD8+ T-cells was decreased by surgery, but the accumulation of MDSC was abrogated.


Results
Patients. Seventeen male and 2 female patients who had liver resections for HCC were included in this study.
The median (interquartile, IQ) age of these patients was 57 (44-66) years with a range from 34 to 70 years. Seventeen patients had hepatitis B and other 2 patients did not have viral hepatitis. Nine patients had cirrhosis and all were in Child-Pugh A classification. Eighteen patients had solitary tumor in the liver and only one patients had two tumors. The median (IQ) diameter of the tumors was 7.5 (3.4-11.2) cm with a range from 1.4 to 16.5 cm. By calculation, the median (IQ) tumor volume was 118 (18-354) cm 3 with a range from 1.23 to 2008.5 cm 3 . All these patients had liver resections to remove the tumors completely. The operations and pre-operative clinical data were listed in Table 1.
CD4 + and CD8 + T-lymphocytes. T-lymphocytes are the most important cells to mediate cellular immunity to cancer cells. To study the alteration of T-lymphocytes before and after liver resection, PBMC was stained with surface CD4 and CD8 molecules, and doubly stained with surface CD4 and CD8 molecules and intracellular cytokine interferon-γ (IFN) to determine the frequency of CD4 + , CD4 + IFN-γ + , CD8 + and CD8 + IFN-γ + cells. The results showed that frequency of CD4 + and CD4 + IFN-γ + were not altered by liver resection (p = 0.819 and 0.359, respectively). The frequency of CD8 + T-cell was 31.7 ± 12.4% before liver resection, decreased to 20.2 ± 10.4% at one week after liver resection (p = 0.006) and returned to 28.3 ± 11.3% at one month after liver resection. The frequency of CD8 + IFN-γ + T-cell was 17.3 ± 12.8% before liver resection, decreased to 11.1 ± 9.5% at one week after liver resection and 10.4 ± 10.0% at one month after liver resection. There was a trend of CD8 + IFN-γ + T-cell decrease after liver resection (p = 0.105) (Fig. 1).

Correlation between tumor sizes and MDSC.
Postoperative survival. After operations, all patients were discharged from the hospital, however, one patient died of myocardiac failure at one month after liver resection. All the other patients were regularly followed up at out-patient clinic. The mean follow up was 25.8 ± 16.4 months. Nine patients had tumor recurrence until now. The 1-and 3-year disease-free survival rates were 73.0% and 56.1%, respectively. The 1-and 3-year overall survival rates were 78.9% and 68.4%, respectively (Fig. 4).

Correlation between tumor sizes and AFP. AFP is a biomarker of HCC and is related to tumor biology.
The higher level is AFP, the worse is the prognosis. To determine whether the serum levels of AFP were correlated to tumor volume, regression analysis between AFP and tumor volume was performed. Because the value of AFP and tumor volume varied greatly, log transformation of AFP and tumor volume was performed to undergo Pearson's correlation test. The result showed that the serum level of AFP had moderately positive correlation to tumor volume. The equation was log AFP (ng/ml) = 0.66 + 0.74 × log tumor volume (cm 3 ) (r = 0.451, Fig. 6a).

Correlation between AFP and MDSC. Because both frequency of MDSC and serum levels of AFP
were correlated to tumor volume, frequency of MDSC and serum levels of AFP may be related closely. Because the value of AFP and MDSC varied greatly, log transformation of AFP and MDSC was performed to undergo Pearson's correlation test. The result showed that the frequency of MDSC in peripheral blood had moderately positive correlation to AFP. The equation was log MDSC = −0.11 + 0.19 × log AFP (ng/ml)) (r = 0.458, Fig. 6b).

