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.

Pathological features associated with metastasis in patients with early invasive (pT1) colorectal carcinoma in colorectal polyps.

AIMS: Colorectal carcinoma (CRC) arising in a colorectal polyp with invasion limited to the submucosa is sufficiently treated by complete endoscopic resection alone in many cases. Histological features of the carcinoma including tumour size, vascular invasion and poor tumour differentiation or evidence of de-differentiation, such as tumour budding, are associated with a higher risk for metastasis such that oncological resection is recommended. However, most malignant polyps with these features do not have lymph node metastases at the time of resection, so there is a need for better refinement of the histological risk features. METHODS AND RESULTS: A total of 437 consecutive colorectal polyps with submucosal invasive carcinoma from a single centre, 57 of which had metastatic disease, were supplemented by 30 cases with known metastatic disease from two additional centres. Clinical and histological features of the polyp cancers were reviewed looking for differences between the 87 cancers with metastatic disease and the remaining cases without metastasis. A subgroup of 204 polyps removed intact was also analysed to ensure maximum histological accuracy. CONCLUSIONS: This study confirmed larger invasive tumour size, vascular invasion and poor tumour differentiation as adverse predictive features. Prominent peritumoral desmoplasia and high cytological grade were additional adverse features. A predictive logistic regression model comprised of (i) presence of any form of vascular invasion; (ii) presence of high tumour budding (BD3); (iii) width of invasive tumour component > 8 mm; (iv) depth of invasive tumour > 1.5 mm; and (v) the finding of prominent expansile desmoplasia located within and beyond the deep invasive edge of the carcinoma, showed excellent performance in predicting metastatic disease.

Esophageal squamous cell carcinoma with basaloid features are genetically and prognostically similar to conventional squamous cell carcinoma.

We compared clinicopathologic and molecular features of esophageal squamous cell carcinoma (SCC) with basaloid features to conventional SCC using surgical resections of treatment naïve esophageal carcinomas and cases available from the TCGA database. Twenty-two cases of SCC with basaloid features were identified in the Mass General Brigham pathology archives, including 9 cases with pure basaloid morphology and 13 cases with mixed other features such as conventional well- or poorly differentiated areas or sarcomatoid areas. Thirty-eight cases of conventional SCC matched by tumor stage were used as controls. HPV infection status was tested by p16 immunohistochemistry and HPV mRNA ISH. Digital slides for 94 cases of esophageal SCC from TCGA found in the Genomic Data Commons (GDC) Data Portal were reviewed. Five cases of SCC with basaloid features were identified. Genomic profiles of SCC with basaloid features were compared to the rest of 89 SCCs without basaloid features. In addition, eight tumor sections from six patients selected from our cohort underwent in-house molecular profiling. Compared to conventional SCC, SCC with basaloid features were more frequently associated with diffuse or multifocal squamous dysplasia (p < 0.001). P16 IHC was positive in 2/13 cases, whereas HPV mRNA ISH was negative in 17/17 cases (including both p16-positive cases). SCC with basaloid features and conventional SCC from TCGA showed similar rates of TP53 mutations, CDKN2A/B deletions, and CCDN1 amplifications. TP53 variants were identified in all in-house samples that had sufficient coverage. Survival analyses between SCC with basaloid features versus conventional SCC (matched for tumor stage) did not reveal any statistically significant differences. In conclusion, esophageal SCC with basaloid features has similar survival and genomic alterations to those of conventional SCC, are more frequently associated with diffuse or multifocal dysplasia, and are not associated with HPV (high-risk strains) infection.

Verrucous carcinoma of the oesophagus is a genetically distinct subtype of oesophageal squamous cell carcinoma.

