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# These authors contributed equally to this study.
Lingli Meng
Footnotes
# These authors contributed equally to this study.
Affiliations
Department of Pathology, Ningbo Diagnostic Pathology Center, Ningbo, ChinaDepartment of Histopathology, Ningbo Clinical Pathology Diagnosis Center, Ningbo, China
# These authors contributed equally to this study.
Qingxiang Zhang
Correspondence
Address correspondence and reprint requests to Qingxiang Zhang, Otolaryngology Head and Neck Surgery, Nanjing Tongren Hospital, School of Medicine, Southeast University, Nanjing, China.
To explore the correlation between detection results of pepsin in vocal fold polyp tissues and the postoperative efficacy.
Methods
The clinical data of 112 patients with vocal fold polyp who received surgical procedures in our hospital from June 2019 to March 2021 were retrospectively analyzed. The vocal fold morphology and vocal acoustic function were assessed at postoperative week 12. Using binary logistic regression, we explored whether the factors, ie, detection result of pepsin in vocal fold polyp tissue, microscopic suturing, the use of CO2 laser, and the history of smoking, affected the postoperative morphological repair of vocal fold polyps. Then, to observe and compare the influence of the detection results of pepsin on the recovery of vocal acoustic function, we divided the enrolled patients into the pepsin group and the pepsin-free group based on the postoperative detection results of pepsin in the polyp tissues by immunohistochemistry,
Results
In the 112 patients with vocal fold polyps, positive staining of pepsin in the postoperative samples was found in 76 patients (67.86%) and negative in 36 (32.14%). Totally 80 patients returned to normal in vocal fold morphology, among whom 32 (88.89%) were in pepsin-free group and 48 (63.16%) in pepsin group. Binary logistic regression showed that pepsin was a clinically significant indicator that affected the postoperative morphological recovery of the vocal fold (P = 0.003). Although hoarse voice was improved in all patients at postoperative week 12, the differences were statistically significant in the proportion of patients with grade, roughness, breathiness, asthenia, strain class G, voice handicap index, maximum phonation time, Jitter, Shimmer and noise-to-harmonic ratio between the pepsin group and the pepsin-free group (P < 0.05), with the pepsin-free group being superior to the pepsin group in the improvement of vocal acoustic function.
Conclusion
Pepsin in vocal fold polyps is a clinically significant indicator affecting the postoperative morphological recovery and acoustic efficacy, and patients with negative pepsin are superior to those with positive pepsin in the postoperative recovery of vocal fold morphology and function.
A vocal fold polyp, common in the Department of E.N.T., is a benign mucosal proliferative lesion in the superficial lamina propria of vocal fold. Its primary clinical manifestation is hoarse voice that seriously impairs language communication. Patients with larger vocal fold polyps often need surgical procedures,
for whom self-retaining laryngoscopic surgical excision under general anesthesia has been applied primarily at present. The postoperative efficacy is not only affected by the surgical modes, but also related to the bad lifestyles including postoperative alcoholic use and smoking, as well as the factors, such as voice rest and voice training, the most important influencing factor of which is the persistent presence of pathogenic factors.
Currently, the role of laryngopharyngeal reflux (LPR) in the occurrence of vocal fold polyp has drawn growing attentions from the laryngological scholars. Jiang Chong-han et al
Reflux symptom index, reflux finding score, esophageal motility and their correlation with laryngopharyngeal reflux in patients with vocal cord polyps.
have reported that some patients have a certain functional or structural disorder in the upper and lower sphincter of the esophagus and have LPR if patients present with vocal fold polyps. When the gastric contents containing gastric juice and pepsin reflux to the throat, they cause chronic stimulations to the epithelium of vocal fold mucosa and induce cell injury, inflammation and tumor cell proliferation,
consequently leading to keratosis, atypical hyperplasia and canceration. Pepsin has been considered as a sensitive and specific biomarker for LPR because it can induce cell injury, inflammation and the proliferation of tumor cells.
have noted that pepsin reflux is a risk factor for the formation of vocal fold polyps, and then have proposed that compared to the 24-hour pH monitoring, the immunohistochemical analysis of pepsin in the polyp biopsy samples is more sensitive and effective for the diagnosis of LPR. However, little is known about the influence of the detection results of pepsin in vocal fold polyp tissues on the postoperative efficacy of vocal fold polyps. Therefore, this study, based on the postoperative vocal fold morphology recovery and vocal fold acoustic effectiveness, was designed to investigate the correlation between the detection results of pepsin and the surgical efficacy.
