Hypoxia-inducible factor-1α mediates reflux-induced epithelial-mesenchymal plasticity in Barrett's oesophagus patients.

INTRODUCTION: Epithelial-mesenchymal plasticity (EMP), the process through which epithelial cells acquire mesenchymal features, is needed for wound repair but also might contribute to cancer initiation. Earlier, in vitro studies showed that Barrett's cells exposed to acidic bile salt solutions (ABS) develop EMP. Now, we have (1) induced reflux oesophagitis in Barrett's oesophagus (BO) patients by stopping proton pump inhibitors (PPIs), (2) assessed their biopsies for EMP and (3) explored molecular pathways underlying reflux-induced EMP in BO cells and spheroids. METHODS: 15 BO patients had endoscopy with biopsies of Barrett's metaplasia while on PPIs, and 1 and 2 weeks after stopping PPIs; RNA-seq data were assessed for enrichments in hypoxia-inducible factors (HIFs), angiogenesis and EMP pathways. In BO biopsies, cell lines and spheroids, EMP features (motility) and markers (vascular endothelial growth factor (VEGF), ZEB1, miR-200a&b) were evaluated by morphology, migration assays, immunostaining and qPCR; HIF-1α was knocked down with siRNA or shRNA. RESULTS: At 1 and/or 2 weeks off PPIs, BO biopsies exhibited EMP features and markers, with significant enrichment for HIF-1α, angiogenesis and EMP pathways. In BO cells, ABS induced HIF-1α activation, which decreased miR-200a&b while increasing VEGF, ZEB1 and motility; HIF-1α knockdown blocked these effects. After ABS treatment, BO spheroids exhibited migratory protrusions showing nuclear HIF-1α, increased VEGF and decreased miR-200a&b. CONCLUSIONS: In BO patients, reflux oesophagitis induces EMP changes associated with increased HIF-1α signalling in Barrett's metaplasia. In Barrett's cells, ABS trigger EMP via HIF-1α signalling. Thus, HIF-1α appears to play a key role in mediating reflux-induced EMP that might contribute to cancer in BO. TRIAL REGISTRATION NUMBER: NCT02579460.

Significance of Crypt Atypia in Barrett's Esophagus: A Clinical, Molecular, and Outcome Study.

BACKGROUND & AIMS: The aim of this study was to characterize baseline morphologic features of crypts in nondysplastic Barrett's esophagus and correlate them with DNA content abnormalities and risk of progression to high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC). METHODS: The morphologic features of nondysplastic crypts in baseline biopsy specimens from 212 BE patients (2956 biopsy specimens) were graded histologically using a 4-point scale (crypt atypia levels, 0-3). DNA content abnormalities were detected using flow cytometry. RESULTS: In patients who had dysplasia in their baseline biopsy specimens, dysplasia was associated significantly with increasing grades of crypt atypia in the background nondysplastic Barrett's esophagus (P < .001). In a subset of patients without dysplasia at baseline (N = 149), a higher grade of crypt atypia was associated with longer Barrett's esophagus segment length (5.5 vs 3.3 cm; P = .0095), and a higher percentage of cells with 4N DNA content (3.67 ± 1.27 vs 2.93 ± 1.22; P = .018). Crypt atypia was associated with the development of any neoplasia (low-grade dysplasia and HGD/EAC). Although no significant association was noted between the grade of crypt atypia and increased 4N, aneuploidy, or progression to HGD/EAC, only patients with grade 2 or 3 crypt atypia showed increased 4N, aneuploidy, or progression to HGD/EAC. CONCLUSIONS: Patients with Barrett's esophagus likely develop dysplasia via a progressive increase in the level of crypt atypia before the onset of dysplasia, and these changes may reflect some alteration of DNA content.

Adjunctive Use of Wide-Area Transepithelial Sampling-3D in Patients With Symptomatic Gastroesophageal Reflux Increases Detection of Barrett's Esophagus and Dysplasia.

