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Study Description

The distal esophagus is an important anatomical area where gastric acid reflux can cause reflux esophagitis (RE), Barrett's esophagus (BE) (intestinal metaplasia), and esophageal adenocarcinoma (EA). The incidence of EA has increased 6-fold in the U.S. since the 1970s, parallel to a significant increase in the prevalence of gastroesophageal reflux diseases (GERD). Although specific host factors might predispose one to disease risk, such a rapid increase in incidence must be predominantly environmental. The cause remains unknown. Our hypothesis is that changes in the foregut microbiome are associated with EA and its precursors, RE and BE in the GERD sequence.

We will conduct a case control study to demonstrate the microbiome-disease association in every stage of GERD sequence as well as analyze the trend in changes in the microbiome along disease progression toward EA.

Specific Aim 1. To conduct a comprehensive population survey of the foregut microbiome and demonstrate its association with GERD sequence, by 16S rRNA gene survey. We will analyze samples of the foregut microbiome at three anatomic loci: mouth, distal esophagus, and gastric corpus. Changes of the microbiota in the distal esophagus will be correlated with the phenotypes. Spatial relationship between the esophageal microbiota and upstream (mouth) and downstream (stomach) foregut microbiotas as well as temporal stability of the microbiome-disease association will also be examined.

Specific Aim 2. To define distal esophageal metagenome and demonstrate its association with GERD sequence, by shotgun metagenomic analysis. We will first classify samples of the metagenome into metagenotypes by between-sample k-mer distance and correlate the metagenotypes with the four phenotypes. Subsequent detailed analyses will include pathway-disease and gene-disease associations. DNA viruses and fungi, if identified, also will be correlated with the phenotypes.

A significant association between the foregut microbiome and GERD sequence, if demonstrated, will be the first step for eventually testing the causal hypothesis that an abnormal microbiome is required for the development of the sequence of phenotypic changes toward EA.

If EA and its precursors represent a microbial ecological disease, treating the cause of GERD might become possible, for example, by normalizing the microbiome through use of antibiotics, probiotics, or prebiotics. Causative therapy for GERD could prevent its progression and reverse the current trend of increasing incidence of EA.

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Study Inclusion/Exclusion Criteria

Inclusion Criteria:
Subjects will be screened by endoscopic examination, histological examination, and 48-hour pH monitoring. A subject will be considered for enrollment into this study if the results of his/her screening examinations are diagnostic for one of the following four phenotypes:

  1. Healthy control: Healthy control will be selected from subjects who present to the GI Clinics with symptoms requiring upper GI endoscopic examination and/or other diagnostic tests. The healthy control should meet all of one of the following sets of criteria: 1) normal endoscopic findings, 2) normal pH range with 48-hour pH monitoring study, and 3) no reflux symptoms, such as heart burn; OR 1) histopathological examination shows no inflammatory infiltrate or only less than 10 lymphocytes per high power field (HPF, 400x magnification) in the squamous epithelium, 2) normal pH range with 48-hour pH monitoring study, and 3) no reflux symptoms, such as heart burn.
  2. Reflux esophagitis (RE): Esophagitis will be determined either: histologically, characterized by basal zone hyperplasia exceeding 20% of the epithelial thickness, papillae extending into the superficial third of the epithelium, and presence of lymphocytes equal to or more than 10 cells/HPF or any numbers of eosinophils or polymorphonuclear leukocytes OR based on endoscopic findings consistent with reflux esophagitis. Symptoms of reflux will also be inquired about at each patient visit, and reflux will be detected by using 48-h pH monitoring.
  3. Barrett's esophagus (BE): esophageal squamous epithelium replaced by intestinal-type epithelial cells and goblet cells.
  4. Esophageal Adenocarcinoma (EA): atypical, infiltrative intestinal-type glands associated with desmoplasia. This phenotype also includes high grade dysplasia (carcinoma in situ).

The final decision on enrollment of a subject will be determined by frequency-match on gender, ethnic background, and age within 10 years.

