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.

Gastrointestinal tissue-based molecular biomarkers: a practical categorisation based on the 2019 World Health Organization classification of epithelial digestive tumours.

Molecular biomarkers have come to constitute one of the cornerstones of oncological pathology. The method of classification not only directly affects the manner in which patients are diagnosed and treated, but also guides the development of drugs and of artificial intelligence tools. The aim of this article is to organise and update gastrointestinal molecular biomarkers in order to produce an easy-to-use guide for routine diagnostics. For this purpose, we have extracted and reorganised the molecular information on epithelial neoplasms included in the 2019 World Health Organization classification of tumours. Digestive system tumours, 5th edn.

Detection of Mutations in Barrett's Esophagus Before Progression to High-Grade Dysplasia or Adenocarcinoma.

BACKGROUND & AIMS: Barrett's esophagus (BE) is the greatest risk factor for esophageal adenocarcinoma (EAC), but only a small proportion of patients with BE develop cancer. Biomarkers might be able to identify patients at highest risk of progression. We investigated genomic differences in surveillance biopsies collected from patients whose BE subsequently progressed compared to patients whose disease did not progress. METHODS: We performed a retrospective case-control study of 24 patients with BE that progressed to high-grade dysplasia (HGD, n = 14) or EAC (n = 10). The control group (n = 73, called non-progressors) comprised patients with BE and at least 5 years of total endoscopic biopsy surveillance without progression to HGD or EAC. From each patient, we selected a single tissue sample obtained more than 1 year before progression (cases) or more than 2 years before the end of follow-up (controls). Pathogenic mutations, gene copy numbers, and ploidy were compared between samples from progressors and non-progressors. RESULTS: TP53 mutations were detected in 46% of samples from progressors and 5% of non-progressors. In this case-control sample set, TP53 mutations in BE tissues increased the adjusted risk of progression 13.8-fold (95% confidence interval, 3.2-61.0) (P < .001). We did not observe significant differences in ploidy or copy-number profile between groups. We identified 147 pathogenic mutations in 57 distinct genes-the average number of pathogenic mutations was higher in samples from progressors (n = 2.5) than non-progressors (n = 1.2) (P < .001). TP53 and other somatic mutations were recurrently detected in samples with limited copy-number changes (aneuploidy). CONCLUSIONS: In genomic analyses of BE tissues from patients with or without later progression to HGD or EAC, we found significantly higher numbers of TP53 mutations in BE from patients with subsequent progression. These mutations were frequently detected before the onset of dysplasia or substantial changes in copy number.

Loss of expression of MLH1 in non-dysplastic crypts is a harbinger of neoplastic progression in sessile serrated adenomas/polyps.

AIMS: Dysplasia in colonic sessile serrated adenomas (SSAs)/sessile serrated polyps often shows loss of MLH1 expression as determined with immunohistochemistry, but the significance of loss of MLH1 expression in non-dysplastic crypts in these polyps is less well studied. The purpose of this study was to evaluate the prevalence of loss of MLH1 expression in non-dysplastic crypts in SSAs, and to evaluate its significance with regard to progression of these polyps. METHODS AND RESULTS: Four hundred SSAs, including 158 SSAs without dysplasia, 219 SSAs with dysplasia (SSAD), and 23 SSAs with invasive adenocarcinoma (SSAC), were evaluated immunohistochemically for loss of MLH1 expression in both non-dysplastic and dysplastic portions of the polyps. Seventy-one of 400 (18%) SSAs showed loss of MLH1 expression in non-dysplastic crypts. The prevalence of MLH1-deficient non-dysplastic crypts was higher in polyps with dysplasia or carcinoma (7%, 22%, and 52% in SSAs, SSADs, and SSACs, respectively; P < 0.0001). When SSAs with MLH1-deficient dysplasia and those with MLH-1-proficient dysplasia were compared, those with MLH1-deficient dysplasia were more likely to have MLH1-deficient non-dysplastic crypts (66% versus 8.1%, P < 0.0001) and a greater number of discrete foci (3.6 foci versus 1.1 foci, P = 0.008). Also, non-dysplastic crypts with loss of MLH1 expression were more likely to be contiguous with the dysplasia when the dysplasia also showed loss of MLH1 expression (26% versus 0%, P = 0.02). CONCLUSIONS: Our results suggest that loss of MLH1 expression in non-dysplastic crypts in SSAs precedes the development of MLH1-deficient dysplasia and adenocarcinoma, and may be a biomarker of an advanced serrated polyp even in the absence of dysplasia.

