Intraluminal pH and Goblet Cell Density in Barrett’s Esophagus

Introduction

Goblet cells in Barrett??s esophagus (BE) vary in their density within the Barrett??s segment. Exposure of Barrett??s epithelium to bile acids is a major stimulant for goblet cell formation. The dissociation of bile acids into forms that penetrate Barrett??s epithelium is known to be pH dependent. We hypothesized that variations in the esophageal luminal pH environment explains the variability in goblet cell density. The aim of this study was to correlate esophageal luminal pH with goblet cell density in patients with BE.

Methods

A customized six-sensor pH catheter was positioned with the most distal sensor in the stomach and the remaining sensors located 1?cm below and 1, 3, 5, and 8?cm above the upper border of the lower esophageal sphincter in five normal subjects and six patients with long-segment BE. The luminal pH was measured by each sensor for 24-h and expressed as median pH. Patients with BE had four quadrant biopsies at levels corresponding to the location of the pH sensors. Goblet cell density was graded from 0 to 3 based on the number per high-power field.

Results

In normal subjects, the median pH values recorded in the sensors within the lower esophageal sphincter (LES) and esophageal body were all above 5. In patients with BE, the median pH recorded by the sensor within the LES was 2.8 and increased progressively to 4.7 in the sensor at 8?cm above the LES. Goblet cell density was significantly lower in the distal Barrett??s segment exposed to a median pH of 2.2 and increased in the proximal Barrett??s segment exposed to a median pH of 4.4 (p?=?0.003).

Conclusion

Patients with BE have a goblet cell gradient that correlates directly with an esophageal luminal pH gradient. This suggests that goblet cell differentiation is pH dependent and likely due to the effect of pH on bile acid dissociation.     

Activation of the BMP4 Pathway and Early Expression of CDX2 Characterize Non-specialized Columnar Metaplasia in a Human Model of Barrett’s Esophagus

Background

A human model of gastroesophageal reflux disease was used to examine the contribution of a non-specialized columnar type of metaplasia (NSCM) and key molecular events (BMP4 and CDX2) in the development of Barrett??s esophagus.

Methods

Biopsies of the remnant esophagus from 18 patients undergoing esophagectomy with gastric preservation were taken at 6?C36-month intervals postoperatively and examined for activation of the BMP pathway (BMP4/P-Smad 1/5/8) and CDX2 and CDX1 expression by imunohistochemistry, quantitative real-time PCR, Western blot, and in situ hybridization.

Results

A short segment (mean 15.6?mm) of NSCM was detected in 10 (56%) patients, with an increasing prevalence from 17% at 6?months to 62% at 36?months. Nuclear expression of P-Smad 1/5/8 in the squamous epithelium close to the anastomosis with strong expression in all epithelial cells of NSCM areas was found. Forty-eight (63%) biopsies with NSCM showed scattered nuclear expression of CDX2. Two cases showed isolated glands at 18, 24, and 36?months that fully expressed CDX2 and co-expressed CDX1. BMP4 mRNA and CDX2 mRNA levels were significantly greater in NSCM than in squamous epithelium.

Conclusions

BMP4 activation in NSCM and early expression of CDX2 are involved in the columnar epithelial differentiation of Barrett??s esophagus.     

Esophageal acid-clearance physiology is altered after Nissen-Collis gastroplasty

Background

Nissen-Collis gastroplasty (NCG) is an effective treatment for short esophagus, but it sometimes is associated with abnormal postoperative esophageal acid exposure. This study was designed to test the hypothesis that NCG prevents gastric reflux and that pathologic distal esophagus acid exposure is due to prolonged acid clearance in the “neoesophagus.”

Methods

The study enrolled 11 normal healthy subjects (ten patients status post—laparoscopic Nissen fundoplication and nine patients status post-NCG). All the participants were age and gender matched, and all were studied via manometry, acid-clearance test, and 24-h pH analysis. The clearance test was performed according to an established protocol. A 15-ml acid bolus (pH 1.2) was rapidly infused (×4) using a nasogastric tube 15 cm proximal to the lower esophageal sphincter, followed by dry swallows every 30 s until the esophageal pH rose above 4.

Results

All the subjects had normal esophageal peristalsis and distal amplitudes. The acid-clearance time was significantly higher with NCG (P < 0.001 vs Nissen and normal subjects). Pathologic esophageal acid exposure occurred in one of ten Nissen patients (10 %) and in two of nine NCG patients (22 %) (nonsignificant difference [NS]). The median distal esophageal acid exposure time during the 24-h pH study was similar in the two groups (NCG, 1.2 %; Nissen, 2.5 %; NS).