MATERIALS AND METHODS
Clinical data
A total of 112 patients with vocal cord polyp who received surgical procedures in Department of Otorhinolaryngology Head and Neck Surgery, Nanjing Tongren Hospital, School of Medicine, Southeast University from June 2019 to March 2021 were selected as the study subjects, among whom there were 46 males and 66 females aged (48.1 ± 11.2), with disease course of 1 month to 10 years (mean: [5.6 ± 2.3] years). All patients were pathologically diagnosed with vocal cord polyp after the surgical procedures. The immunohistochemical pepsin staining of postoperative pathological tissue sections was implemented. LPR seriousness was assessed in all patients by RSI and RFS scales designed by Center for Diagnosis and Treatment of Voice Diseases in Wake Forest University School of Medicine after surgical procedures.
They all could accurately understand the contents in RSI scale and implement the assessment themselves, and the clinical physicians had received uniform training specific to RFS scale.
Patients were excluded if they had 1) acute upper respiratory tract infection; 2) chronic tonsillitis or chronic rhinosinusitis; 3) history of malignant neoplasms; 4) history of anti-reflux medications; 5) incomplete RSI and RFS scores and questionnaire forms; and 6) qutting the clinical study at midway.
Study design
All the patients enrolled received microscopic procedures, 12 weeks after which the vocal fold morphology repair was observed and vocal acoustic function assessed. We evaluated whether such factors as detection result of pepsin in vocal fold tissues, microscopic suturing, the use of CO2 laser, and the history of smoking could affect the postoperative morphological repair of vocal fold. Then, the enrolled patients were assigned into the pepsin group and the pepsin-free group depending on the postoperative immunohistochemical staining results of pepsin in the polyp tissues, based on which to observe and compare the influence of pepsin on the recovery of vocal acoustic function.
Surgical procedures
1) Anesthesia and surgical devices: General anesthesia with endotracheal intubation (tube No. 5.5-6.0) was applied. The surgical devices included suspended self-retaining laryngoscope (Storz, St. Louis) and laryngeal microinstrument, CO2 laser device (AcuPulse 30, Israel) and surgical microscope with distance from laser micro-manipulator (AcuSpot 712-L, Lumenis) connecting to the workstation of 400 mm. The laser spot diameter was 0.8 mm-1.2 mm, continuous output mode was adopted, and the power was set as 1-2 W.
2) Excision of vocal fold polyp: Cold instruments concomitant with CO2 laser excision were applied for all patients. After the glottis was exposed by self-retaining laryngoscope, the margins of the polyp were gently held and raised by the laryngeal micro-forceps and the upper margin of the polyp was cut open by a micro-scissor starting from the base of the polyp directly under the microscope. Then, the polyp was gradually excised along the free margins of the vocal cord under the microscope, and attention should be paid to avoiding damage to the deep ligaments. CO2 laser was used to finely trim the mucosal margins at two ends to avoid residue of coarse mucosal broken edges, maximally make the free margins of vocal fold smooth and neat, and produce hemostasis. For patients with extensive mucosal wound and indirect alignment of the upper and lower margins of vocal cord, 7-0 coated Vicryl absorbable suture was used to suture the mucosal margins (Figure 1).
FIGURE 1Intraoperative findings of vocal fold polyp. A. Bilateral vocal cord polyps were noted after the glottis was exposed under self-retaining laryngoscope. B. 7-0 coated Vicryl absorbable suture was used to suture the mucosal margins after excision of vocal cord polyp. C. The wound margins of vocal cord were smooth after bilateral vocal cords were sutured.
After being dewaxed and then rehydrated, one tissue section was incubated in 0.05 M citrate buffer (pH 6.0) at 100 centigrade for 20 minutes for antigen repair, and then used for anti-pepsin antibody staining. The non-seriate sections from the same tissue mass were repeatedly stained for at least twice. The results were reviewed by two researchers blinded to the study contents.
