Epithelial PD-L2 Expression Marks Barrett's Esophagus and Esophageal Adenocarcinoma

Abstract

Esophageal adenocarcinoma is an increasingly common disease with a dismal 5-year survival rate of 10% to 15%. In the first systematic evaluation of the PD-1 pathway in esophageal adenocarcinoma, we identify expression of PD-L2 in cancer cells in 51.7% of esophageal adenocarcinomas. Epithelial PD-L1 was expressed on only 2% of cases, although PD-L1(+) immune cells were observed in 18% of esophageal adenocarcinomas. We also evaluated expression in the precursor lesion of esophageal adenocarcinoma, Barrett's esophagus, which emerges following gastric reflux-induced esophageal inflammation, and found PD-L2 expression in Barrett's esophagus but not in non-Barrett's esophagus esophagitis. Because the progression from squamous esophagitis to Barrett's esophagus is accompanied by a transition from a TH1 to TH2 immune response, we hypothesized that the TH2 cytokines IL4/IL13 could contribute to PD-L2 induction. We confirmed that these cytokines can augment PD-L2 expression in esophageal adenocarcinoma cell lines. These results suggest that the inflammatory environment in Barrett's esophagus and esophageal adenocarcinoma may contribute to the expression of PD-L2. Furthermore, the potential for PD-1 receptor blockade to be effective in esophageal adenocarcinomas with epithelial PD-L2 or immune cell PD-L1 expression should be evaluated in clinical trials.

©2015 American Association for Cancer Research.

Figures

Figure 1

IHC staining of FFPE esophageal tissues in tissue microarrays (20×). Staining with (A) anti-PD-1 antibody showing PD-1+ TILs; (B) anti-PD-L1 antibody showing PD-L1+ immune cells; (C) negative PD-L2 staining, (D) weak PD-L2 staining in tumor epithelium scored as 1+, (E) moderate PD-L2 staining scored as 2+, and (F) strong PD-L2 staining scored as 3+. High magnification images to show cytologic details for PD-1 and PD-L1.

Figure 2

Validation of IHC results with flow cytometry. A. IHC and flow cytometry results of PD-L2-expressing OE33 and MKN7 cells, and PD-L2 non-expressing ESO26 cells (black, isotype; white, PD-L2). B. PD-L2 mRNA expression in 7 gastro-esophageal cell lines. Data are depicted as mean + standard deviation. C. IHC staining of 2 EAC biopsies with anti-PD-L2 antibody (20×). Staining was scored strong positive for both tumors. Tumor biopsies were implanted in the flanks of nude mice. At time of passage tumors were disaggregated into single-cell suspensions and analyzed. Flow cytometry shows co-expression of EpCAM and PD-L2 in 11.9% and 10.2% of EpCAM+ cells. PE-isotype was used as control. A representative experiment of 2 independent experiments is shown.

Figure 3

PD-L2 expression is detected at the transition of reflux esophagitis to Barrett's Esophagus and can be induced by IL4/IL13 A. IHC staining with hematoxylin and (20×) and anti-PD-L2 antibody (20×) shows PD-L2 expression in Barrett's esophagus tissues with complete intestinal metaplasia (1) and incomplete intestinal metaplasia (2) and not in reflux esophagitis tissues. Induction of PD-L2 mRNA (B) and protein expression (C) of IL4- and IL13-treated MKN7, OE33 and FLO-1 cells. A representative experiment is shown ( n= 3-5). mRNA expression data are depicted as mean + standard deviation. Flow cytometry: black, isotype; white and dashed line, PD-L2 expression 24 hours after adding medium; grey, PD-L2 expression 24 hours after IL4 treatment.