INTRODUCTION: Patients with gastroesophageal reflux (GERD) symptoms undergoing screening upper endoscopy for Barrett's esophagus (BE) frequently demonstrate columnar-lined epithelium, with forceps biopsies (FBs) failing to yield intestinal metaplasia (IM). Repeat endoscopy is then often necessary to confirm a BE diagnosis. The aim of this study was to assess the yield of IM leading to a diagnosis of BE by the addition of wide-area transepithelial sampling (WATS-3D) to FB in the screening of patients with GERD. METHODS: We performed a prospective registry study of patients with GERD undergoing screening upper endoscopy. Patients had both WATS-3D and FB. Patients were classified by their Z line appearance: regular, irregular (<1 cm columnar-lined epithelium), possible short-segment BE (1 to <3 cm), and possible long-segment BE (≥3 cm). Demographics, IM yield, and dysplasia yield were calculated. Adjunctive yield was defined as cases identified by WATS-3D not detected by FB, divided by cases detected by FB. Clinicians were asked if WATS-3D results affected patient management. RESULTS: Of 23,933 patients, 6,829 (28.5%) met endoscopic criteria for BE. Of these, 2,878 (42.1%) had IM identified by either FB or WATS-3D. Among patients fulfilling endoscopic criteria for BE, the adjunctive yield of WATS-3D was 76.5% and absolute yield was 18.1%. One thousand three hundred seventeen patients (19.3%) who fulfilled endoscopic BE criteria had IM detected solely by WATS-3D. Of 240 patients with dysplasia, 107 (44.6%) were found solely by WATS-3D. Among patients with positive WATS-3D but negative FB, the care plan changed in 90.7%. DISCUSSION: The addition of WATS-3D to FB in patients with GERD being screened for BE resulted in confirmation of BE in an additional one-fifth of patients. Furthermore, dysplasia diagnoses approximately doubled.

Benefit of adjunctive wide-area transepithelial sampling with 3-dimensional computer-assisted analysis plus forceps biopsy based on Barrett's esophagus segment length.

BACKGROUND AND AIMS: Wide-area transepithelial sampling with 3-dimensional computer-assisted analysis (WATS-3D) has been shown to increase the diagnostic yield of intestinal metaplasia (IM) and dysplasia within a segment of suspected or known Barrett's esophagus (BE) when used as an adjunct to forceps biopsies. Few data are available regarding how segment length affects WATS-3D yield. The purpose of this study was to evaluate adjunctive WATS-3D use in patients with varying lengths of BE. METHODS: A total of 8471 patients (52.5% male; mean age, 63 years) enrolled in 2 registry studies were included in this study. All patients were being screened or surveyed for BE with both forceps biopsies and WATS-3D. The adjunctive and absolute yield of WATS-3D was calculated according to the length of the patient's BE segment. RESULTS: The overall adjunctive and absolute increased diagnostic yields with WATS-3D were 47.6% and 17.5%, respectively, for detection of IM, and 139% and 2.4% for detection of dysplasia. IM and dysplasia detection both increased with the use of WATS-3D regardless of segment length. Increase in IM diagnostic yield was significantly higher in short- versus long-segment cases but higher in long-segment cases for dysplasia detection. CONCLUSIONS: This study shows that when WATS-3D is added as an adjunct to forceps biopsies, it is effective at increasing the diagnostic yield of both BE and associated dysplasia in patients with both short and long segments of esophageal columnar-lined epithelium.

High rate of missed Barrett's esophagus when screening with forceps biopsies.

BACKGROUND: Screening for Barrett's esophagus (BE) with endoscopy plus forceps biopsy (FB) has poor compliance with the recommended Seattle protocol and fails to sample large areas of mucosa. This statistical modeling study estimates, for the first time, the actual frequency of missed BE cases by FB. METHODS: Published, calibrated models in the literature were combined to calculate the age-specific prevalence of BE in white males with gastroesophageal reflux disease (GERD). We started with estimates of the prevalence of BE and GERD, and applied the relative risk for BE in patients with GERD based on the literature. This created estimates of the true prevalence of BE in white males with GERD by decade of life. The proportion of BE missed was calculated as the difference between the prevalence and the proportion with a positive screen. RESULTS: The prevalence of BE in white males with GERD was 8.9%, 12.1%, 15.3%, 18.7% and 22.0% for the third through eighth decades of life. Even after assuming no false positives, missed cases of BE were about 50% when estimated for patients of ages 50 or 60 years, and over 60% for ages of 30, 40 or 70 years. Sensitivity analysis was done for all variables in the model calculations. For ages 50 and 60 years, this resulted in values from 30.3 to 57.3% and 36.4 to 60.9%. CONCLUSION: Screening for BE with endoscopy and FB misses approximately 50% of BE cases. More sensitive methods of BE detection or better adherence to the Seattle protocol are needed.

An Endoscopic and Histologic Study on Healing of Radiofrequency Ablation Wounds in Patients With Barrett's Esophagus.

