An Ovine Model Yields Histology and Gene Expression Changes Consistent with Laryngotracheal Stenosis.

OBJECTIVES: Animal models for laryngotracheal stenosis (LTS) are critical to understand underlying mechanisms and study new therapies. Current animal models for LTS are limited by small airway sizes compared to human. The objective of this study was to develop and validate a novel, large animal ovine model for LTS. METHODS: Sheep underwent either bleomycin-coated polypropylene brush injury to the subglottis (n = 6) or airway stent placement (n = 2) via suspension microlaryngoscopy. Laryngotracheal complexes were harvested 4 weeks following injury or stent placement. For the airway injury group, biopsies (n = 3 at each site) were collected of tracheal scar and distal normal regions, and analyzed for fibrotic gene expression. Lamina propria (LP) thickness was compared between injured and normal areas of trachea. RESULTS: No mortality occurred in sheep undergoing airway injury or stent placement. There was no migration of tracheal stents. After protocol optimization, LP thickness was significantly increased in injured trachea (Sheep #3: 529.0 vs. 850.8 um; Sheep #4: 933.0 vs. 1693.2 um; Sheep #5: 743.7 vs. 1378.4 um; Sheep #6: 305.7 vs. 2257.6 um). A significant 62-fold, 20-fold, 16-fold, 16-fold, and 9-fold change of COL1, COL3, COL5, FN1, and TGFB1 was observed in injured scar specimen relative to unaffected airway, respectively. CONCLUSION: An ovine LTS model produces histologic and transcriptional changes consistent with fibrosis seen in human LTS. Airway stent placement in this model is safe and feasible. This large airway model is a reliable and reproducible method to assess the efficacy of novel LTS therapies prior to clinical translation. LEVEL OF EVIDENCE: N/A Laryngoscope, 2024.

A Comprehensive Flow Cytometry Panel for Analysis of Idiopathic Subglottic Stenosis.

OBJECTIVE: To present a comprehensive flow cytometry panel for idiopathic subglottic stenosis (iSGS). STUDY DESIGN: Controlled ex vivo cohort study. SETTING: Tertiary care academic hospital in a metropolitan area. METHODS: Flow cytometry and single-cell RNA sequencing were performed on 9 paired normal and scar tissue samples from iSGS patients. Flow cytometry was used to assess the presence of myeloid (CD11b, CD14, CD15, Siglec8), lymphoid (CD3, CD4, CD8, gamma delta [γδ], FOXP3), endothelial (CD31), fibroblast (CD90, SMA), and epithelial (CD326, CK5) markers. RESULTS: On flow cytometry, iSGS scar is characterized by an increased presence of myeloid, lymphoid, endothelial, and fibroblast cell types, but a decreased presence of epithelial cells. In the myeloid lineage, iSGS scar samples demonstrated increased CD11b+ monocytes (P < .001), Siglec8+ eosinophils (P = .03), and CD14+ monocytes (P = .02). In the lymphoid lineage, iSGS scar demonstrated increased CD3+ T-cells (P < .001), CD4+ helper T-cells (P < .001), γδ+ T-cells (P < .001), and FOXP3+ regulatory T-cells (P = .002). iSGS scar exhibited specific increases in CD90+ (P = .04) and SMA+ (P < .001) fibroblasts but decreased CD326+ (E-cadherin) epithelial cells (P = .01) relative to normal samples. CONCLUSION: We present a comprehensive flow cytometry panel for iSGS. This flow panel may serve as a common platform among airway scientists to elucidate the cellular mechanisms underpinning iSGS and other upper airway pathologies. Scar iSGS samples demonstrate a distinct cellular profile relative to normal iSGS specimens, exhibiting increased fibroblast, endothelial, and inflammatory cell types but decreased epithelium.

Factors Associated with Iatrogenic Laryngeal Injury in Recurrent Respiratory Papillomatosis.