AIMS: Oesophageal verrucous carcinoma (VSCC) is a rare and morphologically distinct type of oesophageal squamous cell carcinoma (SCC). Diagnosing VSCC on biopsy material is challenging, given the lack of significant atypia and the presence of keratinising epithelium and exophytic growth. The molecular pathogenesis of VSCC remains unclear. The aim of this study was to characterise the genomic landscape of VSCC in comparison to conventional oesophageal SCC. METHODS AND RESULTS: Three cases of VSCC from the Brigham and Women's Hospital pathology archive were identified. Formalin-fixed, paraffin-embedded (FFPE) tumour tissue was used for p16 immunohistochemistry (IHC), high-risk human papillomavirus (HPV) in-situ mRNA hybridisation (ISH) and DNA isolation. Tumour DNA was sequenced using a targeted massively parallel sequencing assay enriched for cancer-associated genes. Three additional cases of VSCC were identified by image review of The Cancer Genome Atlas (TCGA) oesophageal SCC cohort. VSCC cases were negative for p16 IHC and high-risk HPV ISH. TP53 mutations (P < 0.001) and copy number variants (CNVs) for CDKN2A (P < 0.001), CDKN2B (P < 0.01) and CCND1 (P < 0.01) were absent in VSCC and significantly less frequent in comparison to conventional SCC. Five VSCC cases featured SMARCA4 missense mutations or in-frame deletions compared to only four of 88 conventional SCC cases (P < 0.001). VSCC featured driver mutations in PIK3CA, HRAS and GNAS. Recurrent CNVs were rare in VSCC. CONCLUSIONS: VSCC is not only morphologically but also genetically distinct from conventional oesophageal SCC, featuring frequent SMARCA4 mutations and infrequent TP53 mutations or CDKN2A/B CNVs. Molecular findings may aid in establishing the challenging diagnosis of VSCC.

Magnetic cholecysto-duodenal anastomosis.

INTRODUCTION: There is a growing interest in minimally invasive endoscopic gallbladder drainage therapies. Unlike stenting, a technology based on magnetic compression could theoretically produce a durable drainage anastomosis without residual foreign material. This study aimed to evaluate the safety and technical feasibility of a cholecysto-duodenal magnetic compression anastomosis. MATERIAL AND METHODS: We performed a survival study of two Yorkshire pigs. Duodenal magnets were deployed endoscopically; reciprocal gallbladder magnets were placed laparoscopically, and the magnets were coupled. Pigs underwent serial endoscopy documenting magnet expulsion and evolution of cholecysto-duodenal anastomosis creation. Necropsies and histological evaluation were performed. Primary endpoints were technical success and safety. Secondary endpoints included anastomosis integrity, patency, and histologic characteristics. RESULTS: Magnets were successfully delivered and coupled. Patent, leak-free anastomoses formed by day 4. Magnets were expelled by day 10. All anastomoses were widely patent at one month (mean diameter 15 mm). Necropsy showed the absence of adhesions affecting the anastomosis. Histology showed complete re-epithelialization without inflammation or foreign body reaction. CONCLUSIONS: Magnetic cholecysto-duodenal anastomosis for gallbladder drainage appears safe and feasible in the animal model. The anastomoses are patent, leak-free, and without inflammation from the presence of foreign material. Technical modifications for magnet delivery under endoscopic ultrasound (EUS) guidance are currently underway.

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.

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 SMAD4 protein expression in gastrointestinal and extra-gastrointestinal carcinomas.