INTRODUCTION: Radiofrequency ablation (RFA) of Barrett's esophagus (BE) inflicts a wound spanning 3 epithelial types (stratified squamous, Barrett's metaplasia, gastric epithelium), yet the esophageal injury heals almost completely with squamous epithelium. Knowledge of how this unique wound heals might elucidate mechanisms underlying esophageal metaplasia. We aimed to prospectively and systematically characterize the early endoscopic and histologic features of RFA wound healing. METHODS: Patients with nondysplastic BE had endoscopy with systematic esophageal photographic mapping, biopsy, and volumetric laser endomicroscopy performed before and at 1, 2, and 4 weeks after RFA. RESULTS: Seven patients (6 men; mean age 56.1 ± 10.9 years) completed this study. Squamous re-epithelialization of RFA wounds did not only progress exclusively through squamous cells extending from the proximal wound edge but also progressed through islands of squamous epithelium sprouting throughout the ablated segment. Volumetric laser endomicroscopy revealed significant post-RFA increases in subepithelial glandular structures associated with the squamous islands. In 2 patients, biopsies of such islands revealed newly forming squamous epithelium contiguous with immature-appearing squamous cells arising from esophageal submucosal gland ducts. Subsquamous intestinal metaplasia (SSIM) was found in biopsies at 2 and/or 4 weeks after RFA in 6 of 7 patients. DISCUSSION: RFA wounds in BE are re-epithelialized, not just by squamous cells from the proximal wound margin but by scattered squamous islands in which esophageal submucosal gland duct cells seem to redifferentiate into the squamous progenitors that fuel squamous re-epithelialization. SSIM can be found in most patients during the healing process. We speculate that this SSIM might underlie Barrett's recurrences after apparently successful eradication.

Neonatal Fc receptor expression in dendritic cells mediates protective immunity against colorectal cancer.

Cancers arising in mucosal tissues account for a disproportionately large fraction of malignancies. Immunoglobulin G (IgG) and the neonatal Fc receptor for IgG (FcRn) have an important function in the mucosal immune system that we have now shown extends to the induction of CD8(+) T cell-mediated antitumor immunity. We demonstrate that FcRn within dendritic cells (DCs) was critical for homeostatic activation of mucosal CD8(+) T cells that drove protection against the development of colorectal cancers and lung metastases. FcRn-mediated tumor protection was driven by DCs activation of endogenous tumor-reactive CD8(+) T cells via the cross-presentation of IgG complexed antigens (IgG IC), as well as the induction of cytotoxicity-promoting cytokine secretion, particularly interleukin-12, both of which were independently triggered by the FcRn-IgG IC interaction in murine and human DCs. FcRn thus has a primary role within mucosal tissues in activating local immune responses that are critical for priming efficient anti-tumor immunosurveillance.

Progression of Barrett's esophagus, crypt dysplasia, and low-grade dysplasia diagnosed by wide-area transepithelial sampling with 3-dimensional computer-assisted analysis: a retrospective analysis.

BACKGROUND AND AIMS: Wide-area transepithelial sampling with 3-dimensional computer-assisted analysis (WATS3D) is used as an adjunct to forceps biopsy sampling in Barrett's esophagus (BE). BE-associated crypt dysplasia (CD), which can be detected by WATS3D, involves crypts but not surface epithelium. The risk of neoplastic progression of CD has never been evaluated. The prognosis of WATS3D-diagnosed nondysplastic BE (NDBE) and low-grade dysplasia (LGD) is also unknown. We assessed the risk of progression of WATS3D-reported NDBE, CD, and LGD with high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC). METHODS: We analyzed patients who underwent WATS3D in routine care. Eligible patients had 2 WATS3D ≥12 months apart. Patients were categorized by the initial WATS3D finding as NDBE, CD, or LGD. Patient-years of observation were calculated by multiplying the mean follow-up by the number of patients. Progression, defined as a subsequent finding of HGD/EAC on forceps biopsy sampling, was assessed. The crude progression rate was calculated, and Kaplan-Meier analysis compared progression rates stratified by baseline histology. Bivariate analysis identified progression risk factors. RESULTS: Of 151,224 WATS3D cases, 43,145 (29%) had BE. Of these, 4545 patients had 2 WATS3D separated by ≥12 months. The mean follow-up was 1.97 years (range, 1.0-6.42). In patients with baseline NDBE, progression was .08% per patient-year (95% confidence interval [CI], .02%-.14%). Progression of baseline CD was significantly higher, at 1.42% per patient-year (95% CI, 0%-3.01%). For baseline LGD, progression was 5.79% per patient-year (95% CI, 1.02%-10.55%). Other risk factors for progression were increasing age and BE segment length. CONCLUSIONS: NDBE found on WATS3D has a very low risk of progression. CD reported on WATS3D appears to be a neoplastic precursor lesion, with a risk of progression in this study significantly higher than NDBE but lower than LGD. The clinical utility of CD requires further investigation.

