Detectable levels of pepsin remain in laryngeal epithelia after a reflux event. Pepsin bound there would be enzymatically inactive because the mean pH of the laryngopharynx is pH 6.8. Significantly, pepsin could remain in a form that would be reactivated by a subsequent decrease in pH, such as would occur during an acidic reflux event or possibly after uptake into intracellular compartments of lower pH.

Pepsin was detected in 8 of 9 patients with laryngopharyngeal reflux disease, but not in normal control subjects (0 of 12). The presence of pepsin was associated with CA-III depletion in the laryngeal vocal fold and ventricle. Given the correlation between laryngopharyngeal reflux disease and CA-III depletion, it is highly plausible that CA-III depletion, as a result of pepsin exposure during laryngopharyngeal reflux, predisposes laryngeal mucosa to reflux-related inflammatory damage.

This is the second annual report of an international collaborative research group that is examining the cellular impact of laryngopharyngeal reflux (LPR) on laryngeal epithelium. The results of clinical and experimental studies are presented. Carbonic anhydrase (CA), E-cadherin, and MUC gene expression were analyzed in patients with LPR, in controls, and in an in vitro model. In patients with LPR, we found decreased levels of CAIII in vocal fold epithelium and increased levels in posterior commissure epithelium. The experimental studies confirm that laryngeal CAIII is depleted in response to reflux. Also, cell damage does occur well above pH 4.0. In addition, E-cadherin (transmembrane cell surface molecules, which have a key function in epithelial cell adhesion) was not present in 37% of the LPR laryngeal specimens. In conclusion, the laryngeal epithelium lacks defenses comparable to those in esophageal epithelium, and these differences may contribute to the increased susceptibility of laryngeal epithelium to reflux-related injury.

These findings are perhaps the first to explain why many patients have symptoms and injury associated with nonacidic reflux, and could have important implications for the development of new therapies for reflux, such as pepsin receptor antagonists and/or irreversible inhibitors of peptic activity.

Objective/Hypothesis Laryngopharyngeal reflux (LPR) is thought to be a significant risk factor for laryngeal squamous cell carcinoma (SCC), but causality has never been proven. It is accepted that chronic reflux into the esophagus can induce metaplastic changes in esophageal mucosa with subsequent increased risk of esophageal adenocarcinoma, but no similar associations have been established for LPR and laryngopharyngeal SCC. The objective of this study was to test the hypothesis that reflux of pepsin into the laryngopharynx can promote carcinogenesis. Study Design Translational research study Methods Normal human laryngeal primary epithelial cell cultures and hypopharyngeal FaDu SCC cells were exposed to human pepsin and analyzed by Human Cancer PathwayFinder and miRNA Superarrays, flow cytometry and Western blot to determine the effect of pepsin on carcinogenesis. Laryngeal biopsy specimens, taken from cancer patients and normal control subjects, were analyzed for the presence of pepsin by Western blot. Results Microarray analysis demonstrated that pepsin significantly altered the expression of 27 genes implicated in carcinogenesis and also affected the expression of 22 microRNAs known to be altered in human head and neck cancers. Pepsin increased proliferation in both FaDu SCC cells and cultured normal laryngeal epithelial primary cells by increasing S phase distribution on flow cytometry analysis in a time and dose dependent manner. Furthermore, pepsin was detected in 60% (3/5) human laryngeal cancer biopsies, absent in all (0/5) normal control specimens. Conclusion These data support a role for refluxed pepsin in the promotion of epithelial proliferation and carcinogenesis of the larynx and pharynx.