Analysis of clinical characteristics of 617 patients with benign airway stenosis

Introduction Benign airway stenosis (BAS), namely airway narrowing caused by a variety of benign lesions, can lead to varying degrees of breathing difficulties and even death due to asphyxia. This study aimed to elucidate the clinical characteristics of BAS, including etiology, treatment and pathology, by analyzing the clinical data of BAS patients. Methods A retrospective analysis was conducted using the clinical data of 617 BAS cases from January 2017 to December 2022. The pathological characteristics of the tissues were assessed by hematoxylin–eosin (H&E) and Masson’s staining. Besides, protein expression levels were determined by immunohistochemistry (IHC). Results A total of 617 patients were included (333 females [53.97%] and 284 males [46.03%]), with an average age of 48.93 ± 18.30 (range 14–87). Tuberculosis (n = 306, 49.59%) and trauma (n = 179, 29.02%) were the two leading etiologies of BAS, followed by airway foreign bodies (FB, n = 74, 11.99%), external compression (n = 25, 4.05%) and other etiologies (n = 33, 5.35%). Among 306 tuberculous tracheobronchial stenosis (TBTS) cases, most were females (n = 215, 70.26%), and TBTS mainly occurred in the left main bronchus (n = 97, 31.70%), followed by the right middle bronchus (n = 70 cases, 22.88%). The majority of TBTS patients (n = 259, 84.64%) were treated by interventional therapy. The condition of 179 BAS patients was ascribed to trauma, such as tracheal intubation (n = 92, 51.40%), tracheotomy (n = 69, 38.56%), injury (n = 15, 8.38%) and surgery (n = 3, 1.68%), which mostly took place in the trachea (n = 173, 96.65%). TAS patients mainly received interventional therapy (n = 168, 93.85%) and stent implantation (n = 47, 26.26%). The granulation tissues of BAS primarily featured inflammation, proliferation and fibrosis. IHC indicated the up-regulated expressions of transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA), collagen type I protein (COL-I) and vimentin, and the down-regulated expression of E-cadherin, which indicated fibrosis and epithelial-mesenchymal transition (EMT). Conclusion Tuberculosis was the main etiology, and trauma was the secondary etiology. The granulation tissues of BAS were characterized by inflammation, fibrosis and probably EMT. Comprehensive interventional therapy is an effective method of treating BAS.


Introduction
Benign airway stenosis (BAS) refers to airway narrowing caused by various benign lesions, which can result in varying degrees of breathing difficulties and even death due to asphyxia (1). At present, its mainstream treatment methods include surgery and interventional and drug therapies (2). The therapeutical painful and subsequent complications in surgery or interventional therapy not only bring physical pain to patients but also cause psychological trauma to them and affect their quality of life (3,4). It is necessary to adopt effective measures to prevent the aggravation of airway injury and pathological repair at the early stage, which is the fundamental prevention and treatment of BAS.
In this study, a retrospective analysis was conducted using the clinical data of BAS patients. The pathological characteristics of the tissues were evaluated by hematoxylin-eosin (H&E) and Masson's staining methods. Besides, protein expression levels were detected by immunohistochemistry (IHC) to reveal the change of cytokines and proteins in BAS development to provide a basis for the clinical diagnosis and treatment of BAS.

Case selection
BAS patients in the Second Affiliated Hospital of Guangxi Medical University in Guangxi, China were retrospectively analyzed from January 2017 to December 2022. Data concerning etiology, stenosis site, symptoms and treatments were collected. Selection criteria were as follows: patients who were aged over 14 and confirmed by bronchoscopy, chest computed tomography (CT) or pathology owing to various factors, such as endometrial tuberculosis, endotracheal intubation, tracheotomy, pulmonary surgery, external compression and airway foreign body (FB). Exclusion criteria were as follows: patients who were aged under 14, with unknown etiology unknown and insufficient data.

Pathological analysis
BAS granulations were obtained with bronchoscopy, and normal tissues were obtained from the surgical lobectomy of lung cancer. The specimens were fixed in a 4% paraformaldehyde solution for 48 h, then embedded and sliced for subsequent detection. Adjacent slices received H&E and Masson's staining, and slides were scanned with an inverted microscope (Olympus, Japan). The primary antibodies for IHC, transforming growth factor-β1 (TGF-β1), E-cadherin, α-smooth muscle actin (α-SMA), collagen type I protein (COL-I) and vimentin for IHC were purchased from Servicebio, China. Slices for IHC were stained with TGF-β1, E-cadherin, α-SMA, COL-I and vimentin, then scanned with 3DHistech Digital Pathology System (Bio-One, China). Representative slice areas were shown in screenshots. Subsequently, Image J was used to read and analyze the positivity intensity of the measured areas.

Ethical statement
The study was approved by the Ethics Committee of the Second Affiliated Hospital of Guangxi Medical University, Nanning, China.