Conclusions

The findings showed that NCG is an adequate antireflux procedure but that it is characterized by a delayed esophageal acid clearance.     

Barrett's esophagus: congenital or acquired? An experimental study of esophageal mucosal regeneration in the dog.

The mucosa was removed from the distal esophagus in 35 dogs divided into three groups according to the presence or absence of gastroesophageal reflux and gastric hypersecretion. Partial or complete re-epithelization of the denuded segment by columnar mucus-secreting cells took place. Re-epithelization by squamous epithelium predominated in preparations with a competent inferior esophageal sphincter, while total or nearly total replacement by columnar cells characterized those preparations with gastroesophageal reflux and gastric hypersecretion. Peristalsis persisted in the operated portion of the esophagus as disclosed by esophageal manometry, although contractions were sometimes repetitive and prolonged. Measurement of potential difference was useful in monitoring the re-epithelization process. These experiments support the concept that the columnar-lined distal esophagus ("Barrett's esophagus") may be an acquired condition in which squamous epithelium destroyed by gastroesophageal reflux is replaced, through creeping substitution, by columnar cells of gastric or junctional origin.     

Clinicopathological Significance of BMP7 Expression in Esophageal Squamous Cell Carcinoma

Background

Bone morphogenetic proteins (BMPs) are secreted signaling molecules belonging to the transforming growth factor-?? (TGF-??) superfamily of growth factors. Recent studies have shown that the influence of the expression of BMP7 was altered in several tumors. The purpose of the current study was to examine the expression of BMP7 in esophageal squamous cell carcinoma and to clarify the clinical impact of BMP7 expression in esophageal squamous cell carcinoma (ESCC).

Methods

A total of 180 patients with ESCC who underwent surgical resection from 1991 to 2004 were eligible for this study. The expression of BMP7 in esophageal tumor tissues was examined immunohistochemically.

Results

BMP7 expression was found in the cytoplasm of cancer cells. BMP7 positivity was observed in 61.7% of tumors. The BMP7-positive group had deeper progression, more advanced stages, and greater venous invasion than those without BMP7 expression (p?p?p?p?p?Conclusions Patients with expression of BMP7 in ESCC had high malignant potential. BMP7 could be a useful prognostic marker for patients with ESCC.     

Impact of Inflammation–Metaplasia–Adenocarcinoma Sequence and Inflammatory Microenvironment in Esophageal Carcinogenesis Using Surgical Rat Models

Background and Aims

Chronic inflammation has been demonstrated to correlate with tumor onset and progression. Tumor-associated macrophages (TAMs) play an important role in inflammatory tumor microenvironment. We hypothesized that an inflammatory microenvironment around TAMs may promote the development of esophageal carcinomas when induced by duodenal content reflux without carcinogens.

Animals and Methods

A total gastrectomy followed by esophagojejunostomy was performed on rats in order to induce chronic duodenal content reflux esophagitis. The animals were sacrificed sequentially, at the 20th, 30th, 40th and 50th week after surgery, and their esophagi were examined. The primary antibodies against CD68, CD163, pStat3 and Foxp3 were used. Expression and localization of infiltrated cells was assessed by immunohistochemical analysis.

Results

At 20-weeks’ post-surgery, squamous proliferative hyperplasia (PHP) and Barrett’s metaplasia (BM) were observed. Adenocarcinoma (ADC) associated with BM, and squamous cell carcinoma (SCC) were observed 30–50 weeks’ post-surgery. Numerous CD68 and pStat3-positive cells were identified surrounding PHP and BM after 20 weeks, and around ADC and SCC after 30 weeks. Moderate infiltration of CD163-positive macrophages was seen with BM, ADC, and SCC after 30 weeks. However, very few Foxp3-positive cells were observed around ADC and SCC.

Conclusion

Macrophages infiltrate the esophagus and activate the pStat3 pathway in stromal cells and epithelium. M2 phenotype macrophages infiltrate following infiltration of M1 macrophage and contribute to tumor development through regulatory T cells (Tregs). The involvement of immune cells such as TAMs and Tregs in the inflammatory microenvironment promotes esophageal carcinogenesis.     

Emerging Concepts of Bile Reflux in the Constellation of Gastroesophageal Reflux Disease

Rationale

Reflux of gastric and duodenal contents in patients with gastroesophageal reflux disease (GERD) has been postulated as a major cause of complications, such as Barrett’s esophagus or malignant degeneration.