Exclusion Criteria:

  1. Use of antibioics within 2 weeks before the scheduled endoscopic examination
  2. Previous gastric/esophageal surgery
  3. Active infection of the oral cavity
  4. Active gastric ulcer disease, or gastric cancer
  5. Persons younger than 50 years of age, because GERD and its complications are mainly diseases of older populations
  6. Pregnant women, because the natural history of reflux disease in pregnancy is different from GERD in general populations that leads to metaplasia and/or dysplasia
  7. Persons with an HIV infection will be excluded
  8. For safety reasons, we wll exclude potential subjects with coagulopathy (INR > 2.0) or thrombocytopenia (platelet count < 50) since this study involves repeated mucosal sampling.
  9. We will also exclude potential subjects at high risk for developing endocarditis, such as those with a history of endocarditis, rheumatic heart disease, or heart valve prosthesis who might require antibiotic prophylaxis for the endoscopic examination.
  10. We will exclude potential subjects with a history of intestinal ischemia and those with severe cardiopulmonary disease, or neurological impairment.
  11. We will exclude any subject who has an imperforate anus, recent anal or rectal surgery.
  12. English speakers will be excluded. Our project addresses the increasing incidence of esophageal adenocarcinoma in the US which has increased > 6-fold since the 1970s, but in other parts of the world except Western Europe the trend is stable. Since non-English speakers most likely represent immigrants from foreign countries, their relationship with the increasing incidence of esophageal adenocarcinoma can not be easily defined. Including non-English speakers could introduce unknown factors that may contribute to the change in the foregut microbiome and decrease the power of this demonstration project.

Molecular Data
TypeSourcePlatformNumber of Oligos/SNPsSNP Batch IdComment
Whole Genome Sequencing Roche 454 GS FLX Titanium N/A N/A
Study History

Gastroesophageal reflux disorders (GERD) refer to a sequence of diseases arising from gastric reflux, including reflux esophagitis (RE), Barrett's esophagus (intestinal metaplasia) (BE), and its downstream sequelum esophageal adenocarcinoma (EA). The incidence of EA has increased > 6-fold in the U.S. since the 1970s (1), parallel to an increasing prevalence of RE (2). Despite numerous studies, no explanation has been found for the increase. Although prevalence of RE is increasing in both men and women, strong gender disparity with male predominance is obvious at the stages of BE and EA for reasons unknown (3-5). The current strategy for dealing with the continuum of progression of GERD-related problems is limited to passive observation and symptomatic treatment, such as using proton pump inhibitors, and has been ineffective in preventing the increase in the incidence of EA. GERD, similar to other chronic diseases, likely has a multi-factorial etiology. Although host factors likely play a role, such a rapid increase in incidence must be predominantly environmental. Conventional studies have identified numerous risk factors, but their association with GERD is moderate (Table 1). The main factor(s) remain to be identified. The role of the microbiome in the development of inflammation-related cancers has been suggested by recent studies in some mouse models that mimic inflammatory bowel diseases (6). The development of mucosal inflammation and adenocarcinoma requires both a trigger (chemical or genetic) and the presence of commensal bacteria. Although human exposure to several exogenous pathogens has been monitored, there has been little attention paid to changes in the indigenous microbiome. This is partly due to the complexity and difficulties in culture and analysis of the human microbiome.

Among the environmental agents that have been considered are microbes. The human body can be viewed as a superorganism composed of an amalgam of both microbial and Homo sapiens cells. Our relationship with bacteria can be considered to span a broad spectrum, from mutualism to pathogenicity (7). There are currently two theories to explain bacterial diseases. The classic pathogen theory, largely attributed to Koch, requires the presence of specific pathogens, such as Mycobacterium tuberculosis or Bacillus anthracis (8). Alternatively, the microecological disease or "pathogenic microbial community" theory is a new concept in which the entire community contributes to pathogenicity although no individual community members can be categorized as classic pathogens (9).

Non-pathogenic bacteria do not have classically defined virulence factors but may harm the host by their basic metabolic activities or structural components. The gut microbiota in ob/ob mice, for example, has an increased capacity to harvest energy from the diet and might play a role in the pathophysiology of obesity (10). Also, a structural component of the gram-negative outer membrane from non-pathogenic bacteria, lipopolysaccharide (LPS), can induce abnormal relaxation of the lower esophageal sphincter via activation of the iNOS pathway (11) and (12) delay gastric emptying through the COX-2 pathway. Both effects increase the chance of reflux.

We have endeavored to understand the bacterial-host relationship in GERD in a stepwise way. First, we performed a comprehensive study of the microbiota in the distal esophagus and demonstrated its complexity, despite little prior knowledge (13-14). Surprisingly, the esophageal microbiota is comparable in complexity to those found in the mouth, stomach, colon, vagina, and skin. Collectively, nine phyla were observed, represented by 166 species. The distal esophagus could harbor > 200species, as predicted by the Chao-1 richness estimator. Second, we have demonstrated that in a high-risk population (elderly males) the esophageal microbiota can be classified into two types with the use of both unsupervised and phenotype-directed analyses, and that the type II biota is the strongest (OR >15) amongst all known environmental factors that are associated with the pathological changes related to RE and BE.

Selected publications
Diseases/Traits Related to Study (MESH terms)
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