All procedures performed in studies involving human participants were in accordance with the ethical standards of the ethical committee of Beijing Shijitan Hospital, Capital Medical University, and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent: For the retrospective design, the formal consent was not required.
The retrospective cohort design was carried out to explore the association between PD-L1 expression and prognosis. This study retrospectively recruited 97 NSCLC patients receiving surgical treatment at the Department of Thoracic Surgery, Beijing Shijitan Hospital, Capital Medical University and having pathology diagnosis, consecutively from January 1, 2016, to December 31, 2016.
Expression of PD-1, PD-L1, P53 and ki-67 were detected by immunohistochemistry (IHC) on 4μm-thick formalin fixed paraffin-embedded (FFPE) sections. ALL the monoclonal antibodies were purchased from Beijing Zhong Shan Golden Bridge Biotechnology Co. Ltd. Sections were baked for dehydration at 60°C in an oven for 60 min, dewaxed for 20 min, and washed in 100%, 100%, 95% and 75% alcohol for 2 min respectively; washed with PBS by 5 times, 2 min each time. Antigen retrieval were carried out using the EnVisionTM FLEX Target Retrieval Solutions for 2 min 30 sec, cooled to room temperature for 20 min; washed with PBS by 5 times, 2 min each time; and then incubated with 3% H2O2 at room temperature for 15 min; washed with PBS by 5 times, 2 min each time; sealed with 5% serum at 37℃ for 15 min; discarded and added a moderate primary antibody at 4℃ for a night; washed with PBS by 5 times, 2 min each time; added DAB for 5-10 min and AP-red for 10-15 min. Slides were counterstained with hematoxylin.
Hot-spot area was determined under low-power field for Ki-67 assessment. Then 1000 cells were counted under high-power field and the percentage of nuclear-positive cells was calculated. The p53 gene mutation was defined as nuclear-positive cells more than 70%. More than 1% lymphocytes with positive staining on cytoplasm/membrane was diagnosed as positive expression of PD-1. More than 1% tumor cells with brown staining on cytoplasm/membrane was determined as positive expression of PD-L1.
EGFR Mutation Test
Tumor DNA was extracted from FFPE tissue according to the instructions of DNA extraction kit. EGFR Master Mix containing EGFR Enzyme Mix and Reaction Mix was prepared in separate sterile centrifuge tube, pipetted gently more than 10 times and centrifuged briefly. 35.3μL of each EGFR Master Mix and 4.7μL NTC, PC or sample DNA were added in PCR tube respectively, and centrifuged to the bottom of the tube.
PCR protocol was set according to the cycling parameters recommended.
TNM stage is performed in accordance with eighth edition of the TNM classification of Lung Cancer  .
All data was analyzed by SPSS 19.0 version. Age, tumor size, Ki-67 index, p53 status and AJCC stage were analyzed by Wilcoxon ran-sum test between PD-L1 groups. Pathological type, nerve invasion, blood vessel invasion EGFR mutation status and lymph node metastasis were analyzed by Chi-square test between PD-L1 expression statuses. PD-1 expression status was analyzed by McNemar test with PD-L1 expression status. Kaplan-Meier survival curve with Log-rank test was used to estimate the effects of PD-L1 expression on DFS and OS. The HR and 95% confidence interval (95%CI) were estimated by COX-Hazard Proportion Model with further adjustment of age, sex, pathology type, stage, blood vessel invasion and nerve invasion. All analyses were two-sided test with significant level of 0.05.