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.