Discordance Among Pathologists in the United States and Europe in Diagnosis of Low-Grade Dysplasia for Patients With Barrett's Esophagus.

BACKGROUND & AIMS: There is suboptimal inter-observer agreement, even among expert gastrointestinal pathologists, in the diagnosis of low-grade dysplasia (LGD) in patients with Barrett's esophagus (BE). We analyzed histopathologic criteria required for a diagnosis of LGD using the new subcategories of LGD with inflammatory and dysplastic features. We categorized each diagnosis based on the level of confidence and assessed inter-observer agreement among gastrointestinal pathologists from 5 tertiary centers in the United States and Europe. METHODS: In the first phase of the study, 3 pathologists held a consensus conference at which they discussed the diagnostic criteria for LGD. In the second phase, 79 slides from patients with BE (23 samples of non-dysplastic BE, 22 samples of LGD, and 34 samples of high-grade dysplasia) were identified, randomly assigned to 7 pathologists (4 from the United States and 3 from Europe), and interpreted in a blinded fashion. κ Values were calculated for inter-observer agreement. We performed multinomial logistic regression analysis to assess the weighting of histologic features with the diagnosis. RESULTS: The overall κ value for diagnosis was 0.43 (95% confidence interval [CI], 0.42-0.48). When categorized based on degree of dysplasia, the κ value was 0.22 (95% CI, 0.11-0.29) for non-dysplastic BE, 0.11 (95% CI, 0.004-0.15) for LGD, and 0.43 (95% CI, 0.36-0.46) for high-grade dysplasia. When all pathologists made a diagnosis with high confidence, the inter-observer agreement was substantial among the US pathologists (κ, 0.63; 95% CI, 0.61-0.66) and European pathologists (κ, 0.80; 95% CI, 0.74-0.97). The κ values for all diagnoses made by European pathologists were higher than those made by US pathologists. CONCLUSIONS: In an analysis of criteria used in histopathologic diagnosis of LGD, we did not observe improvement in level of agreement among experienced pathologists, even after accounting for inflammation. The level of inter-observer agreement increased with level of pathologist confidence. There was also a difference in reading of histopathology samples of BE tissues between US and European pathologists.

Clinicopathologic and outcome study of sessile serrated adenomas/polyps with serrated versus intestinal dysplasia.

It is believed that sessile serrated adenomas/polyps lead to the development of microsatellite unstable cancer via a dysplasia-carcinoma sequence. Little is known regarding the morphologic and biologic features, and outcome of sessile serrated adenomas/polyps with dysplasia, or of its specific dysplasia subtypes (intestinal versus serrated). The aims of this study were to analyze and compare the clinical, pathologic, and outcome characteristics of sessile serrated adenomas/polyps with serrated versus intestinal dysplasia. The study included 86 patients with sessile serrated adenomas/polyps with dysplasia (50 serrated dysplasia, 22 intestinal dysplasia, 14 mixed serrated and intestinal dysplasia). The clinical and pathologic features, and the prevalence rate of prior, concurrent, and future neoplastic lesions, were compared between sessile serrated adenomas/polyps with intestinal versus serrated dysplasia and with matched control patients with ≥1 conventional adenoma. The mean age of the patients, polyp size, and prevalence of adenocarcinoma within the polyps were significantly higher in sessile serrated adenomas/polyps with high versus low-grade dysplasia. Sessile serrated adenomas/polyps with intestinal dysplasia showed a significantly higher rate of adenocarcinoma (23%) compared with those with serrated dysplasia (6%, P=0.05), and the high-grade lesions occurred at a significantly younger age in the former compared with the latter (65 versus 76 years, P=0.05). Compared with patients with conventional adenomas, patients with sessile serrated adenomas/polyps with dysplasia showed a significantly higher rate of invasive carcinoma within the polyps (12 versus 0%, P=0.01) and a significantly lower association with prior or future conventional adenomas. Sessile serrated adenomas/polyps with dysplasia should be considered high-risk neoplastic precursor lesions, particularly those with intestinal dysplasia. Cancer may develop from sessile serrated adenomas/polyps with either type of dysplasia.

Mutational landscape of goblet cell carcinoids and adenocarcinoma ex goblet cell carcinoids of the appendix is distinct from typical carcinoids and colorectal adenocarcinomas.

