Fig 2: (A) GAD1 mRNA expression in TET according to the WHO histological classification (A+AB+B1, B2, B3 and TC). The upper and lower ends of the whiskers, the upper and lower edges of the boxes, and the horizontal lines across each box represent the upper and lower extremes, the upper (75th) and lower (25th) quartiles, and the medians, respectively. Data were analyzed via Kruskal Wallis test (P=0.0058) and the Steel-Dwass test. (B) GAD1 mRNA expression between TC and thymomas (A+AB+B1+B2+B3) was 1.85 (range, 0.25–29.76, IQR, 1.21–6.65) vs. 0.38 (range 0.01–4.40, IQR, 0.08–0.86), respectively. (C) Correlation between DNA methylation levels of four CpG sites within the CTCF-3 of GAD1 and GAD1 mRNA expression in 18 thymomas and 12 TC tissues. Data were analyzed via Spearman's rank correlation. (D) Representative images of the immunohistochemically detected GAD1 protein in tumors. (a) Type A thymoma: the cytoplasm of tumor cells was not stained; (b) type AB thymoma: the cytoplasm of tumor cells was strongly stained; (c) type B1 thymoma: the cytoplasm of tumor cells was weakly stained and lymphocytes were not stained; (d) type B2 thymoma: the cytoplasm of tumor cells was not stained; (e) type B3 thymoma: the cytoplasm of tumor cells was strongly stained and lymphocytes were not stained; and (f) TC: the cytoplasm of tumor cells of thymic carcinoma was strongly stained. Scale bar, 50 µm. (E) GAD1 protein expression in TET according to the WHO histological classification. TC, thymic carcinoma; GAD1, glutamate decarboxylase 1; IQR, interquartile range; TET, thymic epithelial tumor; WHO, World Health Organization.

Fig 3: (A) Schematic diagram of the GAD1 structure. The mRNA of GAD1 has 16 exons. Four CGI (black boxes) are located in the promoter and the first three exons of GAD1. Four CTCF-binding sites of GAD1 are shown as vertical lines. The sequences of the four putative CTCF-binding sites at the human GAD1 locus are shown. CpG dinucleotides in the CTCF-binding site are underlined. The CpG site cg15126544 in the CTCF-3 site of GAD1 is shown as a dotted box. (B) DNA methylation levels. The vertical scale indicates average β-values of the DNA methylation level of each CpG site between TC (black bar) and B3 thymomas (white bar). The target region for the pyrosequencing analysis began 10 nucleotides before and 26 nucleotides after cg15126544 (underlined). *P<0.05 TC vs. thymoma. (C) DNA methylation rate of the GAD1 gene in thymomas and paired normal thymic tissues, and in TC and paired normal thymic tissues. Average DNA methylation values are shown as percentages from the quantitative pyrosequencing of 13 thymomas and paired thymic tissues, and 8 TC and paired thymic tissues. (D) DNA methylation rate of the GAD1 gene in thymic epithelial tumors according to the World Health Organization histological classification (A+AB+B1, B2, B3 and TC). The upper and lower ends of the whiskers, the upper and lower edges of the boxes, and the horizontal lines across each box represent the upper and lower extremes, the upper (75th) and lower (25th) quartiles, and the medians, respectively. Data were analyzed using Kruskal Wallis test (P<0.0001) and Steel-Dwass test. (E) Median DNA methylation rate between TC and thymomas (A+AB+B1+B2+B3) was 32.88 (range, 12.25–75.25; IQR, 19.50–51.69) vs. 4.0 (range, 2.5–28.0; IQR, 3.25–5.25), respectively. (F) Receiver operating characteristic curve of the accuracy of the methylation signature for detecting TC among all tumors using GAD1 methylation. CTCF, CCCTC-binding factor; TC, thymic carcinoma; GAD1, glutamate decarboxylase 1; IQR, interquartile range; AUC, area under the curve.

Fig 4: Association between GAD1 protein expression status and prognosis in patients with LADC. (A) Representative images of immunohistochemically detected GAD1 protein in tumors and non‐tumorous lesions of LADC samples and normal lung tissue. Scale bars, 200 μm. The relative GAD1 mRNA expression level of each sample as determined by rqRT‐PCR is also shown. (B) Kaplan‐Meier curves for overall survival, disease‐free survival, and cancer‐specific survival rates of 162 LADC patients according to the immunoreactivity of GAD1. P‐values were calculated using the log‐rank test. Statistically significant P‐values are in boldface type

Fig 5: (A) Relapse-free survival curves of patients with TET with higher and lower levels of DNA methylation in GAD1. The median value (5.375) of the frequency of the DNA methylation of GAD1 was used to divide patients into hypermethylated and hypomethylated groups. P-value was calculated using the log-rank test. (B) Relapse-free survival curves of patients with TET with higher and lower levels of GAD1 mRNA expression. The median value (0.69) of GAD1 mRNA expression was used to divide patients into high and low expression groups. P-value was calculated using the Gehan-Breslow-Wilcoxon test. (C) Relapse-free survival curves of patients with TET with higher and lower levels of GAD1 protein expression. The immunoreactivity (staining score) of GAD1 was used to divide patients into IHC(+) (staining score ≥4) and IHC(−) (staining score <4) groups. P-value was calculated using the log-rank test. GAD1, glutamate decarboxylase 1; IHC, immunohistochemistry; TET, thymic epithelial tumor.