Findings

We present a summary of experimental, clinical, and immunohistochemical studies that show that acid and bile reflux are increased in patients who suffer from GERD, are the key factor in the pathogenesis of Barrett’s esophagus, and are possibly related to the development of esophageal adenocarcinoma.

     

Bile reflux in the constellation of gastroesophageal reflux disease

Gastric acid and bile acids are a particularly noxious combination when they interact with the mucosa of the upper intestinal tract. There is a critical pH range, between 3 and 6, in which bile acids exist in their soluble, un-ionized form, can penetrate cell membranes, and accumulate within mucosal cells. At a lower pH, bile acids are precipitated, and at a higher pH, bile acids exist in their noninjurious ionized form. Experimental, clinical, and immunohistochemical studies show that acid and bile reflux are increased in patients who suffer from GERD, are the key factor in the pathogenesis of Barrett's esophagus, and possibly are related to the development of esophageal adenocarcinoma.     

Role of acid and bile in the genesis of Barrett's esophagus

Clinical and experimental studies have shown that acid and bile reflux are increased in patients who have Barrett's esophagus. The combination of both seems the key factor in the pathogenesis of Barrett's esophagus. This factor has been confirmed by immunohistochemical studies that show that environmental factors, such as acid and bile, are involved in the pathogenesis of Barrett's esophagus. There is a critical pH range between 3 and 6 in which bile acids exist in their soluble, un-ionized form; can penetrate cell membranes; and accumulate within mucosal cells. At a lower pH, bile acids are precipitated, and at a higher pH, bile acids exist in their noninjurious ionized form. Thus incomplete gastric acid suppression, as is the case with most medical treatment regimens for gastroesophageal reflux, may in fact predispose to the development of Barrett's esophagus.     

Symptoms, acid exposure and motility in patients with Barrett's esophagus.

INTRODUCTION: Barrett's esophagus, a syndrome in which the squamous mucosa that normally lines the distal esophagus is replaced with columnar epithelium, is found in a small percentage of patients presenting with gastroesophageal reflux disease (GERD). The columnar epithelium may be protective, guarding people afflicted with Barrett's esophagus from experiencing symptoms related to acid reflux. The purpose of this study was to investigate whether people with Barrett's esophagus subjectively experience fewer symptoms or symptoms of decreased severity, despite sustaining greater acid exposure, than those with GERD but without Barrett's syndrome. METHODS: We conducted a chart review of patients with GERD. Criteria for inclusion in the study were esophagogastroscopy, motility testing and a 24-hour pH study. Fifty-eight patients (29 men, 29 women) fulfilled these criteria. The diagnosis of GERD was based on an abnormal 24-hour pH study (DeMeester score). Of these 58 patients, 21 (14 men, 7 women) were found to have histologically confirmed Barrett's esophagus. A questionnaire to assess the key symptoms of GERD was administered, with a severity score ranging from 0 to 3 (3 being the most severe) for each symptom. RESULTS: Patients with Barrett's esophagus experienced symptoms significantly less severe (p < 0.01) than those with GERD. Patients with Barrett's esophagus also had a greater degree of acid exposure as identified by higher DeMeester scores (p = 0.056), longer episodes of acid exposure, a greater number of long episodes (> 5 min) of acid exposure (p = 0.033) and an increased percentage of time when their pH was less than 4. Patients with Barrett's esophagus had decreased resting lower esophageal sphincter tone, and number and amplitude of peristaltic contractions. CONCLUSIONS: For patients with Barrett's esophagus, the columnar epithelium may serve a protective function in guarding against symptoms of acid reflux. This has implications for the diagnosis and management of this condition.     

Nodal Skip Metastasis is not a Predictor of Survival in Thoracic Esophageal Squamous Cell Carcinoma

Background

The presence of nodal skip metastasis (NSM) has been found to be of clinical importance in non-small cell lung cancer, but the study of this phenomenon in esophageal carcinoma is relatively rare. The purpose of this study was to identify risk factors influencing NSM and to assess its prognostic value in thoracic esophageal squamous cell carcinoma (ESCC).

Methods

A total of 207 patients with thoracic ESCC who underwent three-field lymphadenectomy and who had lymph node metastasis were reviewed. Associations of NSM occurrence with the clinicopathological characteristics of patients and primary tumors were evaluated using logistic regression analysis. The influence of NSM on the overall survival (OS) was assessed by log-rank tests and Cox regression analysis.