AIMS: SMAD4 (DPC4) is a tumour suppressor gene that is dysregulated in various tumour types, particularly pancreaticobiliary and gastrointestinal carcinomas. Corresponding loss of protein expression has been reported in approximately 50% of pancreatic and 25% of colonic adenocarcinomas. In the evaluation of carcinoma of unknown primary site, immunohistochemical loss of SMAD4 expression is often used to suggest pancreaticobiliary origin, but there are limited data on the spectrum of SMAD4 expression in carcinomas of other sites. This study evaluates the frequency of SMAD4 loss in a large cohort of carcinomas from diverse anatomical sites. METHODS AND RESULTS: Immunohistochemistry for SMAD4 was performed on tissue microarrays or whole tissue sections of 1210 carcinomas from various organs: gastrointestinal tract, liver, pancreas/biliary tract, lung, breast, thyroid, kidney, ovary and uterus. Expression was considered lost when there was complete absence of staining in tumour cell nuclei, in the presence of intact staining in non-neoplastic cells. SMAD4 loss was seen in 58% of pancreatic adenocarcinomas, 27% of appendiceal adenocarcinomas, 19% of colorectal adenocarcinomas, 16% of cholangiocarcinomas, 10% of lung adenocarcinomas and <5% of oesophageal, breast, gastric and mucinous ovarian adenocarcinomas. All papillary thyroid, hepatocellular, non-mucinous ovarian, endometrial and renal cell carcinomas showed intact SMAD4 nuclear expression. CONCLUSION: In addition to pancreaticobiliary, appendiceal and colonic tumours, SMAD4 loss is also seen in a small subset of other carcinomas, specifically breast, lung, oesophageal and gastric adenocarcinomas, all of which are typically CK7-positive, similar to pancreaticobiliary carcinoma. Awareness of SMAD4 loss in these other carcinoma types is helpful in the evaluation of carcinomas of unknown or uncertain primary site.

A Case of Disseminated Histoplasmosis in a Patient with Rheumatoid Arthritis on Abatacept.

Biologic agents are effective treatments for rheumatoid arthritis but are associated with important risks, including severe infections. Tumor Necrosis Factor (TNF) α inhibitors are known to increase the risk of systemic fungal infections such as disseminated histoplasmosis. Abatacept is a biologic agent with a mechanism different from that of TNFα inhibitors: It suppresses cellular immunity by competing for the costimulatory signal on antigen-presenting cells. The risk of disseminated histoplasmosis for patients on abatacept is not known. We report a case of abatacept-associated disseminated histoplasmosis and review the known infectious complications of abatacept. While the safety of resuming biologic agents following treatment for disseminated histoplasmosis is also not known, abatacept is recommended over TNFα inhibitors for rheumatoid arthritis patients with a prior serious infection. We discuss the evidence supporting this recommendation and discuss alternative treatments for rheumatoid arthritis patients with a history of a serious infection.

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.

Prevalence and significance of HMGA2 expression in oesophageal adenocarcinoma.

AIMS: Oesophageal adenocarcinoma (EAC) tumorigenesis has been linked primarily to loss-of-function mutations in tumour suppressor genes. Knowledge of specific oncogenes that drive tumour progression, and their relationship to outcomes, is limited. High mobility group AT-hook 2 (HMGA2) has been reported to be amplified in a subset of EACs, but the clinicopathological and prognostic implications of HMGA2 expression in EAC are unknown. METHODS AND RESULTS: We performed HMGA2 immunohistochemistry and fluorescence in-situ hybridization (FISH) in EAC to determine its clinicopathological and prognostic significance. Ninety-one primary EAC resections without neoadjuvant treatment were identified and immunohistochemistry for HMGA2 was performed. The presence or absence of nuclear staining was evaluated and correlated with predetermined clinicopathological parameters and patient outcomes. A selected subset of tumours was subjected to FISH to identify alterations at the HMGA2 locus. HMGA2 expression was present in 25 of 91 (27.4%) tumours. HMGA2-expressing cells were present in solid, poorly differentiated areas of the tumours at the invasive front, or as single infiltrating cells. FISH showed that three to four copies of HMGA2 are frequently present in EAC irrespective of HMGA2 protein expression and that high level HMGA2 amplification is a rare event. HMGA2 expression was associated with numerous adverse clinicopathological parameters, including higher T- and N-stage, the presence of lymphovascular invasion and with a worse recurrence-free and overall survival. CONCLUSION: Our data suggest that HMGA2 is regulated in EAC primarily through non-chromosomal-level alterations that lead to increased HMGA2 expression. HMGA2-positive EAC correlates with adverse pathological features and worse patient outcomes.

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.

A protein and mRNA expression-based classification of gastric cancer.