Statistical analysis
Statistical Package for the Social Sciences (SPSS) statistics software (version 26) was utilized to conduct all statistical analyses. GraphPad Prism 8 software was used for plotting. Qualitative variables were expressed as percent distributions in each category, and quantitative ones were expressed as means ± standard deviations and medians (ranges) for normally and non-normally distributed variables, respectively. The statistical test was an unpaired t-test, and a p < 0.05 was considered to show statistical significance.
Follow-ups were conducted on 535 of these patients by telephone, WeChat or on the site, with a period of 6 months to 5 years, while the
Another representative case report is presented in Figure 1B. In the report, a 23-year-old female patient was also diagnosed with active TBTS, whose main symptoms were cough and dyspnea. Here, the CT scan showed stenosis in the left main bronchus and the enlargement of mediastinal lymph nodes. A bronchoscopy demonstrated the obvious stenosis of the left main bronchus, a large amount of gray and white cheesy necrosis and visible bronchial lymph node fistula. H&E staining revealed cheesy necrosis and inflammatory cell aggregation. Masson staining showed fibrosis in the granulation tissues of TBTS compared with the normal control group (Figure 2).
Here, different treatments are shown in Figure 3. A was a patient treated by interventional therapy, while B, C and D were implanted with V-shape silicone, Y-shape and metallic stents, respectively. Airway stenosis was relieved after interventional therapy or stent plantation. Of 262 TBTS patients followed up, 42 (16.03%) had a complete recovery after treatment, 204 (77.86%) had an incomplete recovery, and 16 (6.11%) died of respiratory failure or lung infection (Table 3).
As depicted in the report of representative cases (Figure 4), a 51-year-old man was diagnosed with post-tracheotomy stenosis and a 27-year-old man with post-intubation stenosis. Their most notable symptoms were dyspnea. CT and bronchoscopy showed severe tracheal stenosis, which resulted from massive granulation tissue hyperplasia. Pathology showed extensive granulation tissue hyperplasia. The staining results revealed the presence of squamous epithelial hyperplasia, the severe thickening of mucosa, hyperplasia, the repair of scar tissues in lamina propria and submucosa, the staggered formation of numerous mature and coarse collagen fibers, the appearance of countless capillaries, frequent vascular dilatation, mild inflammatory reaction, and the infiltration of a few lymphocytes. Masson's staining indicated the proliferation of plentiful blue collagen fibers ( Figure 2). All of them received interventional therapy, and stenosis was relieved.

BAS caused by external compression
A total of 25 BAS cases were attributable to the external compression of surrounding airway tissues (Table 1), including eight males (32.00%) and 17 females (68.00%), with an average age of 62.88 ± 14.04 (range 31-83). Dyspnea was observed to be the main symptom (n = 22, 88.00%), and cough was found in eight cases (32.00%). All 25 cases of stenosis were located in the main trachea, of which 22 (88.00%) were caused by thyroid goiter compression and the other three by mediastinal cyst, bronchial cyst and cervical hematoma compression, respectively. Seven patients received metal stent plantation. Two, 15 and eight patients received interventional therapy, surgical treatment and conservative treatment, respectively. The tracheal mucosa was intact, and the tracheal segment was compressed and deformed under bronchoscopy. Tracheal stenosis can be relieved after surgical treatment in 15 patients. Of the 24 patients we followed, only one died of heart disease.
As depicted in the report of representative cases ( Figure 6), a 58-year-old woman was admitted to hospital due to "cervical mass found for more than one decade and shortness of breath for 2 days. " CT showed huge cervical goiter, and the upper tracheal segment was compressed. Bronchoscopy showed severe external compression stenosis in the trachea, and the tracheal mucous was normal. The tracheal stenosis was improved significantly after the implantation of a metal-coated stent.
Another representative case showed a 64-year-old woman admitted to hospital due to "shortness of breath for more than 1 month" and confirmed to have external compression tracheal stenosis by CT and bronchoscopy. The tracheal stenosis was also improved significantly after the implantation of a metal-coated stent ( Figure 6).

IHC
IHC detection was performed on BAS granulation and normal tissues. Normal tissues were collected from a 57-year-old female patient and a 49-year-old male patient who accepted pulmonary lobectomy on account of lung adenocarcinoma and were diagnosed with lung cancer. BAS granulation tissues were gathered from a 57-year-old male patient diagnosed with TBTS and a 27-year-old male patient diagnosed with TAS. As evidenced in Figure 7, the expression of E-cadherin in BAS granulation tissues was downregulated compared with normal control, while those of vimentin, α-SMA, COL-I, and TGF-β1 were up-regulated, with statistical significance (p < 0.001). These results suggested that EMT, fibrosis and the increased expression of TGF-β1 existed in BAS granulation tissues.

