Hypopharyngeal Pepsin and SEP70 as Diagnostic Markers of Laryngopharyngeal Reflux

Yoshihiro Komatsu, MD, Lori A Kelly, BS, Ali H Zaidi, MD, Christina L Rotoloni, BS, Juliann E Kosovec, BS, Katie Nega, BA, Toshitaka Hoppo, MD, PhD, Blair A Jobe, MD, FACS. Institute for the Treatment of Esophageal and Thoracic Disease, Allegheny Health Network, Pittsburgh, Pennsylvania.

Introduction: The management of patients with laryngopharyngeal reflux (LPR) has been challenging. Hypopharyngeal multichannel intraluminal impedance (HMII) has shown to increase the sensitivity in diagnosing LPR, potentially improving the outcome of antireflux surgery. The objective of this study is to investigate the potential use of pepsin and the heat stress protein Sep70 as diagnostic tools for the detection of LPR in combination with HMII.

Methods and Procedures: Tissue samples of hypopharynx, distal esophagus, and gastric cardia were collected from patients with LPR symptoms regardless of gastroesophageal reflux (GERD) diagnosis and underwent HMII with a specialized catheter to detect LPR and high esophageal reflux (HER: reflux 2cm distal to upper esophageal sphincter) events. Patients were classified into 2 groups based on the presence of abnormal proximal exposure (APE), which was defined as LPR ≥ 1/day and/or HER ≥ 5/day: 1) APE, 2) without APE. Patients with typical GERD symptoms such as heartburn and regurgitation, without LPR symptoms who did not undergo HMII were used as a “normal” GERD group. Protein was isolated from fresh frozen tissue samples and Western blot analysis of pepsin, Sep70, and actin (indigenous control) was performed. Pepsinogen was used as a control to differentiate pepsin from pepsinogen. Relative quantitation was performed using Image Studio Lite Software with normalization against the internal actin of each blot.

Results: From Oct 2012 to Aug 2013, 49 patients underwent HMII. Of 49, 12 patients underwent biopsies from hypopharynx (9 APE [+] and 3 APE [-]). Ten patients with typical GERD symptoms who underwent biopsies from hypopharynx were identified from tissue bank as a “normal” GERD group. Pepsin was detected in distal esophagus in all groups; however patients with APE had decreased expression. In contrast, pepsin levels of hypopharyngeal tissue were 2-fold greater in patients with APE compared to patients without APE, and Sep 70 was significantly downregulated in the patients with APE (p=0.015). The median DeMeester score of APE group and without APE group were 9 (range 2.1-38.6) and 3.5 (range 0.8-27.1), respectively.

Conclusion: Presence of pepsin and depletion of Sep70 in the hypopharyngeal tissue may indicate tissue damage by abnormal reflux exposure to laryngopharynx. However, the normative data for these markers have to be validated. Data set is currently being expanded and will be presented at the conference.

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