Patients and clinicopathological data. This retrospective cohort study consecutively recruited 142 patients from Beijing Shijitan Hospital, Capital Medical University, in Beijing, China between 2013 and 2016. The recruited patients received surgical treatment of esophagectomy and were diagnosed as ESCC. All patients received neither neoadjuvant therapy nor immunotherapy prior to surgical operation. The collected esophageal tissues were fixed in 4% neutral formaldehyde after operation, embedded in paraffin, and stained with hematoxylin and eosin (HE) or used for immunohistochemistry (IHC). The obtained clinical and pathological data included age, gender, tumor location, tumor size, tumor differentiation, nerve invasion, vascular invasion and lymph node metastasis. The pathological data were independently evaluated by two pathologists. The data of relapse and overall survival were followed up every six months till March 2018.
Reagents. CD38: Mouse anti-human monoclonal antibody, Clone number: SPC32, expresses in cytomembrane. PD-1༚Mouse anti-human monoclonal antibody, Clone number: MRQ-22༌expresses in cytoplasm. PD-L1༚Rabbit anti-human monoclonal antibody, Clone number: SP142༌expresses in cytomembrane. CD4༚Rabbit anti-human monoclonal antibody, Clone number: EP204༌expresses in cytomembrane. CD8༚Rabbit anti-human monoclonal antibody, Clone number: SP16༌expresses in cytomembrane. All antibodies were purchased from Beijing Zhongshan Jinqiao Biotechnology co., LTD.
Immunohistochemistry. The paraffin-embedded samples were cut into 4 µm sections, placed on polylysine-coated slides, deparaffinized with xylene and dehydrated in alcohol series. The slides were washed in 3% hydrogen peroxide solution at room temperature for 10 min, and then in 1x phosphate-buffered solution (PBS) for 5 min for three times. Slides were repaired in citrate buffer (pH6.0) by microwave for 20 min, cooled to room temperature, and washed in PBS for 5 min for three times. Slides were incubated with the primary antibody in a moist chamber at 4 °C overnight, and then washed with PBS for 5 min for three times. The slides were incubated with the corresponding secondary antibody for 20 min at room temperature and then washed with PBS for 5 min for three times. Sav-HRP conjugates were added to the sections, incubated in a humidified chamber at room temperature for 30 min and then washed with PBS for 5 min for three times. Finally, 100 µl DAB substrate solution was added to the sections to reveal the color of antibody staining and washed with PBS for 5 min by three times. Slides were counterstained with hematoxylin.
Assessment of CD38. Tumor tissue sections stained with anti-CD38 antibody with at least 100 tumor cells and 100 immune cells were selected, and myeloma tissue sections were used as the positive controls. The positive cells were defined as the cells had partial or complete cytomembrane brown staining. The expression rate of CD38 was calculated by counting at least 100 cells in three high-power fields (HPFs). The immunohistochemically stained slides were examined by two pathologists independently.
Assessment of PD-L1, PD-1, CD4 and CD8. Tumor tissue sections stained with anti- PD-L1, PD-1, and CD8 antibodies with at least 100 tumor cells and 100 immune cells were selected, and tonsil tissues were used as the positive controls. Two pathologists independently examined the Immunohistochemically stained slides. The expression rate of PD-L1 on tumor cells and immune cells was evaluated. The Assessment of tumor-infiltrating immune cells (TILs) evaluation was conducted according to recommendations by an International TILs Working Group 2014 . An average number of TILs were counted in 10 random HPF (400×) in IHC sections. The proportions of PD-1+TILs, CD4+TILs and CD8+TILs were calculated by the percentage of cells with positive PD-1, CD4 and CD8 expression, respectively, in 100 random TILs from three HPFs with number of TILs around the mean.
Statistical analyses. All analyses were processed by SPSS version 19.0. The expression rate of CD38 was measured by median and inter-quartile range (IQR). Spearman correlation test was used to analysis the correlation between age, tumor location, differentiation, stage and CD38. Lymph node metastasis, neural invasion and blood vessel invasion were analyzed with CD38 expression by Wilcoxon rank-sum test. The CD38 expression rate was categorized by median. Kaplan-Meier survival curve was used to estimate the difference of disease-free survival (DFS) and overall survival (OS). Cox-Hazard Proportion regression model was used to estimate the hazard ratio (HR) and 95% confidence interval (95%CI), with further confounder adjustment. All analyses were two-tailed and significant level was 0.05.