Patients and methods
This study was a retrospective review of patients with thymoma and was approved by the Institutional Review Board in the first affiliated hospital of SAN-YAT-SUN university. Seventy patients who had undergone surgical resection for thymomas at the first affiliated hospital of SAN-YAT-SUN university between January 2017 to December 2018 were enrolled, and their paraffin-embedded specimens were used to assess the expression of PD-L2 by immunohistochemistry (IHC). Clinical data were obtained from patients’ medical charts, including patient age, sex, tumor size, World Health Organization histologic type, status of myasthenia gravis, Masaoka-Koga stage, ectopic thymus. For the patients with MG, Myasthenia Gravis Foundation of America (MGFA) Clinical Classification at diagnosis were obtained.
Immunohistochemistry Studies Immunohistochemistry studies were performed on formalin-fixed, paraffin-embedded tissue sections from each tumor. Each section was cut to a 4-mm thickness. Anti-PD-L2 mouse immunoglobulin G monoclonal antibody (clone UMAB223; ZSGB Bioscience) were diluted to 1:100 and used as a primary antibody. The IHC staining was performed automatically with the Ventana BenchMark XT Stainer (Roche Diagnostics, Basel, Switzerland).
Scoring of PD-L2 Positivity Two pathologists evaluated the expression of PD-L2 on tumor cells. They reviewed each sample independently then discussed the findings of each sample and determined the proportion of PD-L2 positive tumor cells together. According to a previous consensus, a tumor cell was defined as “PD-L2 positive” when the cell membranes were partially or completely stained [12] (Fig 1). In contrast, PD-L1 staining in the cytoplasm of a tumor cell was defined as “negative.” The PD-L2 positive immune cells, such as lymphocytes and macrophages, were excluded from the cell counts. Tumor cells were quantified by evaluating the ratio of stained to unstained tumor cells. The PD-L2 positivity was evaluated based on based on the proportion of PD-L2 positive tumor cells. A PD-L2 expression rate of 1% or greater was defined as PD-L2 positive, and all other cases were PD-L2 negative in the present study. The intensity of PD-L2 (from 0 to 3: 0, negative; 1, very weak; 2, moderate; and 3, strong) in tumor cells were evaluated for each core. And mean PD-L2 expression scores were calculated by multiplying the percentage of tumor area stained by the staining intensity.
Descriptive statistics were used to describe the patients’ baseline. Univariate analysis was conducted using non-parametric test, the Chi square (χ2) test and the Student t-test. If necessary, fisher’s exact test was conducted for the categorical variables. The logistic regression model was used for the analysis of the correlation between patients’ characteristics and PD-L2 expression. The p values of< 0.05 were defined as significant.