The overall survival of gastric carcinoma patients remains poor despite improved control over known risk factors and surveillance. This highlights the need for new classifications, driven towards identification of potential therapeutic targets. Using sophisticated molecular technologies and analysis, three groups recently provided genetic and epigenetic molecular classifications of gastric cancer (The Cancer Genome Atlas, 'Singapore-Duke' study, and Asian Cancer Research Group). Suggested by these classifications, here, we examined the expression of 14 biomarkers in a cohort of 146 gastric adenocarcinomas and performed unsupervised hierarchical clustering analysis using less expensive and widely available immunohistochemistry and in situ hybridization. Ultimately, we identified five groups of gastric cancers based on Epstein-Barr virus (EBV) positivity, microsatellite instability, aberrant E-cadherin, and p53 expression; the remaining cases constituted a group characterized by normal p53 expression. In addition, the five categories correspond to the reported molecular subgroups by virtue of clinicopathologic features. Furthermore, evaluation between these clusters and survival using the Cox proportional hazards model showed a trend for superior survival in the EBV and microsatellite-instable related adenocarcinomas. In conclusion, we offer as a proposal a simplified algorithm that is able to reproduce the recently proposed molecular subgroups of gastric adenocarcinoma, using immunohistochemical and in situ hybridization techniques.

Association of Acute Gastroesophageal Reflux Disease With Esophageal Histologic Changes.

IMPORTANCE: The histologic changes associated with acute gastroesophageal reflux disease (GERD) have not been studied prospectively in humans. Recent studies in animals have challenged the traditional notion that reflux esophagitis develops when esophageal surface epithelial cells are exposed to lethal chemical injury from refluxed acid. OBJECTIVE: To evaluate histologic features of esophageal inflammation in acute GERD to study its pathogenesis. DESIGN, SETTING, AND PARTICIPANTS: Patients from the Dallas Veterans Affairs Medical Center who had reflux esophagitis successfully treated with proton pump inhibitors (PPIs) began 24-hour esophageal pH and impedance monitoring and esophagoscopy (including confocal laser endomicroscopy [CLE]) with biopsies from noneroded areas of distal esophagus at baseline (taking PPIs) and at 1 week and 2 weeks after stopping the PPI medication. Enrollment began May 2013 and follow-up ended July 2015. INTERVENTIONS: PPIs stopped for 2 weeks. MAIN OUTCOMES AND MEASURES: Twelve patients (men, 11; mean age, 57.6 year [SD, 13.1]) completed the study. Primary outcome was change in esophageal inflammation 2 weeks after stopping the PPI medication, determined by comparing lymphocyte, eosinophil, and neutrophil infiltrates (each scored on a 0-3 scale) in esophageal biopsies. Also evaluated were changes in epithelial basal cell and papillary hyperplasia, surface erosions, intercellular space width, endoscopic grade of esophagitis, esophageal acid exposure, and mucosal impedance (an index of mucosal integrity). RESULTS: At 1 week and 2 weeks after discontinuation of PPIs, biopsies showed significant increases in intraepithelial lymphocytes, which were predominantly T cells (median [range]: 0 (0-2) at baseline vs 1 (1-2) at both 1 week [P = .005] and 2 weeks [P = .002]); neutrophils and eosinophils were few or absent. Biopsies also showed widening of intercellular spaces (confirmed by CLE), and basal cell and papillary hyperplasia developed without surface erosions. Two weeks after stopping the PPI medication, esophageal acid exposure increased (median: 1.2% at baseline to 17.8% at 2 weeks; Δ, 16.2% [95% CI, 4.4%-26.5%], P = .005), mucosal impedance decreased (mean: 2671.3 Ω at baseline to 1508.4 Ω at 2 weeks; Δ, 1162.9 Ω [95% CI, 629.9-1695.9], P = .001), and all patients had evidence of esophagitis. CONCLUSIONS AND RELEVANCE: In this preliminary study of 12 patients with severe reflux esophagitis successfully treated with PPI therapy, stopping PPI medication was associated with T lymphocyte-predominant esophageal inflammation and basal cell and papillary hyperplasia without loss of surface cells. If replicated, these findings suggest that the pathogenesis of reflux esophagitis may be cytokine-mediated rather than the result of chemical injury. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01733810.

