An Investigation of Biomarkers in Laryngopharyngeal Reflux

Author: John Wood

Wood, John, 2013 An Investigation of Biomarkers in Laryngopharyngeal Reflux, Flinders University, School of Medicine

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Laryngopharyngeal reflux (LPR) is an increasingly diagnosed disease in Otolaryngology, however it is a highly controversial topic. There is no gold standard diagnostic test and despite a wealth of articles, there is little understanding of the pathophysiological mechanisms underlying laryngeal damage. In addition, the response to anti-reflux medical treatment is highly variable, with a notable proportion failing to have any response. The lack of comprehension of the pathophysiology and definitive diagnosis limits the ability to conduct adequate investigation of treatment options. This study aimed to identify known and novel biomarkers in patients with LPR. Given evidence suggesting that LPR biomarker expression may vary across different areas of the larynx, biopsies were collected and analysed from sub-regions of the larynx. Recruited patients completed the Reflux Symptom Index and the Reflux Finding Score was assessed at the time of biopsy collection under general anaesthetic. Biopsies were collected from 4 anatomically distinct locations in the larynx in both LPR and non-refluxing control patients. Sections were sent for histological examination and qRT-PCR analysis was conducted on 20 genes identified as being related to reflux and inflammation, including interleukins 6 (IL-6) and 8 (IL-8), prostaglandin-endoperoxide synthase-2, cytokeratins 8 and 14, mucin genes MUC1, 2, 3B, 4, 5B, 6, 7, and carbonic anhydrase III. In patients with LPR, site-specific differences in gene expression were noted. The medial arytenoid area of the larynx was more susceptible to alterations in gene expression. Statistically significant differences were noted in genes related to intrinsic defences and inflammation, including CD1d, TGFβ-1 and mucins. Mucins play an important role in protecting the epithelium from fluctuations of pH, ionic concentration and hydration. They are also implicated in renewal and differentiation of the epithelium and modulation of cell-cycle progression. In patients with LPR, this study demonstrated significantly lower expression of the secreted gel-forming mucin genes in the medial arytenoid region (MUC2 and MUC5B) and the posterior commissure (MUC5B). Carbonic anhydrase (CA) is an integral component of laryngeal defence, increasing the pH of the mucosal surface. Expression of CA I, II and III are present in the normal larynx. Expression of CA-III is known to vary in the larynx between different locations in response to refluxate. CA3 gene expression was lower in the false cord region in LPR patients, however this was not significantly different. There is also evidence of an inflammatory process, with changes in CD1d expression, which is known to be decreased in epithelial inflammation and increase in CRNN and TGFβ-1 noted in the medial arytenoid sub-site. Consequently, there is significant evidence of molecular changes in laryngeal epithelium between patients with LPR compared to normal controls. This study identifies that these changes vary according to the sub-site of the larynx. Whilst the posterior commissure is most commonly identified as the area demonstrating macroscopic change consistent with LPR, this study has identified that the medial arytenoid is the area most likely to demonstrate a molecular change. With identified molecular changes in mucin expression (MUC2 and 5B), cytokeratin 14 and molecular markers of inflammation, this study provides increasing evidence for the diagnosis of LPR and potential markers for therapeutic monitoring.

Keywords: Laryngopharyngeal reflux,Gastroesophageal reflux,Laryngitis,Mucins,Pepsin

Subject: Surgery thesis, Medicine thesis

Thesis type: Masters
Completed: 2013
School: School of Medicine
Supervisor: Professor Simon Carney, Dr Damian Hussey