High Goblet Cell Count Is Inversely Associated with Ploidy Abnormalities and Risk of Adenocarcinoma in Barrett’s Esophagus

RESEARCH ARTICLE High Goblet Cell Count Is Inversely Associated with Ploidy Abnormalities and Risk of Adenocarcinoma in Barrett’s Esophagus Amitabh Srivastava1☯, Kevin L. Golden1☯¤, Carissa A. Sanchez2,3‡, Karen Liu4‡, Pui Yee Fong2‡, Xiaohong Li2,3‡, David S. Cowan2,3‡, Peter S. Rabinovitch5‡, Brian J. Reid2,3,6,7‡, Patricia L. Blount2,6‡, Robert D. Odze1☯* a11111 OPEN ACCESS Citation: Srivastava A, Golden KL, Sanchez CA, Liu K, Fong PY, Li X, et al. (2015) High Goblet Cell Count Is Inversely Associated with Ploidy Abnormalities and Risk of Adenocarcinoma in Barrett’s Esophagus. PLoS ONE 10(7): e0133403. doi:10.1371/journal. pone.0133403 Editor: John P. Lynch, University of Pennsylvania, UNITED STATES Received: December 24, 2014 Accepted: June 25, 2015 Published: July 31, 2015 Copyright: © 2015 Srivastava et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by National Institute of Health P01 CA91955, BJR. Competing Interests: The authors have declared that no competing interests exist. 1 Department of Pathology, Brigham & Women's Hospital, Boston, Massachusetts, United States of America, 2 Department of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 3 Department of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 4 Department of Vaccine Infectious Disease Institute, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 5 Department of Pathology, University of Washington, Seattle, Washington, United States of America, 6 Department of Medicine, University of Washington, Seattle, Washington, United States of America, 7 Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America ☯ These authors contributed equally to this work. ¤ Current address: Laboratory Medicine Consultants, Las Vegas, Nevada, United States of America. ‡ These authors also contributed equally to this work. * Abstract Purpose Goblet cells may represent a potentially successful adaptive response to acid and bile by producing a thick mucous barrier that protects against cancer development in Barrett's esophagus (BE). The aim of this study was to determine the relationship between goblet cells (GC) and risk of progression to adenocarcinoma, and DNA content flow cytometric abnormalities, in BE patients. Experimental Design Baseline mucosal biopsies (N=2988) from 213 patients, 32 of whom developed cancer during the follow up period, enrolled in a prospective dynamic cohort of BE patients were scored in a blinded fashion, for the total number (#) of GC, mean # of GC/crypt (GC density), # of crypts with  1 GC, and the proportion of crypts with 1 GC, in both dysplastic and non-dysplastic epithelium separately. The relationship between these four GC parameters and DNA content flow cytometric abnormalities and adenocarcinoma outcome was compared, after adjustment for age, gender, and BE segment length. PLOS ONE | DOI:10.1371/journal.pone.0133403 July 31, 2015 1 / 16 Goblet Cells and Adenocarcinoma in BE Results High GC parameters were inversely associated with DNA content flow cytometric abnormalities, such as aneuploidy, ploidy >2.7N, and an elevated 4N fraction > 6%, and with risk of adenocarcinoma. However, a Kaplan-Meier analysis showed that the total # of GC and the total # crypts with 1 GC were the only significant GC parameters (p<0.001 and 0.003, respectively). Conclusions The results of this study show, for the first time, an inverse relationship between high GC counts and flow cytometric abnormalities and risk of adenocarcinoma in BE. Further studies are needed to determine if GC depleted foci within esophageal columnar mucosa are more prone to neoplastic progression or whether loss of GC occurs secondary to underlying genetic abnormalities. Introduction In North America, Barrett’s esophagus (BE) is defined as columnar metaplasia, with goblet cells (GC), in the distal esophagus, although in some parts of the world, GC are not required for this diagnosis [1, 2]. There is abundant evidence to suggest that adenocarcinoma in BE develops via a columnar metaplasia/dysplasia/carcinoma pathogenic sequence [1,3,4]. However, recent population-based studies have reported that the risk of progression from BE to esophageal adenocarcinoma is substantially lower than earlier estimates, [5,6], and most patients with BE die of unrelated causes [1]. Columnar metaplasia occurs as a result of chemical/toxic damage secondary to reflux of gastric acid and bile into the distal esophagus, combined with release of inflammatory mediators [1]. Most patients with endoscopically recognizable columnar metaplasia have GC, and in the majority of patients with adenocarcinoma, the cancer arises in a background of neoplastic columnar mucosa with GC [7–10]. However, the precise role of GC in BE is uncertain [3,11,12]. It has been proposed that BE-associated metaplastic columnar epithelium represents a successful adaptation against the noxious effects of acid and bile [1,13–21]. This hypothesis has been based on the results of a number of discovery studies that have shown that Barrett’sassociated metaplastic epithelium secretes a thick adherent layer of mucus, as well as anions and bicarbonate, that decreases reflux-related injury [13,16]. Barrett's epithelium also possesses claudin-18 tight junctions that provides improved protection against acid permeation [17], and a crypt architecture that is believed to be tumor suppressive [18]. Metaplastic columnar cells have been shown to maintain intracellular pH following prolonged and repeated acid exposure [19]. One expression study, and another combined expression and proteomic study, concluded that Barrett's epithelium overexpresses genes involved in defense and repair of reflux-related injury [20,21]. All of these studies were appropriately designed for discovery research, but they involved small numbers of patients and did not directly evaluate the critical hypothesis of whether or not these mucosal adaptations modulate cancer development in BE and did not distinguish goblet from non-goblet columnar epithelium. We hypothesized that increasing numbers, density, and distribution of GC at baseline endoscopy protects against progression to adenocarcinoma and DNA content flow cytometric abnormalities (tetraploidy and aneuploidy), the latter of which are associated with an increased risk of progression. Therefore, the PLOS ONE | DOI:10.1371/journal.pone.0133403 July 31, 2015 2 / 16 Goblet Cells and Adenocarcinoma in BE aims of this discovery study were to eva (...truncated)