Discussion
MDSC is an immunosuppressive cell and attracts attention for cancer and chronic diseases in a recent decade. This study showed that the frequency of MDSC in peripheral blood of HCC patients was linearly correlated to tumor volume and could be calculated by a formula using tumor volume. It was known in murine study that MDSC appeared in the mice bearing large tumors 22 17 . Clinically, it was reported that the population of MDSC in PBMC of HCC patients was much higher than in healthy normal control and non-tumor cirrhotic patients 20,24 . This study showed that the frequency of MDSC in peripheral blood could be quantitatively estimated according to the equation between the frequency of MDSC and tumor volumes. The frequency of MDSC could be reduced by excision of tumor. When we focused on the patients with large tumors and high frequency of MDSC, the frequency of MDSC was reduced to normal level at one month after liver resection. In animal study, the population of MDSC was reduced after the tumor was removed and the survival of the mice was prolonged 25 . Dr. Gabrilovich described that granulocyte colony-stimulating factor, macrophage colony-stimulating factor, granulocyte/macrophage colony-stimulating factor), stem cell factor, vascular endothelial growth factor and other cytokines might be the factors to expand MDSC 10 . As the frequency was expanded by tumor, some of these cytokines might be released by tumors. Excision of HCC became the most direct way to block the stimulatory factors and returned the frequency of MDSC back normal.
The disease-free and overall survival of the patients with large tumors were much worse than those patients with small-sized tumors. Tumor size is a well-known risk factor for patients with liver resection for HCC 7 . In this study, when the patients were divided into two groups according median tumor volume 118 cm 3 , the patients with tumor volume >118 cm 3 had much worse disease-free and overall survival rates than the patients with tumor volume ≤118 cm 3 . As the results of population of MDSC directly related to tumor sizes in this study, the patients  with large-sized tumors had high population of MDSC and low anti-tumor immunity. Subsequently, the patients with large tumors had higher incidence of tumor recurrence and worse postoperative survival. When the tumors were resected to decrease tumor burden, the population of MDSC was decreased but the low anti-tumor immunity could not be reversed completely. Although many factors were well-known and contributed to worse prognosis after liver resection for HCC, the high frequency of MDSC might be one of them, but rarely be mentioned. T-lymphocytes are the direct effector cells to attack cancer cells, particularly CD8 + T-cells. Helper CD4 + T-cells were not altered before and after liver resection, however, CD8 + T-cells were significantly decreased at one week after liver resection and recovered at one month. It was clearly show that surgery itself was immunosuppressive. When we looked at CD8 + IFN-γ + T-cells, this effective cytotoxic CD8 + IFN-γ + T-cells had the tendency to decrease after liver resection and did not recover at one month after liver resection. It implied that both tumor and surgery were immunosuppressive. Hosts' immunity was not fully recovered although the tumors were totally removed.
Although only two-third of HCC patients have elevated serum level of AFP, AFP is recognized as a biomarker of HCC. In this study, serum AFP level had moderately positive correlation to tumor volume and MDSC. High level of AFP was a poor prognostic factor. Dr. Peng et al. mentioned that high level of AFP was associated with lower 10-year survival rate, particular in large tumors 26 . AFP is also recognized as a marker of biology of HCC, particular in liver transplantation setting. In liver transplantation, AFP is a risk factor of tumor recurrence after liver transplantation and high level of AFP is even not suitable to undergo liver transplantation 27,28 . Obviously, AFP level reflects aggressive biology of HCC with poor prognosis even after the tumors were removed.
Regulatory T-cells are another immunosuppressive cells. Regulatory T-cells were not changed before and after liver resection in this study. Hoechst et al. reported that regulatory T-cells were induced by MDSC when autologous T-cells were co-cultured with MDSC 20 . In our previous report, we found that the frequency of regulatory T-cells was higher the in tumor tissues of large tumors than small tumors, but was not different in peripheral blood 12 . In this study, we had the similar data that the frequency of regulatory T-cells in peripheral blood was not related to MDSC linearly. It did not show direct relationship between regulatory T-cells and tumor volume in this study, either.
The limitation of this study was the limited number of patients. Because the number of the patients were limited, some significant changes before and after liver resection could not be demonstrated. However, the population of MDSC and serum level of AFP correlated to tumor volume still could be well-expressed.
In conclusion, the frequency of MDSC was well correlated to tumor volume of HCC. Removal of tumors could reduce the population of MDSC. However, the remained unsatisfied prognosis of the patients with large tumor implied the low immunity of the patients with large tumors would not recover completely after tumor resection. To improve prognosis, enhancement of anti-tumor immunity has to be performed after surgery. Further studies are needed to meet the unmet.

Methods and Materials
Patients. Nineteen patients, 17males and 2 females, who received liver resection for early stage HCC from Dec. 2013 to Nov. 2016 were included in this study. All the patients signed informed consent to joint this clinical study. After informed consent was obtained, 10 cc of peripheral blood sample was withdrawn before liver resection, and one week and one month after liver resection for immune cell analysis. Clinical characteristics of the    Characteristics of tumors. The final diagnosis of HCC was based on pathological reports. Tumor sizes were documented as those were measured during gross-examination by pathologists. The tumor volume was calculated by the formula: tumor volume = 0.52 × width 2 × length 29 . The microscopic examination of tumor cell differentiation, encapsulation of tumors and microvascular invasion were all recorded.
Follow up. The patients were followed up regularly after liver resection. Tests of liver function and α-fetoprotein, and liver ultrasonography were performed every 3 months. Dynamic computed tomography (CT) of the liver was performed if deemed necessary. Tumor recurrence was defined when CT detected tumors with typical HCC imaging pattern in the liver or extrahepatic tumors. Disease-free survival was measured from the date of surgical treatments to tumor recurrence. Overall survival was measured from the date of surgical treatments to date of last following up or patients' death. Hospital mortality was defined that patients were not discharged and died in hospital after surgery.
Biostatistics analysis. The comparisons of categorical variables were determined by Chi-square Tests. The significance of the differences between different groups was determined by unpaired or paired Student's t-test. The disease-free and overall survival rates were calculated using the Kaplan-Meier method and compared between groups using the log-rank test. While the data of MDSC or α-FP varied greatly, Log transformation of MDSC or α-FP was performed to pass Shapiro-Wilk test. The relationship between tumor volume and MDSC or α-FP was performed by Pearson's correlation. The statistical analyses were all performed with SigmaPlot 12.3 software for Windows (Systat Softwave, Inc., San Jose, CA, USA). P value below 0.05 was considered to be significantly different.