OBJECTIVE: To describe iatrogenic laryngeal injury and identify its risk factors in recurrent respiratory papillomatosis (RRP) patients receiving surgical care. STUDY DESIGN: Case-control. SETTING: Tertiary care academic hospital in a metropolitan area. METHODS: Charts of patients with RRP seen at our institution from January 2002 to December 2022 were reviewed. Patients were separated into 2 cohorts based upon whether they experienced any form of iatrogenic laryngeal injury-including anterior commissure synechiae, vocal cord scar, reduced vocal fold pliability, vocal fold motion impairment, and glottic and/or subglottic stenosis. Adjusted logistic regressions were performed to identify factors associated with iatrogenic laryngeal injury. RESULTS: Of 199 RRP patients, 133 (66.8%) had identifiable iatrogenic laryngeal injury. The most common injuries were anterior commissure synechiae (n = 67; 50.4%) and reduced vocal fold pliability (n = 54; 40.6%). On a multivariate logistic regression, patients with diabetes mellitus (adjusted odds ratio [aOR] [95% confidence interval [CI]]: 2.99 [1.02, 8.79]; P = .04) and who received at least 10 surgeries lifetime (aOR [95% CI]: 14.47 [1.70, 123.19]; P = .01) were at increased risk for iatrogenic laryngeal injury, whereas receiving less than 5 surgeries (aOR [95% CI]: 0.21 [0.09, 0.51]; P < .001) was found to be protective. When treating the lifetime number of surgeries as a continuous variable, a greater number of surgeries was a significant risk factor for iatrogenic laryngeal injury (aOR [95% CI]: 1.32 [1.14, 1.53]; P < .001). CONCLUSION: These results suggest the importance of strict glucose control for diabetic patients receiving RRP surgical care, and emphasize the clinical need to identify medical therapies to decrease RRP surgical frequency for patients.

Sirolimus-eluting airway stent reduces profibrotic Th17 cells and inhibits laryngotracheal stenosis.

Laryngotracheal stenosis (LTS) is pathologic fibrotic narrowing of the larynx and trachea characterized by hypermetabolic fibroblasts and CD4+ T cell-mediated inflammation. However, the role of CD4+ T cells in promoting LTS fibrosis is unknown. The mTOR signaling pathways have been shown to regulate the T cell phenotype. Here we investigated the influence of mTOR signaling in CD4+ T cells on LTS pathogenesis. In this study, human LTS specimens revealed a higher population of CD4+ T cells expressing the activated isoform of mTOR. In a murine LTS model, targeting mTOR with systemic sirolimus and a sirolimus-eluting airway stent reduced fibrosis and Th17 cells. Selective deletion of mTOR in CD4+ cells reduced Th17 cells and attenuated fibrosis, demonstrating CD4+ T cells' pathologic role in LTS. Multispectral immunofluorescence of human LTS revealed increased Th17 cells. In vitro, Th17 cells increased collagen-1 production by LTS fibroblasts, which was prevented with sirolimus pretreatment of Th17 cells. Collectively, mTOR signaling drove pathologic CD4+ T cell phenotypes in LTS, and targeting mTOR with sirolimus was effective at treating LTS through inhibition of profibrotic Th17 cells. Finally, sirolimus may be delivered locally with a drug-eluting stent, transforming clinical therapy for LTS.

Myeloid Phenotypes in Tracheostomy-Associated Granulation Tissue.

OBJECTIVE(S): Tracheostomy-associated granulation tissue is a common, recurrent problem occurring secondary to chronic mucosal irritation. Although granulation tissue is composed of predominantly innate immune cells, the phenotype of monocytes and macrophages in tracheostomy-associated granulation tissue is unknown. This study aims to define the myeloid cell population in granulation tissue secondary to tracheostomy. METHODS: Granulation tissue biopsies were obtained from 8 patients with tracheostomy secondary to laryngotracheal stenosis. Cell type analysis was performed by flow cytometry and gene expression was measured by quantitative real-time polymerase chain reaction. These methods and immunohistochemistry were used to define the monocyte/macrophage population in granulation tissue and were compared to tracheal autopsy control specimens. RESULTS: Flow cytometry demonstrated macrophages (CD45+CD11b+) and monocytes (CD45+FSClow SSClow ) represent 23.2 ± 6% of the granulation tissue cell population. The M2 phenotype (CD206) is present in 77 ± 11% of the macrophage population and increased compared to the M1 phenotype (p = 0.012). Classical monocytes (CD45+CD14high CD16low ) were increased in granulation tissue compared to controls (61.2 ± 7% and 30 ± 8.5%, p = 0.038). Eighty-five percent of macrophages expressed pro-inflammatory S100A8/A9 and 36 ± 4% of macrophages co-localized CD169, associated with tissue-resident macrophages. M2 gene expression (Arg1/CD206) was increased in granulation tissue (3.7 ± 0.4, p = 0.035 and 3.5 ± 0.5, p = 0.047) whereas M1 gene expression (CD80/CD86) was similar to controls (p = 0.64, p = 0.3). Immunohistochemistry of granulation tissue demonstrated increased cells co-localizing CD11b and CD206. CONCLUSIONS: M2 macrophages are the dominant macrophage phenotype in tracheostomy-associated granulation tissue. The role of this cell type in promoting ongoing inflammation warrants future investigation to identify potential treatments for granulation tissue secondary to tracheostomy. LEVEL OF EVIDENCE: 3 Laryngoscope, 133:2346-2356, 2023.

