Ascl2 and Lgr5 expression on whole slides
The clinicopathological characteristics of 35 cases in WS are summarized in Table 1. Briefly, the age range was 38 to 78 years old (mean age of 65.5 years), and males were the predominant sex (89%). 20% of the patients received neoadjuvant chemotherapy. The pathological stage was based on the 8th edition AJCC/UICC staging of cancers of the esophagus and esophagogastric junction [23].
Based on IHC scores, 18 cases (51%) were Ascl2high, and 17 cases (49%) were Ascl2low. Ascl2 was localized to the cytoplasm of tumor cells. Ascl2 expression tended to be correlated to histological grade (p=0.086) but did not have any significant correlation with any other clinicopathological factors (Table 1). For our study population (WS), the median overall survival (OS) time was 1.8 years (95% CI,0.7-7.4), and progression-free survival (PFS) time was 0.9 years (95% CI, 0.7-1.4). Ascl2 showed statistical significance in the KM analyses for OS and PFS, p=0.0168, and p=0.000638, respectively (Figure 1). Univariable Cox PH analyses showed Ascl2 significance in both OS and PFS (Table 2 and 3; OS HR=2.83, 95% CI=1.16-6.9, p=0.022; PFS HR=3.93, 95% CI=1.69-9.12, p=0.0014). In multivariate analysis adjusting for pathological stage, Ascl2 remained statistically significant for PFS (HR=3.13, 95% CI=1.28-7.65, p=0.012) but not for OS (HR=1.78, 95% CI=0.66-4.76, p=0.25) (Table 2 and 3).
Lgr5 was localized to the cytoplasm and membrane of tumor cells. Twenty cases (57%) were Lgr5high, and 15 cases (43%) were Lgr5low. Lgr5 was not significantly correlated to any of the clinicopathological factors (Table 1). Lgr5 did not show statistical significance in KM analyses for survival (OS: p=0.449, PFS: p=0.459; Data not shown). Univariable Cox PH analyses also failed to show Lgr5 significance in OS and PFS (Table 2 and 3: OS HR=0.74, 95% CI=0.31-1.77, p=0.5; PFS HR=0.75, 95% CI=0.34-1.66, p=0.48).
Ascl2 and Lgr5 expression on TMA slides
Sixty-four EAC patients were included in the TMA slides. Briefly, the age range was 38 to 86 years old (mean age of 64.6 years), and males were the predominant sex (84%). 31% of the patients received neoadjuvant chemotherapy (Table 4).
Twenty cases were Ascl2low, and 44 cases were Ascl2high. The representative picture of Ascl2high is shown in Figure 2a. High Ascl2 expression was associated with a more significant number of positive lymph nodes (p=0.027). No association was observed between Ascl2 expression and age, sex, pathological stage, neoadjuvant therapy, or histological differentiation.
For the cases in TMA, the median overall survival (OS) time was two years (95% CI,1.4-3.2), and progression-free survival (PFS) time was 0.9 years (95% CI, 0.7-1.3). The median OS time for Ascl2high was 1.4 years (95% CI, 0.9-2.3) compared to the much longer OS time for Ascl2low at 5.2 years (95% CI, 2.6-8.2). The results of the KM analysis for OS is shown in Figure 3a (p=0.0276). Figure 3b shows the significant association of Ascl2 with PFS. The median PFS time for Ascl2high was 0.8 years (95% CI, 0.5-1.3), compared to the 1.3 years for Ascl2low (95% CI 1-8.2, p=0.0466). The results of the univariate and multivariate analyses for OS (Table 5) and PFS (Table 6) support the results of KM analysis. Of note, multivariate analysis indicated that Ascl2 was an independent predictor of prognosis in line with gender and stage.
Fifty-six cases (88%) were Lgr5high; 8 cases (13%) were Lgr5low. Representative picture of Lgr5high is shown in Figure 2b. 6 out of 8 Lgr5low cases (75%) were pathological stage IV. Lgr5low had significantly more positive lymph nodes compared to Lgr5high (p=0.039). No other association was observed between Lgr5 expression and other clinicopathological factors. In KM analysis, Lgr5 was not a predictive factor of OS (Lgr5low 1.8years 95% CI 0.7-2.8; Lgr5high 2years 95% CI 0.7-7.4 p=0.393) nor PFS (Lgr5low 0.9years 95% CI 0.5-1.4; Lgr5high 1.2years 95%CI 0.5-14.7 p=0.62). Univariable Cox PH analyses also failed to show Lgr5 significance in OS and PFS (Table 5, 6: OS HR=1.27, 95% CI=0.52-3.09, p=0.59; PFS HR=1.69, 95% CI=0.67-4.21, p=0.26).
No significant correlation was seen between the expressions of Ascl2 and Lgr5 (p=0.42). 37 out of 64 cases (57.8%) were Ascl2high/Lgr5high; 19 cases (29.7%) Ascl2low/Lgr5high; 7 cases (10.9%) Ascl2high/Lgr5low; and one case (1.6%) Ascl2low/Lgr5low.
Ascl2 protein expression is significantly increased in esophageal adenocarcinoma
Adjacent 43 normal squamous epithelium, 22 BE, and six glandular dysplasia of the TMA patients were included in the TMA. EAC showed significantly increased Ascl2 expression compared to the normal squamous epithelium (p<0.001) and BE (p<0.001) and a trend towards higher expression compared to glandular dysplasia (p=0.0686, Figure 4a). Expression of Lgr5 in the normal squamous epithelium was significantly lower compared to the tumor (p<0.0001), BE (p<0.0001), and dysplasia (p<0.0001) but tumor, BE and dysplasia showed similar expression (Figure 4b).
Further validation of ASCL2 in TCGA datasets
Significance of Ascl2 protein expression in both WS and TMA prompted us to analyze the TCGA dataset. 86 cases of EAC revealed that ASCL2 mRNA was correlated with nodal status. ASCL2 mRNA was not significantly correlated to survival using KM analysis (Figure 5). No mutation in the ASCL2 gene was identified in the TCGA dataset, but two cases showed deep deletion of the ASCL2 gene.