Quantification of LgR5 Immunohistochemistry Furthermore, we analyzed positivity of all counted cells according to the precursor lesion and tumor entity. LgR5 expression was significantly upregulated in BE (n = 41, Median 33%, IQR 14.75% – 45.0%; 95% CI 24.761 – 39.954%; p < 0.05; Figure 2a) but was decreased

in adjacent EAC (n = 41, Median 15%, IQR 13.0% – 18.0%; 95% CI 13.761 – 17.0%; p < 0.05; Figure 2a) and EAC without BE (n = 19, Median 13%, IQR 4.75% - 23.0%; 95% CI 6.346 - 22.436%; p < 0.05; Figure 2a; p < 0.05 for LgR5 expression of BE with adjacent EAC and EAC with and without BE). No differences of LgR5 expression were found between different degrees in high-grade and low-grade intraepithelial neoplasia within Barrett's mucosa and did not significantly differ from EAC. Median LgR5 expression of all EACs (n = 60) was 15%, IQR 11.0% - 18.0%; 95% CI 13.0 find more – 16.061%. For adenocarcinomas without BE, the results find protocol of LgR5 expression were comparable with the lower expression levels of adenocarcinomas from BE (Figure

2a, Table 1 and 2). Stainings from the OE-33 adenocarcinoma cancer cell line in cytospins served as additional positive controls for LgR5 expression and showed 25% positive cells (Figure 2b). Preincubation with LgR5 blocking peptide completely abolished LgR5 immunoreactivity (Figure 1b). Figure 2 Immunohistochemical analysis, staining and gene expression of LgR5. In comparison to BE (1) a significantly (p < 0.05) decreased expression of LgR5 was observed in associated EACs (2) and EACs without BE (3). ESCC showed no LgR5 expression (4). Analysis refers to percentages of positivity of all counted cells. Grey lines show 95% confidence intervals. Statistically significant values from BE to EACs and ESCC are indicated with asterisks (a). LgR5 staining in cytospins from the OE-33 adenocarcinoma cancer cell line served

as additional positive control (left, top) and showed 25% positive cells; Preincubation with LgR5 blocking peptide completely abolished LgR5 immunoreactivity (right, bottom) (b). Increased expression of LgR5 (c) was observed in early BE (arrows). Adjacent normal tissue stained negative for LgR5 (asterisk). Single staining of LgR5 in BE was confirmed by immunohistochemical double staining (d), showing Cdx-2 (nuclear staining pattern, Mannose-binding protein-associated serine protease Fast red) and LgR5 (membranous staining pattern, brown). Significantly decreased LgR5 expression was observed in adenocarcinomas compared to BE. Staining was observed in putative stem cell niches at the bottom of EACs (arrows) (e). Original magnification × 200. Gene expression of LgR5 in human BE and EAC (x-fold difference mRNA). LgR5 gene expression in BE-associated EAC (1) was significantly (p = 0.0159) higher in comparison to EAC without BE (2). Grey lines show 95% confidence intervals. Statistically significant value is indicated with an asterisk. Normal tissue is considered as one-fold (f).