Persistent immune microenvironment changes associated with progression risk in Barrett's esophagus

Barrett's esophagus (BE) is the principal risk factor for esophageal adenocarcinoma (EAC). Since only a small number of patients with BE progress to high-grade dysplasia (HGD) or EAC, biomarkers of risk are in high demand. Our previous work identified alterations in the relative abundance of lamina propria lymphocyte and macrophage subsets in BE patients at initial diagnosis. In this study, we extended these findings by analyzing serial surveillance samples from these patients to assess the changes of these immune microenvironments over time. Formalin-fixed, paraffin-embedded tissue samples from patients with non-dysplastic BE undergoing endoscopic biopsy were examined. Initial diagnostic samples (n=58) and serial surveillance samples (n=78) from 58 unique patients were examined, among whom 25 were subsequently diagnosed with HGD or EAC (“progressors”), while 33 patients did not progress during at least 5 years of surveillance (“non-progressors”). Immunohistochemical staining was performed with antibodies specific for CD3 and CD8 (lymphocytes), CD68/CD86 co-positive (“M1- like”) macrophages, and CD68/CD206 co-positive (“M2-like”) macrophages. Regions of interest (ROI) selected from the lamina propria in the region of BE were subjected to digital image analysis, and subgroup comparisons of immune cell densities were carried out using Welch's t-test for the outcome of progression. In total, 3540 ROIs from non-progressors (1526 initial, 2014 follow-up) and 2994 ROIs from progressors (1077 initial, 1917 follow-up) were examined. Relative to non-progressors, progressor biopsies contained a markedly greater density of CD3+ lymphocytes (4586 vs. 2633, p=0.0019) and a significantly lower density of CD8+ lymphocytes (798 vs. 1359, p=0.002). Concomitantly, M1-like macrophages were significantly elevated in progressors (84.7 vs. 34.6, p=0.003), while M2-like macrophages were markedly decreased (35.5 vs. 80.3, p<0.0001) relative to non-progressors. The difference persisted at follow-up biopsy, (CD3+:1566 vs. 1228, p<0.0001; M1: 244 vs. 48.4, p<0.0001), whereas other immune cells changed (CD8+: 350 vs. 396, p=0.096 vs. M2: 66.5 vs 37.6, p=0.0066). The immune cell density suggests that there is an enduring feature of the BE microenvironment.The BE immune microenvironment demonstrates substantial differences in lamina propria lymphocyte and macrophage populations in patients who are destined to progress to HGD or EAC, relative to those who are not. These findings, which we show here to persist in serial biopsies over time, warrant further investigation to deepen our understanding of this altered immune microenvironment, both as a biomarker of progression risk and a potential target for immunomodulatory prevention strategies.