SARS-CoV-2 Nasopharyngeal Swab as a Foreign Body: A Case Report.

The SARS-CoV-2 pandemic response utilizes nasopharyngeal swabbing as a prolific testing method for presence of viral RNA. The depth of the swab to the nasopharynx coupled with breakpoints along the shaft leads to a risk for foreign body retention. Here, we present a case of a nasopharyngeal swab that became a retained foreign body during routine swabbing to test for the SARS-CoV-2 virus. Bedside flexible fiberoptic endoscopy was performed and did not reveal a foreign body in the nasopharynx or larynx. Subsequent computed tomography (CT) scan demonstrated the radiopaque retained foreign body at the distal gastroesophageal junction. The patient remained asymptomatic and did not have any upper airway or gastrointestinal symptoms. This unique case demonstrates a potential risk associated with SARS-CoV-2 nasopharyngeal swab testing and highlights management strategies that serve the patient while adequately protecting health care providers. A standardized approach to evaluation optimally includes bedside flexible endoscopy with appropriate personal protective equipment, prompt airway evaluation if aspiration is suspected, and noncontrasted CT imaging if the known foreign body is not identified via other modalities.

Phenotypic Epithelial Changes in Laryngotracheal Stenosis.

OBJECTIVE: Characterize and quantify epithelium in multiple etiologies of laryngotracheal stenosis (LTS) to better understand its role in pathogenesis. STUDY DESIGN: Controlled in vitro cohort study. METHODS: Endoscopic brush biopsy samples of both normal (non-scar) and scar were obtained in four patients with idiopathic subglottic stenosis (iSGS) and four patients with iatrogenic LTS (iLTS). mRNA expression of basal, ciliary, and secretory cell markers were evaluated using quantitative PCR. Cricotracheal resection tissue samples (n = 5 per group) were also collected, analyzed using quantitative immunohistochemistry, and compared with rapid autopsy tracheal samples. RESULTS: Both iSGS and iLTS-scar epithelium had reduced epithelial thickness compared with non-scar control epithelium (P = .0009 and P = .0011, respectively). Basal cell gene and protein expression for cytokeratin 14 was increased in iSGS-scar epithelium compared with iLTS or controls. Immunohistochemical expression of ciliary tubulin alpha 1, but not gene expression, was reduced in both iSGS and iLTS-scar epithelium compared with controls (P = .0184 and P = .0125, respectively). Both iSGS and iLTS-scar had reductions in Mucin 5AC gene expression (P = .0007 and P = .0035, respectively), an epithelial goblet cell marker, with reductions in secretory cells histologically (P < .0001). CONCLUSIONS: Compared with non-scar epithelium, the epithelium within iSGS and iLTS is morphologically abnormal. Although both iSGS and iLTS have reduced epithelial thickness, ciliary cells, and secretory cells, only iSGS had significant increases in pathological basal cell expression. These data suggest that the epithelium in iSGS and iLTS play a common role in the pathogenesis of fibrosis in these two etiologies of laryngotracheal stenosis. SETTING: Tertiary referral center (2017-2020). LEVEL OF EVIDENCE: NA Laryngoscope, 132:2194-2201, 2022.

Identifying Phenotypically Distinct Fibroblast Subsets in Type 2 Diabetes-Associated Iatrogenic Laryngotracheal Stenosis.

OBJECTIVE: Iatrogenic laryngotracheal stenosis (iLTS) is the pathologic narrowing of the glottis, subglottis, and/or trachea secondary to intubation or tracheostomy related injury. Patients with type 2 diabetes mellitus (T2DM) are more likely to develop iLTS. To date, the metabolomics and phenotypic expression of cell markers in fibroblasts derived from patients with T2DM and iLTS are largely unknown. STUDY DESIGN: Controlled in vitro cohort study. SETTING: Tertiary referral center (2017-2020). METHODS: This in vitro study assessed samples from 6 patients with iLTS who underwent surgery at a single institution. Fibroblasts were isolated from biopsy specimens of laryngotracheal scar and normal-appearing trachea and compared with controls obtained from the trachea of rapid autopsy specimens. Patients with iLTS were subcategorized into those with and without T2DM. Metabolic substrates were identified by mass spectrometry, and cell protein expression was measured by flow cytometry. RESULTS: T2DM iLTS-scar fibroblasts had a metabolically distinct profile and clustered tightly on a Pearson correlation heat map as compared with non-T2DM iLTS-scar fibroblasts. Levels of itaconate were elevated in T2DM iLTS-scar fibroblasts. Flow cytometry demonstrated that T2DM iLTS-scar fibroblasts were associated with higher CD90 expression (Thy-1; mean, 95%) when compared with non-T2DM iLTS-scar (mean, 83.6%; P = .0109) or normal tracheal fibroblasts (mean, 81.1%; P = .0042). CONCLUSIONS: Scar-derived fibroblasts from patients with T2DM and iLTS have a metabolically distinct profile. These fibroblasts are characterized by an increase in itaconate, a metabolite related to immune-induced scar remodeling, and can be identified by elevated expression of CD90 (Thy-1) in vitro.

