H+/K+ATPase Expression in the Larynx of Laryngopharyngeal Reflux and Laryngeal Cancer Patients.

OBJECTIVES: The gastric H+/K+ ATPase proton pump has previously been shown to be expressed in the human larynx, however its contribution to laryngopharyngeal reflux (LPR) signs, symptoms and associated diseases such as laryngeal cancer is unknown. Proton pump expression in the larynx of patients with LPR and laryngeal cancer was investigated herein. A human hypopharyngeal cell line expressing the proton pump was generated to investigate its effects. STUDY DESIGN: In-vitro translational. METHODS: Laryngeal biopsies were obtained from three LPR and eight LSCC patients. ATP4A, ATP4B and HRPT1 were assayed via qPCR. Human hypopharyngeal FaDu cell lines stably expressing proton pump were created using lentiviral transduction and examined via transmission electron microscopy and qPCR for genes associated with inflammation or laryngeal cancer. RESULTS: Expression of ATP4A and ATP4B was detected in 3/3 LPR, 4/8 LSCC-tumor and 3/8 LSCC-adjacent specimens. Expression of ATP4A and ATP4B in FaDu elicited mitochondrial damage and expression of IL1B, PTGS2, and TNFA (P < .0001); expression of ATP4B alone did not. CONCLUSIONS: Gastric proton pump subunits are expressed in the larynx of LPR and LSCC patients. Mitochondrial damage and changes in gene expression observed in cells expressing the full proton pump, absent in those expressing a single subunit, suggest that acid secretion by functional proton pumps expressed in upper airway mucosa may elicit local cell and molecular changes associated with inflammation and cancer. LEVEL OF EVIDENCE: NA Laryngoscope, 131:130-135, 2021.

Presence of pepsin in laryngeal tissue and saliva in benign and malignant neoplasms.

OBJECTIVES: The current study was performed to determine the presence of pepsin in saliva and laryngeal tissue among participants with benign and malignant laryngeal neoplasms. STUDY DESIGN: Case-control study included three groups of patients with: (1) benign laryngeal neoplasms, (2) malignant laryngeal neoplasms and (3) control subjects without symptoms or signs of laryngopharyngeal reflux (LPR). METHODS: Eighty-one voluntary participants were included into study. They were recruited from a group of patients with histologically proven benign and malignant laryngeal neoplasms and in case of control subjects among patients with nasal septum deformation without symptoms of LPR. Morning saliva samples were collected preoperatively. Tumor biopsies were collected by directoscopy of larynx and the control samples from interarytenoid unit of larynx. All samples were analyzed by Enzyme-Linked Immunosorbent Assay (ELISA) and Immunohistochemistry. RESULTS: Pepsin was found in all samples of saliva and tissue biopsies in groups with malignant and benign neoplasms. The highest concentration of pepsin was found in a group of patients with malignant laryngeal neoplasms. Patients with benign laryngeal neoplasms had lower concentrations and the control subjects presented with the lowest concentration of pepsin measured from their saliva. Differences were not statistically significant. Immunohistochemical (IHC) analysis showed the largest number of high positive samples in the group of malignant lesions. CONCLUSION: These results suggest that pepsin and LPR can contribute to the development of benign and malignant laryngeal neoplasms. Further prospective studies, with far more patients, are necessary to prove the role of pepsin in multifactorial etiology of laryngeal neoplasms.

Association Between Pepsin in the Saliva and the Subjective Symptoms in Patients With Laryngopharyngeal Reflux.

OBJECTIVES: Our study was designed to further evaluate the relationships between the saliva pepsin level and the symptoms and quality of life of patients with laryngopharyngeal reflux (LPR). STUDY DESIGN: A prospective cohort study without controls. SETTING: Tertiary teaching hospital. SUBJECTS AND METHODS: We analyzed 50 patients diagnosed with LPR by 24-hour multichannel intraluminal impedance pH monitoring. All subjects were instructed to collect saliva samples upon waking in the morning. The saliva pepsin levels were analyzed using enzyme-linked immunosorbent assay. The Reflux Symptom Index, Reflux Finding Score, Laryngopharyngeal Reflux-Health-Related Quality of Life, and Short Form 36 survey were administered. RESULTS: The pepsin was detected in the saliva of 41 patients with LPR (17.15 ± 20.42 ng/mL). Nine patients did not have pepsin in the saliva. There were no significant associations between the pepsin level in the saliva and Reflux Symptom Index, Laryngopharyngeal Reflux-Health-Related Quality of Life, or Short Form 36 of patients with LPR. CONCLUSION: The saliva pepsin level is not significantly correlated with LPR symptoms or quality of life in LPR patients. It may be true that there is no association between pepsin levels and LPR symptoms, but this lack of association does not prove the lack of pathophysiological effect.