Results

NSM were present in 58 (26 %) patients. No factor was significantly associated with the incidence of NSM except for the location of primary tumor. There were no NSMs in the 29 patients from our study with upper thoracic ESCC, and the rates of tumors occurrence in the middle and lower third of the esophagus were 38.9 and 14.9 %, respectively. The median OS was 30 months, and no significant difference in OS was found between the patients with or without NSM (p = 0.767). Only N status was found to be the independent risk factor for OS by Cox multivariate analysis.

Conclusions

NSM is common in thoracic ESCC, especially in patients with tumors located in the middle and lower third of the esophagus. However, the presence of NSM did not predict prognosis.     

Sleeve Gastrectomy and Development of “De Novo” Gastroesophageal Reflux

Background

Sleeve gastrectomy (SG) is currently gaining popularity due to an excellent efficacy combined to minimal anatomic changes. However, some concerns have been raised on increased risk of postoperative gastroesophageal reflux disease (GERD) due to gastric fundus removal, section of the sling muscular fibers of gastroesophageal junction, reduced antral pump function, and gastric volume. We undertook the current study to evaluate by means of high-resolution impedance manometry (HRiM) and combined 24-h pH and multichannel intraluminal impedance (MII-pH) the impact of SG on esophageal physiology.

Methods

In this study, 25 consecutive patients had HRiM and MII-pH before and after laparoscopic SG. The following parameters were calculated at HRiM: lower esophageal sphincter (LES) pressure and relaxation, peristalsis, number of complete esophageal bolus transit, and mean total bolus transit time. The acid and non-acid GER episodes were assessed by MII-pH with the patient in both upright and recumbent positions.

Results

At a median follow-up of 13 months, HRiM showed an unchanged LES function, increased ineffective peristalsis, and incomplete bolus transit. MII-pH showed an increase of both acid exposure of the esophagus and number of non-acid reflux events in postprandial periods.

Conclusions

Laparoscopic SG is an effective restrictive procedure that creates delayed esophageal emptying without impairing LES function. A correctly fashioned sleeve does not induce de novo GERD. Retrograde movements and increased acid exposure are probably due to stasis and postprandial regurgitation.     

Loss of Alkalization in Proximal Esophagus: a New Diagnostic Paradigm for Patients with Laryngopharyngeal Reflux

Introduction

Cervical esophageal pH monitoring using a pH threshold of <4 in the diagnosis of laryngopharyngeal reflux (LPR) is disappointing. We hypothesized that failure to maintain adequate alkalization instead of acidification of the cervical esophagus may be a better indicator of cervical esophageal exposure to gastric juice. The aim of this study was to define normal values for the percent time the cervical esophagus is exposed to a pH ≥7 and to use the inability to maintain this as an indicator for diagnosis of LPR.

Material and Methods

Fifty-nine asymptomatic volunteers had a complete foregut evaluation including pH monitoring of the cervical esophagus. Cervical esophageal exposure to a pH <4 was calculated, and the records were reanalyzed using the threshold pH ≥7. The sensitivity of these two pH thresholds was compared in a group of 51 patients with LPR symptoms that were completely relieved after an antireflux operation.

Results

Compared to normal subjects, patients with LPR were less able to maintain an alkaline pH in the cervical esophagus, as expressed by a lower median percent time pH?≥?7 (10.4 vs. 38.2, p?<?0.0001). In normal subjects, the fifth percentile value for percent time pH?≥?7 in the cervical esophagus was 19.6%. In 84% of the LPR patients (43/51), the percent time pH?≥?7 were below the threshold of 19.6%. In contrast, 69% (35/51) had an abnormal test when the pH records were analyzed using the percent time pH?<?4. Of the 16 patients with a false negative test using pH?<?4, 11 (69%) were identified as having an abnormal study when the threshold of pH?≥?7 was used.

Conclusion

Normal subjects should have a pH ≥7 in cervical esophagus for at least 19.6% of the monitored period. Failure to maintain this alkaline environment is a more sensitive indicator in the diagnosis of the LPR and identifies two thirds of the patients with a false negative test using pH <4.     