The positive cell count in each sample was quantitatively assessed, which was scored in the following. 1% cells were stained (0 point); 2%-25% (1 point); 26%-50% (2 points); 51%-75% (3 points); and >75% (4 points). The staining intensity was scored below. 0 point: no staining; 1 point: weak staining; 2 points: moderate staining; and 3 points: strong staining. Total score (0-12 points) = score of stained cell count × staining intensity. The staining was classified into negative (–, 0-1 point), weakly positive (+, 2-4 points), moderately positive (++, 5-8 points), and strongly positive (+++, 9-12 points).
Efficacy assessment
The voice quality assessment and electric laryngoscopy or stroboscopic laryngoscopy were implemented in advance and 12 weeks after the surgical procedures.
1) Voice quality: Voice assessment was based on the following. 1) Grade, Roughness, Breathiness, Asthenia, Strain (GRBAS) scale was used for assessment of the preoperative voice function classified to grades 0-3: G0 represented normal voice, G1 mild hoarse voice, G2 moderate hoarse voice, and G3 serious hoarse voice; 2) self-assessment was done using voice handicap index (VHI) (0-30: mild voice handicap; 31-60: moderate voice handicap: 61-120: serious voice handicap); and 3) the voice acoustic function was assessed in a room with noise <45 dB, and the patient was placed 20 cm away from the microphone and asked to make tones as required, so as to obtain stable waveforms. Sound collector (CSLmodel 4150, KayElemetrics, USA) and Docterspeech 4.1 acoustic analytic software (Shanghai Tiger, China) were used to test and analyze the acoustic noise parameters including harmonic to noise ratio (NHR), Jitter and Shimmer, and maximum phonation time (MPT) was implemented as required to assess the voice function.
2) Laryngoscopy: In our study there were two postoperative morphologies of the laryngoscopic vocal folds: 1) recovered morphology with smooth vocal fold, without notable scar (Figure 2); and 2) unrecovered morphology, including the scar or edema of the vocal fold (FIGURE 3, FIGURE 4); 3) polyp recurrence (Figure 5).
FIGURE 2Electric laryngoscopic and immunohistochemical results before and after the surgical procedures in a patient whose vocal fold recturned to normal. A. Preoperative electric laryngoscopy showed that vocal fold polyp was located in the anterior and middle 1/3 of left vocal fold, with mild oedema of vocal fold mucosa. B. At postoperative week 12, electric laryngoscopy exhibited that the vocal folds were smooth with normal morphology. C. The immunohistochemical staining of vocal fold polyp revealed the presence of negative pepsin.
FIGURE 3The stroboscopic laryngoscopic and immunohistochemical results before and after the surgical procedures in a patient with vocal fold morphology unrecovered (I). A. The preoperative stroboscopic laryngoscope shows nodular vocal fold polyp in the middle of left vocal fold, with laryngeal mucosal oedema and notable hypertrophy of posterior commissure mucosa. B. At postoperative week 12, the stroboscopic laryngoscope exhibits a localized scar in the left surgical field of vocal fold. C. The immunohistochemical test of vocal fold polyp tissue reveals weakly the presence of pepsin.
FIGURE 4The electric laryngoscopic and immunohistochemical results before and after the surgical procedures in a patient with vocal fold morphology unrecovered (II). A. The preoperative electric laryngoscope shows that the vocal fold polyp is located in the anterior left vocal fold nears to anterior commissure, the laryngeal ventricle disappears, with vocal fold oedema, diffuse laryngeal oedema, and hypertrophy of posterior commissure mucosa. B. At postoperative week 12, the electric laryngoscope exhibits that the vocal fold is smooth, with bilateral vocal fold oedema and laryngeal ventricle disappeared. C. The immunohistochemical staining of vocal cord polyp tissue reveals the presence of pepsin.
FIGURE 5The electric laryngoscopic and immunohistochemical results before and after the surgical procedures in a patient with vocal fold morphology unrecovered (III). A. The preoperative electric laryngoscopy showed vocal fold polyps in the middle of bilateral vocal cords, associated by mucous secretions in the throat, edema and hypertrophy of vocal folds, and hypertrophy of posterior commissure mucosa. B. At postoperative week 12, the electric laryngoscopy suggested scarring in the right surgical field of vocal fold and polyp recurrence in the left surgical field. C. The immunohistochemical staining of vocal fold polyp tissue revealed the presence of pepsin.