Mitochondrial STAT3 contributes to transformation of Barrett's epithelial cells that express oncogenic Ras in a p53-independent fashion.

Metaplastic epithelial cells of Barrett's esophagus transformed by the combination of p53-knockdown and oncogenic Ras expression are known to activate signal transducer and activator of transcription 3 (STAT3). When phosphorylated at tyrosine 705 (Tyr705), STAT3 functions as a nuclear transcription factor that can contribute to oncogenesis. STAT3 phosphorylated at serine 727 (Ser727) localizes in mitochondria, but little is known about mitochondrial STAT3's contribution to carcinogenesis in Barrett's esophagus, which is the focus of this study. We introduced a constitutively active variant of human STAT3 (STAT3CA) into the following: 1) non-neoplastic Barrett's (BAR-T) cells; 2) BAR-T cells with p53 knockdown; and 3) BAR-T cells that express oncogenic H-Ras(G12V). STAT3CA transformed only the H-Ras(G12V)-expressing BAR-T cells (evidenced by loss of contact inhibition, formation of colonies in soft agar, and generation of tumors in immunodeficient mice), and did so in a p53-independent fashion. The transformed cells had elevated levels of both mitochondrial (Ser727) and nuclear (Tyr705) phospho-STAT3. Introduction of a STAT3CA construct with a mutated tyrosine phosphorylation site into H-Ras(G12V)-expressing Barrett's cells resulted in high levels of mitochondrial phospho-STAT3 (Ser727) with little or no nuclear phospho-STAT3 (Tyr705), and the cells still formed tumors in immunodeficient mice. Thus tyrosine phosphorylation of STAT3 is not required for tumor formation in Ras-expressing Barrett's cells. We conclude that mitochondrial STAT3 (Ser727) can contribute to oncogenesis in Barrett's cells that express oncogenic Ras. These findings suggest that agents targeting STAT3 might be useful for chemoprevention in patients with Barrett's esophagus.

Radiofrequency ablation for Barrett's-associated intramucosal carcinoma: a multi-center follow-up study.

BACKGROUND: Radiofrequency ablation (RFA), with or without endoscopic mucosal resection (EMR), has been validated as a safe, effective and durable treatment option for dysplastic Barrett's esophagus. Its durability in eradicating Barrett's-associated intramucosal carcinoma (IMC), however, is unclear. We set out to assess the long-term safety and efficacy of RFA for IMC. METHODS: Retrospective review of two tertiary care facility records for patients undergoing RFA, with or without EMR, for biopsy-proven IMC. Our primary outcome of interest was to quantify the rate of durable complete eradication for intestinal metaplasia and for IMC and associated dysplasia. A multi-variate regression analysis was performed to identify features which correlate with durable eradication of IMC/dysplasia. Our secondary outcome of interest was treatment-related complications. RESULTS: 36 patients (26 male; mean age 64 ± 12 years), with a mean Barrett's length of 3.5 ± 2.5 cm, underwent RFA for biopsy-proven IMC. EMR was performed in 31 (86%) prior to or during RFA. Complete eradication of IMC/dysplasia was achieved in 32/36 (89%) and patients required a mean of 1 ± 1 EMR and 2 ± 1 RFA sessions to achieve eradication. During a mean follow-up period of 24 ± 19 months, durable complete eradication of IMC/dysplasia was achieved in 29/36 (81%) patients. On multi-variate regression analysis, undergoing an EMR prior to RFA was associated with an increased likelihood of maintaining durable eradication of IMC/dysplasia (p = 0.03). Treatment-related complications included: bleeding (3%) and stricture formation (19%). CONCLUSION: RFA is an effective and durable treatment option for Barrett's-associated IMC. Greater than 80% of patients will achieve and maintain complete eradication of IMC at a mean of 2 years follow-up.

Transcriptionally Active Human Papillomavirus Infection in a Minority of Esophageal Squamous Cell Carcinomas in North America.

