A Research On Expression Of PD-1, PD-L1 In Liver Cancer Tissue And Their Significance

Primary liver cancer, one ofthe common malignancies with higher incidence, has a rising incidence and morality around the globe and it is the second leading cause of deaths from cancers, only next to lung cancer. In China, about 347 thousand people develop liver cancer each year, accounting for 55% of the global rate. Hepatic cancer has a complicated pathogenesis and its occurrence, progression and metastasis are associated with multi-gene mutationsas well as immune escapeand abnormalities of cell signal transduction pathways of tumor cells. The primary mechanism of tumor cell immune escape is the induced apoptosis of CD8+Tlymphocyte. Activation or suppression of CD8+T lymphocyte in the body is regulated by positive signals and negative signals,but the cell signal pathway system of PD-1 and PD-L1 is negativeimmune regulation signal. When combined, they transmitinhibitive signals, suppressing the proliferationof cytotoxic T-lymphocyte cells and inducing apoptosis of the cells.Sorafenib is the only drug currently used in China for liver cancer chemotherapy. SHARP tests and the tests in Asian-Pacific region indicate that the median survival time of liver cancer patients treated with Sorafenib in the world is only 10.9 months and the median survival time of liver cancer patients in Asia treated with Sorafenib is only 5.5 months. The development of targeted drugs PD-1 and PD-L1 will provide brand new choices for the treatment of liver cancer patients, which is likely to prolong their survival time. Current studies specific to programmed death1(PD-1)/programmed death ligand-1(PD-L1) in the field of liver cancer are mainly cell experiments and animal experiments. This study is aimed to explore the correlation between target spots PD-1 and PD-L1 and liver cancer taking clinical samples as the subjects. ObjectiveTo detect the protein expression of PD-1 and PD-L1 in liver cancer tissue and investigate their relationship with different pathological classification, clinical stages of liver cancer, liver function, age and AFP; also, detect the expression levels of PD-1, PD-L1,NGF, AFP, MET, RAF1, VEGFR2 and PDGFRB in m RNA in liver cancer tissue, compare the difference and relevance so as to provide theoretical and experimental bases for immune treatment of liver cancer. Methods:Immunohistochemistry(IHC) was used to detect the positive expression of PD-1 and PD-L1 in the paraffin-embedded liver cancer tissue specimens of 63 patients to compare the differential of expression of PD-1 and PD-L1 in experiment group and control group as well as 5 the differential of expression in different pathological classification, clinical stages of liver cancer, liver function,age and AFP levels.For 10 of the patients, florescent real-time quantitative PCR was applied to their paraffin-embedded tissue specimens for detecting the m RNA expression levels of PD-1, NGF, AFP, MET, RAF1, VEGFR2, PDGFRB and the differences and relevance were compared; Statistical treatment: for the immunohistochemical results, CHISS 2004 software was used for statistical analysis of measurement data, which showed a normal distribution or approximately normal distribution, expressed as x±s. Paired t test was adopted for difference comparison between 2 groups of measurement data(equal inter-group variance); one-way analysis of variance was adopted for difference comparison among multiple groups of measurement data(equal inter-group variance), and q test was adopted for multiple comparisons. The difference was considered statistically significant when P<0.05. SAS 9.2 was used for Pearsoncorrelation analysis of q-PCRresults.All the 63 liver cancer specimens were from inpatients with HBV-related liver cancer receiving operation at 302 Military Hospital of China. All of the patients were positive for HBs Ag, and postoperative pathological analysis showed that all the 63 patients had HCC. Of the 63 patients, 57 were male and 6 were female, all aged between 30 and 70. All the patients had complete clinical data. The 63 patients were divided into three groups – high-differentiation group(n=20), moderate-differentiation group(n=25) and low-differentiation group(n=18) according to the results of pathological examination. 10 of the patients conductedflorescent real-time quantitative PCR to detect the m RNA expression levels of PD-1, NGF, AFP, MET, RAF1, VEGFR2, PDGFRB in the paraffin-embedded liver tissue specimens. The 16 paraffin-embeddedliver tissue specimens in the control group were all from healthy donors at 302 Military Hospital of China, all of whom were males, with an age of 30 to 42 years old. All the liver tissue specimens were fixed with 10% Formalin solution and then embedded with paraffin for preparation of sections. WHO Classification of Tumors of the Digestive System(4th edition, 2012) was adaptedfor classification of HCC. Results:The positive rates of protein expression of PD-1 and PD-L1 in liver cancer tissue specimens were higher than that in the normal control. The positive expression rate of PD-1 and PD-L1 in the liver cancer tissue varied with the pathological classification, being the highest in moderately differentiated liver cancer tissue. Barcelona Clinic Liver Cancer(BCLC) classification was relevant to clinical indicators, and the increase in stage C was the most significant(P<0.05).The differences in the positive expression rates of PD-1 and PD-L1 in the liver cancer tissue specimens of the patients with a different Child-Pugh class were not statistically significant; the differences in the positive expression rates of PD-1 and PD-L1 in liver cancer tissue specimens were statistically significant between patients with a serum AFP level > 400 μg/L and those with a serum AFP level < 400μg/L, in which the rates in the former were higher than those in the latter; however, among the patients aged over 50 and those aged under 50, the differences in the positive expression rates were not statistically significant. PD-1, NGF, AFP, MET, RAF1, VEGFR2, PDGFRB gene expression was detected in liver cancer tissues, and the expression intensity of NGF, AFP, VEGFR2, PDGFRB showed a strong positive correlation with the expression intensity of PD-1. Conclusions: 1. Patients with moderately differentiated hepatocellular carcinomaand patients in Barcelona stage C are suitable subjects of PD-1 or PD-L1 antibody therapy. 2. The expression of PD-1 and PD-L1 was not related to the age or HCC patients and Child-Pugg(P > 0.05)3. VEGFR2 may have synergistic effects with PD-1/PD-L1 in the immune escape of Hepatocellular Carcinoma. 4. The AFPconcentration in the blood may be used as a screening index for PD-1 or PD-L1 antibody treatment.