Diagnosis of digestive system tumours.

The WHO Classification of Tumours provides the international standards for the classification and diagnosis of tumours. It enables direct comparisons to be made between different countries. In the new fifth edition, the series has gone digital with the launch of a website as well as a series of books, known widely as the WHO Blue Books. The first volume to be produced is on the classification of Digestive System tumours, replacing the successful 2010 version. It has been rewritten and updated accordingly. This article summarises the major diagnostic innovations that have occurred over the last decade and that have now been incorporated in the classification. As an example, it incorporates the recently proposed classification of neuroendocrine tumours, based on the recognition that neuroendocrine tumours and carcinomas differ substantially in the genetic abnormalities that drive their growth, findings relevant to treatment selection and outcome prediction. Several themes have emerged during the production process. One is the importance of the progression from hyperplasia to dysplasia to carcinoma in the evolution of the malignant process. Advances in imaging techniques and endoscopy have resulted in enhanced access to precancerous lesions in the gastrointestinal and biliary tract, necessitating both changes in classification schema and clinical practice. Diagnosis of tumours is no longer the sole purview of pathologists, and some patients now receive treatment before tissue is obtained, based on clinical, radiological and liquid biopsy results. This makes the classification relevant to many disciplines involved in the care of patients with tumours of the digestive system.

Acidic Bile Salts Induce Epithelial to Mesenchymal Transition via VEGF Signaling in Non-Neoplastic Barrett's Cells.

BACKGROUND & AIMS: Metaplastic glands buried under squamous epithelium are frequently detected in patients with Barrett esophagus (BE). This subsquamous intestinal metaplasia might be responsible for cancers that develop despite endoscopic surveillance and for metaplasia recurrences after endoscopic ablation. To determine whether reflux induces BE cells to undergo an epithelial-to-mesenchymal transition (EMT) that produces subsquamous intestinal metaplasia, we assessed EMT in BE cells exposed to acidic bile salts and in rat and human esophageal tissues. METHODS: We compared markers of EMT and cell motility in trans-well and 3-dimensional organotypic culture systems among dysplastic BE epithelial cell lines, nondysplastic telomerase-immortalized BE cell lines (BAR-T), and BAR-T cells exposed acutely or for 20 weeks to acidic bile salts. Vascular endothelial growth factor (VEGF) A was inhibited with a neutralizing antibody or CRISPR-Cas9n and VEGF receptor 2 was inhibited with SU1498 or shRNA, and cells were analyzed by immunohistochemistry, quantitative polymerase chain reaction, or immunoblotting for markers of VEGF signaling and EMT; cell motility was assessed by trans-well assay. We used immunohistochemistry and quantitative polymerase chain reaction to assess EMT markers in the columnar-lined esophagus of rats with surgically induced reflux esophagitis and in esophagectomy specimens from patients with BE. RESULTS: We detected features of EMT (decreased cadherin 1 [CDH1]; increased fibronectin 1, vimentin, and matrix metalloproteinase 2; and increased motility) in dysplastic BE epithelial cell lines and in BAR-T cells exposed for 20 weeks, but not in unexposed BAR-T cells. Acute acidic bile salt exposure induced expression of zinc finger E-box binding homeobox 1 and 2 (ZEB1/2) in BAR-T cells, which decreased their expression of CDH1 and increased motility; inhibitors of VEGF signaling blocked these effects. Columnar-lined esophagus of rats with reflux esophagitis had increased expression of ZEB1/2 and decreased expression of CDH1 compared with controls. Dysplastic BE tissues also had significantly increased levels of ZEB1 and significantly decreased levels of CDH1 compared with nondysplastic BE tissues. CONCLUSIONS: In BE cell lines, acidic bile salts induce EMT by VEGF signaling, which increases expression of ZEB1/2, repressors of CDH1. These observations suggest that reflux induces EMT in metaplastic BE tissues, which promotes development of subsquamous intestinal metaplasia.