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.

Further exploration of the role of MUC5B in the pathophysiology of OME holds promise for development of novel, targeted therapies to reduce effusion viscosity, facilitation of effusion clearance, and prevention of disease chronicity and hearing loss in patients with OME.

Objective Toll-like receptor signaling activated by bacterial otitis media pathogens in the middle ear has been shown to play a key role in OM susceptibility, pathogenesis and recovery. Recent studies implicate microRNA 146 (miR-146) in regulation of inflammation via negative feedback of toll-like receptor signaling (TLR) in a wide variety of tissues, however its involvement in otitis media is unknown. Methods Human middle ear epithelial cells were stimulated with proinflammatory cytokines, interluekin 1 beta or tumor necrosis factor alpha, for two to twenty-four hours. Middle ear biopsies were collected from children with otitis media with effusion (n=20), recurrent otitis media (n=9), and control subjects undergoing cochlear implantation (n=10). miR-146a, miR-146b expression was assayed by quantitative PCR (qPCR). Expression of miR-146 targets involved in TLR signaling, IRAK1 and TRAF6, was assayed by qPCR in middle ear biopsies. Middle ear biopsies were cryosectioned and epithelial thickness measured by a certified pathologist. Results Proinflammatory cytokines induced expression of miR-146 in middle ear epithelial cells in vitro. Middle ear miR-146a and miR-146b expression was elevated in otitis media patients relative to control subjects and correlated with middle ear epithelial thickness. A trend towards inverse correlation was observed between miR-146 and TRAF6 expression in the clinical population. Conclusions This report is the first to assess miRNA expression in a clinical population with OM. Findings herein suggest miR-146 may play a role in OM.

OBJECTIVES:To determine the frequency of tracheal pepsin in ventilated neonates and whether the angle of head elevation was associated with tracheal pepsin. STUDY DESIGN: Serial trachael samples (at 3, 7, 14, 21 and 28 days of ventilation) were obtained from intubated, ventilated very low birth weight infants. Presence of tracheal pepsin was determined by Western blot analysis using a specific anti-human pepsin antibody. RESULTS: Tracheal pepsin was detected in 35/66 (53%) of the ventilated neonates (birthweight: 798 ± 268 grams [mean ± standard deviation]). Neonates whose head elevation was in the upper quartile (≥14 degrees) during the first sampling time (day 3) were less likely (4/16 vs 9/10, P = 0.0013) to have tracheal pepsin when compared to neonates whose head elevation was in the lowest quartile (≤8 degrees). CONCLUSIONS: Pepsin, a marker for gastric secretion aspiration, was detected in 53% of ventilated low birth weight neonates; early elevation of the head of the bed was associated with a lower rate of tracheal pepsin.in micro-aspiration of gastric contents [5]. Farhath et al. [5] showed that pepsin, a relatively new marker of gastric contents, was frequently present in the trachea of mechanically ventilated neonates. Tracheal pepsin concentrations were increased among ventilated neonates receiving feedings [5] and the presence of tracheal pepsin increased the risk of bronchopulmonary dysplasia [6].

Squamous cell carcinoma (SCC) of the head in neck is currently the 5th most common cancer in the United States, with over 35,000 cases diagnosed in 2009. In addition to tobacco and alcohol, laryngopharyngeal reflux (LPR) is thought to be a significant risk factor for laryngopharyngeal SCC, but definitive associations between LPR and carcinogenesis are lacking. The purpose of this study was to investigate a role for refluxed pepsin in the promotion of SCC of the laryngopharynx. We recently discovered that inactive but stable pepsin (at pH7, in non-acidic reflux) is actively taken up by laryngeal epithelial cells by endocytosis in a receptor mediated manner and leads to intracellular damage and pro-inflammatory cytokine gene expression changes. This reveals a novel mechanism by which pepsin can cause cell/tissue damage and thereby promote carcinogenesis in patients with LPR. These studies were therefore done to specifically study the effect of neutral pH pepsin on laryngopharyngeal SCC proliferation. Pepsin levels were measured in posterior laryngeal biopsy specimens from patients with clinically diagnosed LPR (n = 5) and normal control subjects (n = 5) by Western blot analysis. Human hypopharyngeal SCC FaDu cells and normal human primary laryngeal epithelial cells were exposed to affinity purified human pepsin (0.1mg/ml, pH7) and gene expression levels measured on a Human Cancer PathwayFinder SuperArray by real-time RT-PCR. Cell proliferation was measured by growth curve, propidium iodide staining, and Click-iT Edu assay. Pepsin was detected in 3/5 patient laryngeal cancer biopsies, and was absent in all normal control laryngeal biopsies (0/5). Pepsin (0.1mg/ml, pH7) significantly altered the expression level of 27 genes implicated in carcinogenesis on SuperArray analysis with a pattern promoting generalized increased cellular proliferation. Flow cytometry analysis with propidium iodide staining demonstrated an increase in S phase distribution following exposure to pepsin in a time and dose dependent manner. Growth curve and Click-iT EdU proliferation assays also demonstrated increased proliferation following pepsin exposure. Pepsin increases cell proliferation and alters the expression of multiple genes implicated in carcinogenesis. Pepsin also appears to be selectively present in human laryngeal tumors and not in control specimens. Thus, uncontrolled chronic reflux of pepsin as in LPR may increase risk for laryngopharyngeal SCC development. Studies are ongoing to investigate the effect of pepsin on cell transformation, clone-forming ability, resistance to anoikis, DNA damage and reactive oxygen species levels. New therapies to prevent pepsin uptake and damage, such as pepsin inhibitors and receptor antagonists, are also being tested for patients with LPR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2726. doi:10.1158/1538-7445.AM2011-2726