The role of Human papillomavirus (HPV) infection in esophageal squamous cell carcinoma (ESCC) is a topic of ongoing debate. This study used two screening approaches to look for evidence of HPV infection in esophageal squamous cell carcinoma. We initially checked for HPV infection in a randomly selected group of 53 ESCC cases. We did not detect any tumors positive for high-risk HPV. However, during clinical practice, we identified an HPV-positive ESCC in the distal esophagus, which tested positive for HPV16. This index case was TP53 wild-type, as determined by next-generation DNA sequencing (NGS). Since TP53 mutations are rare in other HPV-driven cancers, we improved our screening method by limiting our screen to a subset of ESCC cases without TP53 mutations. A second screen of 95 ESCCs (from 93 patients) sequenced by NGS revealed an additional 7 ESCCs with TP53 wild-type status (7.3% of the total). Of the 7 cases, 2 cases were found to be high-risk HPV positive. Both patients also tested positive for circulating cell-free HPV DNA and had a complete response to neoadjuvant chemoradiation. The index patient had microscopic residual tumor following neoadjuvant therapy. The patient underwent adjuvant immunotherapy and remained disease free after 22 months of surveillance. This study affirms the transcriptionally active status of high-risk HPV in a minority of ESCC patients in North America.

Genomic signatures of past and present chromosomal instability in Barrett's esophagus and early esophageal adenocarcinoma.

The progression of precancerous lesions to malignancy is often accompanied by increasing complexity of chromosomal alterations but how these alterations arise is poorly understood. Here we perform haplotype-specific analysis of chromosomal copy-number evolution in the progression of Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC) on multiregional whole-genome sequencing data of BE with dysplasia and microscopic EAC foci. We identify distinct patterns of copy-number evolution indicating multigenerational chromosomal instability that is initiated by cell division errors but propagated only after p53 loss. While abnormal mitosis, including whole-genome duplication, underlies chromosomal copy-number changes, segmental alterations display signatures of successive breakage-fusion-bridge cycles and chromothripsis of unstable dicentric chromosomes. Our analysis elucidates how multigenerational chromosomal instability generates copy-number variation in BE cells, precipitates complex alterations including DNA amplifications, and promotes their independent clonal expansion and transformation. In particular, we suggest sloping copy-number variation as a signature of ongoing chromosomal instability that precedes copy-number complexity. These findings suggest copy-number heterogeneity in advanced cancers originates from chromosomal instability in precancerous cells and such instability may be identified from the presence of sloping copy-number variation in bulk sequencing data.

Use of Real Time Specimen Radiography to Evaluate the Number of Stereotactic Core Biopsy Specimens Containing Calcifications Required for Diagnosis.

OBJECTIVE: To determine the minimum number of stereotactic biopsy specimens containing calcifications sufficient for pathologic diagnosis and the minimum number of specimens containing calcifications sufficient for immunohistochemistry (IHC) in cases of malignancy. METHODS: In this IRB-exempt quality assurance initiative, individual specimens from 126 patients with 129 calcified targets retrieved using a stereotactic system with real time specimen imaging were prospectively analyzed. Pathology was reported independently for each specimen containing calcifications. In every case, the pathologist reported which specimen containing calcifications was sufficient for diagnosis and, in cases of malignancy, which calcified specimen was sufficient for diagnosis and IHC. RESULTS: A diagnosis was made from the first calcified specimen in 74% of cases (95/129), from the first two calcified specimens in 92% (119/129) of cases, and from the first three calcified specimens in 100% of cases. Pathology was benign in 66% (85/129) of cases, with the diagnosis made from the first calcified specimen in 78% (66/85) of cases. High-risk lesions were the primary pathology in 8% (11/129) of cases, with 55% (6/11) diagnosed from the first calcified specimen. Pathology was malignant in 26% (33/129) of cases. The first calcified specimen was sufficient for diagnosis and IHC in 73% (24/33) of malignancies and the first three calcified specimens were sufficient for diagnosis and IHC in all cases of malignancy. CONCLUSION: Three cores verified to contain calcifications on real time specimen imaging were sufficient to make a diagnosis in all cases and to make a diagnosis and obtain IHC in nearly all cases of malignancy.