There is limited data on the spectrum of molecular alterations in goblet cell carcinoids and adenocarcinoma ex goblet cell carcinoids of the appendix. We used next generation sequencing to determine mutations of potential pathogenetic and therapeutic significance in this rare group of tumors. Adequate DNA was successfully extracted in 34/46 cases and the final group included 18 goblet cell carcinoids and 16 adenocarcinoma ex goblet cell carcinoids. Illumina TruSeq™ was used for sequencing exons of a custom 282 gene panel using an Illumina HiSeq 2000. All cases had a minimum coverage depth of at least 50 reads. After filtering through the Exome Sequencing Project, the number of mutations per case ranged from 0-9 (mean:3). The mutational burden in adenocarcinoma ex goblet cell carcinoids was significantly higher than goblet cell carcinoids (mean 5 vs. 3; p < 0.05) but the spectrum of alterations overlapped between the two groups. The most frequent mutations included ARID1A (4/34), ARID2 (4/34), CDH1 (4/34), RHPN2 (4/34), and MLL2 (3/34). Some mutations typically seen in conventional colorectal adenocarcinomas were also identified but with much lower frequency (APC :4/34; KRAS :2/34). MLL2 and KRAS mutations were only seen in adenocarcinoma ex goblet cell carcinoids and TP53 mutations were limited to poorly differentiated adenocarcinoma ex goblet cell carcinoids (2/34). Copy number changes could be evaluated in 15/34 cases and showed low copy number gains in CDKN1B (6/15) and NFKBIA (6/15), among others. The overlapping molecular alterations suggest that goblet cell carcinoids and adenocarcinoma ex goblet cell carcinoids are best considered two grades of differentiation of the same tumor rather than two distinct histological types. Mutations in TP53, CDH1 and MLL2 mutations were predominantly present in the adenocarcinoma ex goblet cell carcinoid group consistent with transformation to a higher grade lesion. The unique mutational profile also offers an explanation for the poor chemosensitivity in these tumors and highlights the need for developing new targeted therapies.

Hypoxia-inducible factor-2α plays a role in mediating oesophagitis in GORD.

OBJECTIVE: In an earlier study wherein we induced acute reflux by interrupting proton pump inhibitor (PPI) therapy in patients with reflux oesophagitis (RO) healed by PPIs, we refuted the traditional concept that RO develops as an acid burn. The present study explored our alternative hypothesis that RO results from reflux-stimulated production of pro-inflammatory molecules mediated by hypoxia-inducible factors (HIFs). DESIGN: Using oesophageal biopsies taken from patients in our earlier study at baseline and at 1 and 2 weeks off PPIs, we immunostained for HIF-1α, HIF-2α and phospho-p65, and measured pro-inflammatory molecule mRNAs. We exposed human oesophageal squamous cell lines to acidic bile salts, and evaluated effects on HIF activation, p65 function, pro-inflammatory molecule production and immune cell migration. RESULTS: In patient biopsies, increased immunostaining for HIF-2α and phospho-p65, and increased pro-inflammatory molecule mRNA levels were seen when RO redeveloped 1 or 2 weeks after stopping PPIs. In oesophageal cells, exposure to acidic bile salts increased intracellular reactive oxygen species, which decreased prolyl hydroxylase function and stabilised HIF-2α, causing a p65-dependent increase in pro-inflammatory molecules; conditioned media from these cells increased T cell migration rates. HIF-2α inhibition by small hairpin RNA or selective small molecule antagonist blocked the increases in pro-inflammatory molecule expression and T cell migration induced by acidic bile salts. CONCLUSIONS: In patients developing RO, increases in oesophageal HIF-2α correlate with increased pro-inflammatory molecule expression. In oesophageal epithelial cells, acidic bile salts stabilise HIF-2α, which mediates expression of pro-inflammatory molecules. HIF-2α appears to have a role in RO pathogenesis. TRIAL REGISTRATION NUMBER: NCT01733810; Results.

In oesophageal squamous cells, nitric oxide causes S-nitrosylation of Akt and blocks SOX2 (sex determining region Y-box 2) expression.