Detecting Laryngopharyngeal Reflux by Immunohistochemistry of Pepsin in the Biopsies of Vocal Fold Leukoplakia.

Laryngopharyngeal reflux (LPR) may contribute to the development of laryngeal diseases including vocal fold leukoplakia. Clinical methods of determining LPR are limited. Pepsin, as an exogenous protein, is considered as a biomarker of LPR. The aim of the current study was, therefore, to detect pepsin by immunohistochemistry in the biopsies from patients with vocal fold leukoplakia, and by which, to determine the potential association of LPR and vocal leukoplakia. A total of 26 biopsies from patients with vocal fold leukoplakia were examined in comparison with 20 vocal fold biopsies from control subjects. We found that 2 out of 26 patients (7.7%) were strongly positive, 4 of the 26 (15.4%) patients were positive, 11 of the 26 (42.3%) patients were weakly positive, and 9 of the 26 (34.6%) were negative staining for pepsin. In contrast, only 4 of the 20 (20.0%) control subjects were weakly positive and the rest (16; 80.0%) were negative staining for pepsin. There was significant difference between the two groups in terms of positivity of pepsin staining (χ2 = 24.181, P <0.001). These findings suggest that pepsin immunohistochemical staining could be a biomarker of LPR and that LPR may be a risk factor for the development of vocal fold leukoplakia.

New aspects in the pathomechanism and diagnosis of the laryngopharyngeal reflux-clinical impact of laryngeal proton pumps and pharyngeal pH metry in extraesophageal gastroesophageal reflux disease.

AIM: To determine the laryngeal H+K+-ATPase and pharyngeal pH in patients with laryngopharyngeal reflux (LPR)-symptoms as well as to assess the symptom scores during PPI therapy. METHODS: Endoscopy was performed to exclude neoplasia and to collect biopsies from the posterior cricoid area (immunohistochemistry and PCR analysis). Immunohistochemical staining was performed with monoclonal mouse antibodies against human H+K+-ATPase. Quantitative real-time RT-PCR for each of the H+K+-ATPase subunits was performed. The pH values were assessed in the aerosolized environment of the oropharynx (DxpH Catheter) and compared to a subsequently applied combined pH/MII measurement. RESULTS: Twenty patients with LPR symptoms were included. In only one patient, the laryngeal H+K+-ATPase was verified by immunohistochemical staining. In another patient, real-time RT-PCR for each H+K+-ATPase subunit was positive. Fourteen out of twenty patients had pathological results in DxpH, and 6/20 patients had pathological results in pH/MII. Four patients had pathological results in both functional tests. Nine out of twenty patients responded to PPIs. CONCLUSION: The laryngeal H+K+-ATPase can only be sporadically detected in patients with LPR symptoms and is unlikely to cause the LPR symptoms. Alternative hypotheses for the pathomechanism are needed. The role of pharyngeal pH-metry remains unclear and its use can only be recommended for patients in a research study setting.

Clinical evaluation of pepsin for laryngopharyngeal reflux in children with otitis media with effusion.

OBJECTIVE: We sought to evaluate the clinical role of pepsin for laryngopharyngeal reflux (LPR) in children with otitis media with effusion (OME). METHODS: Pepsin/pepsinogen and fibrinogen were analyzed in fifty effusion and blood samples of children with OME using enzyme linked immunosorbent assay (ELISA). Ambulatory 24-h dual-probe pH monitoring was additionally performed in 31 children divided into two groups according to response of medical treatment. RESULTS: The effusion levels of pepsin/pepsinogen ranged from 8.5 to 1512 µg/dl and were up to 4-540 times higher than the concentrations found in plasma samples. The effusion levels of fibrinogen ranged from 0.05 to 4.1g/dl. Some effusion samples showed fibrinogen concentrations did not exceed 10 times higher than the concentrations found in plasma samples and others showed lower concentrations. The pH of effusion samples was 7.13 to 8.72. Dual-probe pH monitoring showed that 22/31 (71%) of the studied children had significant acid reflux documented by either the esophageal probe or the pharyngeal probe and all of them had LPR. There is a significant positive correlation between the level of pepsin assayed in the effusions and the number of pharyngeal reflux episodes measured by pH monitoring. CONCLUSIONS: Analysis of pepsin/pepsinogen in effusion samples of children with OME, using ELISA, can be considered as a reliable biochemical marker for assessment of laryngopharyngeal reflux.