Glutamine Inhibition Reduces Iatrogenic Laryngotracheal Stenosis.

OBJECTIVE/HYPOTHESIS: Glutamine inhibition has been demonstrated an antifibrotic effect in iatrogenic laryngotracheal stenosis (iLTS) scar fibroblasts in vitro. We hypothesize that broadly active glutamine antagonist, DON will reduce collagen formation and fibrosis-associated gene expression in iLTS mice. STUDY DESIGN: Prospective controlled animal study. METHODS: iLTS in mice were induced by chemomechanical injury of the trachea using a bleomycin-coated wire brush. PBS or DON (1.3 mg/kg) were administered by intraperitoneal injection (i.p.) every other day. Laryngotracheal complexes were harvested at days 7 and 14 after the initiation of DON treatment for the measurement of lamina propria thickness, trichrome stain, immunofluorescence staining of collagen 1, and fibrosis-associated gene expression. RESULTS: The study demonstrated that DON treatment reduced lamina propria thickness (P = .025) and collagen formation in trichrome stain and immunofluorescence staining of collagen 1. In addition, DON decreased fibrosis-associated gene expression in iLTS mice. At day 7, DON inhibited Col1a1 (P < .0001), Col3a1 (P = .0046), Col5a1 (P < .0001), and Tgfß (P = .023) expression. At day 14, DON reduced Co1a1 (P = .0076) and Tgfß (P = .023) expression. CONCLUSIONS: Broadly active glutamine antagonist, DON, significantly reduces fibrosis in iLTS mice. These results suggest that the concept of glutamine inhibition may be a therapeutic option to reduce fibrosis in the laryngotracheal stenosis. LEVEL OF EVIDENCE: N/A Laryngoscope, 131:E2125-E2130, 2021.

Laryngotracheal Mucosal Surface Expression of Candidate Biomarkers in Idiopathic Subglottic Stenosis.

OBJECTIVES: Idiopathic subglottic stenosis (iSGS) is an inflammatory process leading to fibrosis and narrowing of the laryngotracheal airway. There is variability in patient response to surgical intervention, but the mechanisms underlying this variability are unknown. In this pilot study, we measure expression of candidate targets at the mucosal surface of the subglottis in iSGS patients. We aim to identify putative biomarkers for iSGS that provide insights into the molecular basis of disease progression, yield a gene signature for the disease, and/or predict a response to therapy. STUDY DESIGN: In vitro comparative study of human cells. METHODS: Levels of candidate transcripts and proteins were measured in healthy and stenotic laryngotracheal tissue specimens taken from the mucosal surface in 16 iSGS patients undergoing endoscopic balloon dilation. Pre- and post-operative pulmonary function test and patient reported voice and breathing outcomes were also assessed. Unsupervised clustering was used to define patient subgroups based on expression profile. RESULTS: Pulmonary function and voice and breathing outcome metrics demonstrated significant post-operative improvement. Transcript levels of αSMA, CCL2, COL1A1, COL3A1, FN1, IFNG, and TGFB1 and protein levels of CCL2, IFNG, and IL-6 were significantly upregulated in stenotic as compared to healthy tissues. Marked heterogeneity was observed in the patterns of expression of candidate markers across individuals and tissue types. Patient subgroups defined by expression profile did not show a statistically significant difference in dilation interval. CONCLUSION: Pro-inflammatory and pro-fibrotic pathways are significantly upregulated along the mucosal surface of stenotic laryngotracheal tissues, and CCL2 and IFNG merit further investigation as potential iSGS biomarkers. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:342-349, 2021.

Characterization of Fibroblasts in Iatrogenic Laryngotracheal Stenosis and Type II Diabetes Mellitus.