SPSS25.0 software was applied for data analysis. Based on the preoperative and postoperative data, measurement data with normal distribution were represented by mean ± standard deviation (x ± s), binary logistic regression was used to analyze the influencing factors for vocal fold morphology repair, and GRBAS, VHI, MPT, Jitter, Shimmer and NHR were compared using paired t test before and after the surgical procedures, The voice quality was compared using independent-sample t test between pepsin group and pepsin-free group. A confidence interval of 0.95 was chosen for statistical evaluation. P < 0.05 was considered as statistically significant.
RESULTS
General data
In the immunohistochemical analysis, gastric mucosa was selected for positive control, with brown granules in the cytoplasm detected in the primary cells. As to negative control, phosphate buffer was used instead of anti-pepsin primary antibody for the laryngeal mucosa test, without brown granules in the cytoplasm detected. In the 112 patients, 76 patients (67.86%) had pepsin detected in the samples, among whom 20 patients (17.86%) had moderately level of pepsin and 56 patients (50.00%) had low level of pepsin; 36 patients (32.14%) did not have the presence of pepsin.
Comparison of the postoperative efficacy
Vocal fold morphology recovered in totally 80 patients, with 32 cases (88.89%) in pepsin negative group and 48 (63.16%) in pepsin group. Among the factors including detection of pepsin in vocal fold tissues, microscopic suturing, use of CO2 laser, and history of smoking, pepsin was a clinically significant indicator affecting the postoperative morphological recovery of the vocal fold (P = 0.003) (Table 1).
TABLE 1Influencing Factors for Vocal Fold Morphology Repair
There was significant difference in preoperative MPT between pepsin-free group and pepsin group (P < 0.05). At postoperative week 12, the hoarse voice was improved in all patients, and the differences was statistically significant in the proportion of patients with GRBAS class G, VHI, MPT, Jitter, Shimmer ,and NHR before and after surgical procedures (P < 0.05), and between pepsin-free group and pepsin group (P < 0.05) (Table 2).
TABLE 2Comparison of Voice Quality Between Pepsin Group and Pepsin-Free Group Before and After Surgical Procedures (x ± s)
Vocal fold polyp is one of the common diseases in Department of E.N.T., possibly causing voice handicap. Its etiology is complicated because inflammation can increase vascular permeability in the vocal fold, topical edema, anoxia, degeneration and fibrosis.
LPR is defined as the reflux of gastric contents into upper respiratory tract and digestive tract as well as the throat, which is more likely to induce many chronic laryngeal irritative symptoms including cough, repeat throat clearing, hoarse voice, sensation of foreign bodies in pharynx, dyspnea, and dysphagia. Several studies have revealed that 75% patients with vocal fold polyp have LPR,
The results of this study demonstrated that in all patients, 67.86% patients had pepsin detected in the samples, which is consistent with the study results of Lechien et al,
indicating that the attack of vocal fold polyp was possibly related to the pepsin (one of the components in the refluxed contents) detected in the samples. The laryngeal mucosal epithelium has been verified to be more sensitive to reflux-induced injury than the esophagus, and more than 50 refluxes of gastric juice (pH: <4.0) into the esophagus daily has been considered as normal, but 3 refluxes per week will cause serious laryngeal mucosal injury.
The otolaryngologic manifestations of gastroesophageal reflux disease (GERD): a clinical investigation of 225 patients using ambulatory 24-hour pH monitoring and an experimental investigation of the role of acid and pepsin in the development of laryngeal injury.
The reason is that compared with the esophageal mucosa, the laryngeal mucosae do not have protective mechanism specific to the gastric juice, such as mucous coating, acid clearance, and defending function of the epithelial cells, so that the laryngeal mucosae are more likely to be impaired by acid and pepsin.
The gastric reflux contents contain hydrochloric acid, protease and bile acid, with pH of 1.5-2.0, and they are harmful to the internal environment in the respiratory and upper digestive tracts if the pH is 6.8-7.0.