Discussion
In the present study, the most common cause of BAS was found to be tuberculosis, and the other causes included trauma, FB and external compression. This finding was consistent with other research in China (5, 6), which was possibly ascribed to the higher incidence of tuberculosis in this country (7,8). China has a high incidence of tuberculosis owing to its underdeveloped economy and poor medical conditions. However, the etiology of BAS reported abroad differs from that reported in China (9). For instance, the main cause of 40 BAS patients reported by the University of California was lung transplantation (32.5%), followed by tracheotomy or tracheal intubation (25.0%) (10). Follow-ups were conducted on 535 of these patients in 5 years. It was found that most of them had partial recovery, while some died of respiratory failure, lung infection or other underlying diseases. It was shown that BAS was a chronic disease with low cure and death rates.
TBTS mainly occurred in the left main bronchus and the right middle bronchus, which could be put down to the anatomical features in these sites (2). It was less common in multiple sites or the trachea. Its common symptoms included cough and dyspnea, and some patients appeared to have fever and loss of weight. Most TBTS patients were females, whose mean age was less than TAS patients. This was in line with the other research, which may imply that TBTS was related to estrogens (11). Typical pathological characteristics like cheesy necrosis and acid-fast bacillus were noted in TBTS granulation tissues, but fibrosis and collagen hyperplasia mainly appeared in posttuberculous cicatricial airway stenosis tissues (12). Most of the patients had received comprehensive interventional therapy, including balloon dilatation, high-frequency electric, laser and argon plasma thermal ablation, snare resection, etc. due to less trauma and low cost (2,5). Some TBTS patients received stent implantation, including V-shaped silicone, Y-shaped silicone and metal stents. Y-shaped silicone and metal stents are widely used in BAS by virtue of low mobility (13). V-shaped silicone stents are a new type of stents with the advantages of low mobility, easy removal and few complications (14). TAS was caused mainly by tracheal intubation and tracheotomy in this and other research, and sometimes by surgery or injury (15). The majority of TAS patients were complicated with cerebrovascular disease, hypertension and diabetes in that they were more likely to require long-term intubation or tracheotomy. Typical pathological   IHC of benign airway stenosis granulations and normal control tissues (20X  (18). Stent implantation included straight silicone, Y-shape silicone, Montgomery T-tube and metallic stents.
Montgomery T-tube implantation is a safe, viable and effective tracheal forming method for airway stenosis caused by tracheotomy (19).
In the present study, organic FBs were shown to be the most common airway FBs that resulted in BAS, including bones, nuts and other food. Most of the patients had a history of definite choking (20). Iatrogenic FBs are also common in adults' airway FBs, almost caused by dental operation (21). The most common site was the right bronchus because of its anatomical characteristics, which were aligned with other studies (22,23). Timely and effective interventional therapy is an important way to prevent BAS caused by airway FB (23).
In general, BAS caused by external compression is triggered by thyroid goiter, lymphoma, granuloma, esophageal tumor or large vascular malformation, etc. The airway mucosa is mostly normal, and only the compression of surrounding tracheal tissues gives rise to the occurrence of lumen stenosis and cartilage deformation (24,25). For BAS patients caused by thyroid goiter, most were relieved by surgery, while some may have comorbidities and require a multidisciplinary approach to management owing to complications like tracheomalacia or respiratory failure after surgery (24). The other etiologies included benign airway tumor, congenital airway stenosis, oxygen inhaling and tracheobronchomalacia (26,27).
IHC analysis revealed that the expression of E-cadherin in BAS granulation tissues was down-regulated, while those of vimentin, α-SMA, COL-I, and TGF-β1 were up-regulated compared with the normal control group. This suggested that EMT, fibrosis and the increased expression of TGF-β1 existed in BAS granulation tissues, which accorded with other studies (12,28,29). TGF-β1 is of importance to the occurrence and development of BAS and is activated under stress like inflammation and injury. It promotes the occurrence and development of EMT, fibroblast-to-myofibroblast transformation, and fibrosis through various pathways. In addition, TGF-β1 facilitates the mesenchymal transformation of epithelial cells, the differentiation of fibroblasts into myofibroblasts, and the proliferation and migration of myofibroblasts (30). Some research has demonstrated that EMT appears in BAS, usually at the early stage of the disease (12,28).
In this study, tuberculosis was the most common cause of BAS. Therefore, the prevention and timely treatment of tuberculosis became critical to preventing BAS. Likewise, managing patients with tracheal intubation and tracheotomy is also a top priority in BAS prevention.

Conclusion
The clinical characteristics of 617 BAS patients caused by different etiologies were analyzed and summarized. Tuberculosis was the main etiology, and trauma was the secondary etiology. The granulation tissues of BAS were characterized by inflammation, fibrosis and probably EMT. Comprehensive interventional therapy is an effective method of treating BAS, while airway stent plantation and surgery are used to supplement those without responding to conventional interventional therapy. This study is limited because it only includes cases from one hospital. It may contribute to the development of more personalized approaches to the diagnosis and management of BAS in the future.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
The study was approved by the Ethics Committee of the Second Affiliated Hospital of Guangxi Medical University, Nanning, China. Approval number [No.2021-KY(0172)] and was conducted according to the principles outlined in the Declaration of Helsinki. Informed consent was obtained from all the subjects. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.