STAT1 Activation-Induced Apoptosis of Esophageal Squamous Cell Carcinoma Cells In Vivo

Background The induction of apoptosis might be a promising treatment for cancers refractory to conventional therapies, such as esophageal cancer. In this study, we examined whether epidermal growth factor–induced growth inhibition results from apoptosis of esophageal squamous cell carcinoma (SCC) cells as a result of STAT1 activation and evaluated whether interferon gamma (IFN-γ) can induce apoptosis of cancer cells in vivo. Methods To assess the function of STAT1, we established stable transfectants expressing dominant-negative STAT1. Apoptosis was assessed by several experimental techniques, including flow cytometry. Differentiation was evaluated by Western blot test with involucrin used as a marker. In vivo, cancer cells were injected into male BALB/c nu/nu mice. Two weeks later, the mice started to receive injections of IFN-γ or saline into a tail vein four times per week. Concentrations of IFN-γ in the tumors were analyzed by enzyme-linked immunosorbent assay. Apoptosis was evaluated by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining. Results Epidermal growth factor inhibited the growth of esophageal SCC cells by causing apoptosis through several pathways involving STAT1 activation. IFN-γ induced the apoptosis of cancer cells, but it also promoted the differentiation (not apoptosis) of primary cultured cells derived from normal esophageal epithelium. IFN-γ also inhibited the growth of xenograft tumors of esophageal SCC cells in vivo. Conclusions Our results suggest that IFN-γ is one candidate for cytokine-based therapy of cancer. IFN-γ–induced STAT1 activation might be involved in the apoptosis of esophageal SCC cells and in the terminal differentiation of normal squamous cells. Further studies of STAT1 signaling pathways may provide the basis for new targeted therapeutic strategies for esophageal SCC.     

Bile reflux in benign and malignant barrett’s esophagus: Effect of medical acid suppression and nissen fundoplication

Bile reflux has been implicated in the pathogenesis and malignant degeneration of Barrett’s esophagus, but clinical studies in patients with adenocareinoma arising in Barrett’s esophagus are lacking. Ambulatory esophageal measurement of acid and bile reflux was performed with the previously validated fiberoptic bilirubin monitoring system (Bilitec) combined with a pH probe in 20 asymptomatie volunteers, 19 patients with gastroesophageal reflux disease (GERD) but no mucosal injury, 45 patients with GERD and erosive esophagitis, 33 patients with GERD and Barrett’s esophagus, and 14 patients with early adenocarcinoma arising in Barrett’s esophagus. Repeat studies were done in 15 patients under medical acid suppression and 16 patients after laparoscopie Nissen fundoplication. The mean esophageal bile exposure time showed an exponential increase from GERD patients without esophagitis to those with erosive esophagitis and benign Barrett’s esophagus and was highest in patients with early carcinoma in Barrett’s esophagus (P <0.01). Pathologic esophageal bile exposure was documented in 18 (54.5%) of 33 patients with benign Barrett’s esophagus and 11 (78.6%) of 14 patients with early adenoearcinoma in Barrett’s esophagus. Nissen fundoplieation but not medical acid suppression resulted in complete suppression of bile reflux. Bile reflux into the esophagus is particularly prevalent in patients with Barrett’s esophagus and early cancer. Bile reflux into the esophagus can be completely suppressed by Nissen fundoplication but not medical acid suppression alone. (J GASTROINTEST SURG 1998;2:333-341.) Presented at the Thirty-Eighth Annual Meeting of The Society for Surgery of the Alimentary Tract, Washington, D.C., May 11–14, 1997     

Correlation Between gp96 Expression and the Surgical Outcome in Patients With Esophageal Squamous Cell Carcinoma

Background

Heat-shock protein gp96 plays an important role in antitumor immunoreactions. Gp96 has a close relationship with antitumor immunity. This study evaluated the correlation between gp96 expression and the prognosis in esophageal squamous cell carcinoma.

Methods

Seventy-eight patients with primarily resected esophageal squamous cell carcinoma were enrolled onto this study, and gp96 expression was evaluated by immunohistochemical staining. The association of clinicopathological factors and patients’ survival was calculated by univariate (log rank test) and multivariate (Cox proportional hazard regression method) analyses.

Results

Fifty-seven (73%) of 78 cases were gp96 positive, and 21 were negative (27%). The survival of patients with gp96-negative disease was significantly shorter (5-year survival, 22.9 months) than with gp96-positive disease (45.8 months; P = 0.049), and the multivariate analysis showed that gp96 negativity is an independent risk factor for poor survival (hazard ratio, 2.577; P = 0.040). Gp96-negative cases had more metastatic lymph nodes than did negative cases, especially in T1 cases (4.8 in gp96-negative cases vs. 0.84 in gp96-positive cases; P = 0.064)

Conclusions

The downregulation of gp96 expression is closely correlated with poor survival in esophageal squamous cell carcinoma.