A total of 123 patients were included in the analysis. Among them, 17 had paired metachronous metastatic tumors for which MSLN expression could be assessed. Patient characteristics are presented in Table 1. The median age was 45 years (range, 26–72 years). There were 67 (54.5%), 27 (22.0%), 13 (10.6%), 12 (9.8%), and 4 (3.3%) patients with SCC, AC, adenosquamous carcinoma (ASC), GAS, and NEC, respectively. Ninety-six patients (78%) had FIGO stage I disease, while 27 (22%) had FIGO stage II disease. Lymph node metastasis was observed in 45 patients (36.6%). For postoperative treatment, 44 patients (35.8%) received postoperative radiotherapy, whereas 13 (10.6%) received postoperative chemoradiotherapy.
MSLN expression and differences in histological types
MSLN expression was observed in 98.4% (121/123) of the patients. High MSLN expression was observed in 63.4% (78/123) of primary tumor samples. Figure 1A and 1B show staining intensity patterns for MSLN expression in SCC and AC, respectively. Supplementary Figure 1 shows the representative micrographs of MSLN expression in each histological type.
High MSLN expression was not associated with age, tumor size, FIGO stage, or lymph node metastasis; however, it was associated with histological type and lymphovascular space invasion. High MSLN expression was more frequent in non-SCC than in SCC (80.4% [45/56] in non-SCC vs. 49.2% [33/67] in SCC, p < 0.001) (Table 1). Figure 2 shows H-score distribution with respect to histology. The median H-scores for AC/ASC and GAS were significantly higher than those for SCC (200 [152.5–270] for AC/ASC vs. 110 [20–220] for SCC, p < 0.01; 225 [147.5–270] for GAS vs. 110 [20–220] for SCC, p < 0.01).
MSLN expression in primary and metastatic tumors
Analysis of the paired primary and metastatic tumors of 17 patients who had paired metachronous metastatic tumors showed a significant correlation between MSLN expression in primary and metastatic lesions (Spearman’s rank correlation coefficient; Rs = 0.557, p = 0.015, Figure 3). Four of these patients (23.5%) showed a discordance in MSLN expression between primary and metastatic tumors. Three patients with high MSLN expression in their primary tumors showed low MSLN expression in their metastatic tumors. One patient showed low MSLN expression in the primary tumor and high MSLN expression in the metastatic tumor. Supplementary Figure 2 shows a representative case in which MSLN expression changed between primary and metastatic tumors.
Association between MSLN expression and survival
We evaluated the association between MSLN expression in primary tumors and survival. The median follow-up period was 68.9 months (95% confidence interval [CI], 63.3–76.2). The median RFS and OS were not significantly different between the high and low MSLN expression groups in all populations (RFS: hazard ratio [HR], 1.26; 95% CI, 0.68–2.43; p = 0.46; OS: HR, 2.39; 95% CI, 0.95–7.27; p = 0.08) (Supplementary Figure 3A and 3B).
As GAS and NEC are associated with a significantly poorer prognosis, we subsequently evaluated the association between MSLN expression and survival in 107 patients with common histological types, i.e., SCC, AC, and ASC. RFS tended to be shorter in the high MSLN expression group than in the low MSLN expression group (HR, 1.34; 95% CI, 0.68–2.79; p = 0.41) (Figure 4A). OS was shorter in the high MSLN expression group than in the low MSLN expression group (HR, 3.53; 95% CI, 1.16–15.3; p = 0.03) (Figure 4B). In 67 patients with SCC, the median RFS and OS tended to be shorter in the high MSLN expression group than in the low MSLN expression group (RFS: HR, 1.96; 95% CI, 0.79–5.27; p = 0.15; OS: HR, 3.82; 95% CI, 0.94–25.49; p = 0.06) (Supplementary Figure 4A and 4B). In 40 patients with AC and ASC, the median RFS and OS were not significantly different between the high and low MSLN expression groups (RFS: HR, 0.54; 95% CI, 0.19–1.75; p = 0.29; OS: HR, 1.53; 95% CI, 0.25–29.3; p = 0.69) (Supplementary Figure 4C and 4D).
In the multivariate analyses adjusting for age, histology, FIGO stage, lymph node metastasis, and presence of post-operative radiotherapy and chemoradiotherapy, MSLN expression was significantly associated with OS (HR, 3.70; 95% CI, 1.09–17.6; p = 0.034), but not RFS (HR, 0.98; 95% CI, 0.47–2.15; p = 0.96) (Supplementary Table 1).