OBJECTIVE: Barrett's metaplasia might develop if GORD causes oesophageal squamous cells to convert into columnar cells. Acid and bile exposures upregulate columnar differentiation genes like CDX2 in oesophageal squamous cells, but it is not known if such exposures downregulate squamous differentiation genes like SOX2. In addition to acid and bile, patients with GORD also have high oesophageal concentrations of nitric oxide (NO). This study aims to determine how acid, bile salts and NO affect genes that influence oesophageal cell phenotype. DESIGN: Oesophageal squamous cells from patients with Barrett's oesophagus were exposed to acidic bile salts or NOC-9 (an NO donor). SOX2, p63 (squamous transcription factor) and CDX2 mRNAs were measured by quantitative RT-PCR. SOX2 and its regulatory Akt pathway proteins were evaluated by western blotting. S-nitrosylation by NO was blocked by dithiothreitol. Immunohistochemistry for SOX2 was performed on the oesophagus of rats with surgically induced GORD which were fed diets with and without nitrite supplementation. RESULTS: In oesophageal squamous cells, NO profoundly decreased SOX2 protein and caused a significantly greater decrease in SOX2 mRNA than did acidic bile salts. NO also decreased p63 and increased CDX2 expression. NO caused S-nitrosylation of Akt, blocking its phosphorylation. Akt pathway inhibition by LY294002 or Akt siRNA reduced SOX2 mRNA. Rats fed with nitrite-supplemented diets exhibited weaker SOX2 oesophageal staining than rats fed with normal diets. CONCLUSIONS: In oesophageal squamous cells, NO blocks SOX2 expression through Akt S-nitrosylation. NO also increases CDX2 and decreases p63 expression. By triggering molecular events preventing squamous differentiation while promoting intestinal differentiation, NO might contribute to Barrett's pathogenesis.

Colorectal cancer in Crohn's colitis is comparable to sporadic colorectal cancer.

PURPOSE: It is now recognized that Crohn's disease (CD), similar to ulcerative colitis (UC), carries an up to 20-fold higher cancer risk, and the development of colorectal carcinoma (CRC) is a major long-term complication. Once CRC is present, molecular profiling is one of the components in selecting appropriate treatment strategies; however, in contrast to UC, genetic alterations in Crohn's colitis-associated CRC are poorly understood. METHODS: In a series of 227 patients with Crohn's colitis, we identified 33 cases of CRC (~14 %) and performed targeted mutational analysis of BRAF/KRAS/NRAS and determined microsatellite status as well as immunophenotype of the tumors. RESULTS: In the CRC cohort, the median age at time of cancer diagnosis was 58 (range 34-77 vs. 59.5 in sporadic; P = 0.81) and the median CD duration was 29 years (range 6-45). As a group, CRC complicating Crohn's colitis is BRAF (97 %) and NRAS (100 %) wild type and the vast majority is microsatellite stable (94 %); KRAS-mutations were found in six cases (18 %). Stage grouping, anatomic distribution, and overall survival were similar to sporadic CRC; however, long-standing CD (≥25 years) as well as gastric-immunophenotype (MUC5AC+) was associated with significantly shorter overall survival (P = 0.0029; P = 0.036, respectively). CONCLUSION: In summary, the clinicopathological and molecular profile of CD-associated CRC is similar to that observed in sporadic CRC.

NSAIDs modulate clonal evolution in Barrett's esophagus.

Cancer is considered an outcome of decades-long clonal evolution fueled by acquisition of somatic genomic abnormalities (SGAs). Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to reduce cancer risk, including risk of progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA). However, the cancer chemopreventive mechanisms of NSAIDs are not fully understood. We hypothesized that NSAIDs modulate clonal evolution by reducing SGA acquisition rate. We evaluated thirteen individuals with BE. Eleven had not used NSAIDs for 6.2±3.5 (mean±standard deviation) years and then began using NSAIDs for 5.6±2.7 years, whereas two had used NSAIDs for 3.3±1.4 years and then discontinued use for 7.9±0.7 years. 161 BE biopsies, collected at 5-8 time points over 6.4-19 years, were analyzed using 1Million-SNP arrays to detect SGAs. Even in the earliest biopsies there were many SGAs (284±246 in 10/13 and 1442±560 in 3/13 individuals) and in most individuals the number of SGAs changed little over time, with both increases and decreases in SGAs detected. The estimated SGA rate was 7.8 per genome per year (95% support interval [SI], 7.1-8.6) off-NSAIDs and 0.6 (95% SI 0.3-1.5) on-NSAIDs. Twelve individuals did not progress to EA. In ten we detected 279±86 SGAs affecting 53±30 Mb of the genome per biopsy per time point and in two we detected 1,463±375 SGAs affecting 180±100 Mb. In one individual who progressed to EA we detected a clone having 2,291±78 SGAs affecting 588±18 Mb of the genome at three time points in the last three of 11.4 years of follow-up. NSAIDs were associated with reduced rate of acquisition of SGAs in eleven of thirteen individuals. Barrett's cells maintained relative equilibrium level of SGAs over time with occasional punctuations by expansion of clones having massive amount of SGAs.