It is a proteolytic enzyme generated in the stomach, and is primarily secreted into pepsinogen in the zymogen form by the primary cells of stomach. A previous study disclosed that acid alone will not damage the integrity of epithelial barrier, but the combination of pepsin and acid can trigger significant damage to the laryngeal mucosa.
Moreover, some studies have demonstrated that pepsin transferred into the epithelial cells and reactivated by the intracellular structures dictyosomes and lysosomes (with pH of 5.0 and 4.0), which will further damage the connection structure between laryngeal mucosal epithelia cells, thus leading to intracellular injury and laryngeal mucosal inflammatory responses and affecting the repair of damaged vocal fold.
Pepsin promotes activation of epidermal growth factor receptor and downstream oncogenic pathways, at slightly acidic and neutral pH, in exposed hypopharyngeal cells.
Pepsin has been confirmed as a biomarker and potential treatment target for LPR since it can be detected in many organs and tissues in the upper digestive and respiratory tracts.
found in a dog study that the dogs’ vocal folds exposed to gastric juice will be notably thickened to irregular appearance, after which ulcer and granulation tissues gradually appear, suggestive of pathological evidence of chronic inflammation at post-exposure week 4. Kantas et al
reported that the symptoms and signs of patients with LPR-related vocal fold polyps could be notably improved after anti-reflux treatment is added following the surgical procedures as opposed to the control group, and they believed that LPR might be associated with postoperative epithelial repair and recurrence of vocal fold polyps.
To date, the 24-hour dual-probe pH monitoring has been the golden standard for the diagnosis of LPR.
However, there may be some false negative or positive results due to inaccurate probe location in the clinical practice. Additionally, this method has not been accepted by many patients partly because it is invasive and laborious, and partly because it may cause significant discomforts and high costs. In the clinical practice, most clinical physicians mainly depend on the patients’ symptoms and laryngoscopic results for judgment.
The present study revealed that in the 36 (32.14%) patients without pepsin detected in the samples of 112 patients with vocal fold polyps, 88.89% patients had vocal fold morphology recovered after procedures and nonexperienced recurrence; the GRBAS, MPT, jitter, shimmer, VHI, and NHR improved more significantly in pepsin-free group than those in pepsin group; and there were significant differences between two groups in vocal fold morphology recovery and ojective acoustic results. It is therefore suggested that pepsin detected in the vocal polyp tissue might have affected the postoperative repair of the surgical injury, and absence of pepsin in the vocal fold polyp tissues was beneficial to the morphological and functional recovery in the postoperative vocal fold. As concerns patients with pepsin detected, anti-reflux drugs, such as PPIs and other treatments including H2 receptor blockers and gastrointestinal prokinetic agents should be positively given.
This study also has some limitations: This study confirmed the relationships between detection results of pepsin in the vocal fold polyp tissues and the surgical efficacy. However, a certain causality between LPR (especially pepsin) and vocal fold polyps has not been adequately verified, and the clinical significance of the pepsin staining expression intensity needs to be further investigated in clinical practice in the future.
CONCLUSION
Pepsin in vocal fold polyp tissues is a significant clinical indicator influencing the postoperative acoustic effect and morphology recover of vocal fold, and patients with absence of pepsin in vocal fold polyps are superior to those with pepsin detected in terms of morphological and functional recovery of the postoperative vocal fold.
Conflict of interest
The authors of this study declare no conflict of interest.
Authors' contributions
Lingli Meng and Jie Meng completed the data analysis and wrote the manuscript. Yaqun Liu and Dewang Wang conducted the statistical analysis. Qingxiang Zhang revised the clinical data and the manuscript.
Reflux symptom index, reflux finding score, esophageal motility and their correlation with laryngopharyngeal reflux in patients with vocal cord polyps.
The otolaryngologic manifestations of gastroesophageal reflux disease (GERD): a clinical investigation of 225 patients using ambulatory 24-hour pH monitoring and an experimental investigation of the role of acid and pepsin in the development of laryngeal injury.
Pepsin promotes activation of epidermal growth factor receptor and downstream oncogenic pathways, at slightly acidic and neutral pH, in exposed hypopharyngeal cells.
Statistically significant difference.Abbreviations: CI, confidence interval; OR, odds ratio; NA, not applicable.
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