OBJECTIVES: Iatrogenic laryngotracheal stenosis (iLTS) is the pathological narrowing of the glottis, subglottis, and/or trachea due to scar tissue. Patients with type 2 diabetes mellitus (T2DM) are over 8 times more likely to develop iLTS and represent 26% to 53% of all iLTS patients. In this investigation, we compared iLTS scar-derived fibroblasts in patients with and without T2DM. STUDY DESIGN: Controlled ex vivo study. METHODS: iLTS scar fibroblasts were isolated and cultured from subglottic scar biopsies in iLTS patients diagnosed with or without type 2 diabetes (non-T2DM). Fibroblast proliferation, fibrosis-related gene expression, and metabolic utilization of oxidative phosphorylation (OXPHOS) and glycolysis were assessed. Contractility was measured using a collagen-based assay. Metabolically targeted drugs (metformin, phenformin, amobarbital) were tested, and changes in fibrosis-related gene expression, collagen protein, and contractility were evaluated. RESULTS: Compared to non-T2DM, T2DM iLTS scar fibroblasts had increased α-smooth muscle actin (αSMA) expression (8.2× increased, P = .020), increased contractility (mean 71.4 ± 4.3% vs. 51.7 ± 16% Δ area × 90 minute-1 , P = .016), and reduced proliferation (1.9× reduction at 5 days, P < .01). Collagen 1 (COL1) protein was significantly higher in the T2DM group (mean 2.06 ± 0.19 vs. 0.74 ±.44 COL1/total protein [pg/µg], P = .036). T2DM iLTS scar fibroblasts had increased measures of OXPHOS, including basal respiration (mean 86.7 vs. 31.5 pmol/minute/10 µg protein, P = .016) and adenosine triphosphate (ATP) generation (mean 97.5 vs. 25.7 pmol/minute/10 µg protein, P = .047) compared to non-T2DM fibroblasts. Amobarbital reduced cellular contractility; decreased collagen protein; and decreased expression of αSMA, COL1, and fibronectin. Metformin and phenformin did not significantly affect fibrosis-related gene expression. CONCLUSION: T2DM iLTS scar fibroblasts demonstrate a myofibroblast phenotype and greater contractility compared to non-T2DM. Their bioenergetic preference for OXPHOS drives their increased contractility, which is selectively targeted by amobarbital. LEVEL OF EVIDENCE: NA Laryngoscope, 131:1570-1577, 2021.

Inhibition of glutaminase to reverse fibrosis in iatrogenic laryngotracheal stenosis.

OBJECTIVES/HYPOTHESIS: Glutamine metabolism is a critical energy source for iatrogenic laryngotracheal stenosis (iLTS) scar fibroblasts, and glutaminase (GLS) is an essential enzyme converting glutamine to glutamate. We hypothesize that the GLS-specific inhibitor BPTES will block glutaminolysis and reduce iLTS scar fibroblast proliferation, collagen deposition, and fibroblast metabolism in vitro. STUDY DESIGN: Test-tube Lab Research. METHODS: Immunohistochemistry of a cricotracheal resection (n = 1) and a normal airway specimen (n = 1) were assessed for GLS expression. GLS expression was assessed in brush biopsies of subglottic/tracheal fibrosis and normal airway from patients with iLTS (n = 6). Fibroblasts were isolated and cultured from biopsies of subglottic/tracheal fibrosis (n = 6). Fibroblast were treated with BPTES and BPTES + dimethyl α-ketoglutarate (DMK), an analogue of the downstream product of GLS. Fibroblast proliferation, gene expression, protein production, and metabolism were assessed in all treatment conditions and compared to control. RESULTS: GLS was overexpressed in brush biopsies of iLTS scar specimens (P = .029) compared to normal controls. In vitro, BPTES inhibited iLTS scar fibroblast proliferation (P = .007), collagen I (Col I) (P < .0001), collagen III (P = .004), and α-smooth muscle actin (P = .0025) gene expression and protein production (P = .031). Metabolic analysis demonstrated that BPTES reduced glycolytic reserve (P = .007) but had no effects on mitochondrial oxidative phosphorylation. DMK rescued BPTES inhibition of Col I gene expression (P = .0018) and protein production (P = .021). CONCLUSIONS: GLS is overexpressed in iLTS scar. Blockage of GLS with BPTES significantly inhibits iLTS scar fibroblasts proliferation and function, demonstrating a critical role for GLS in iLTS. Targeting GLS to inhibit glutaminolysis may be a successful strategy to reverse scar formation in the airway. LEVEL OF EVIDENCE: NA Laryngoscope, 2020.

The role of inflammatory cytokines in the development of idiopathic subglottic stenosis.

Idiopathic subglottic stenosis (iSGS) is a debilitating extrathoracic obstruction involving the lower laryngeal and upper tracheal airway. It arises without a known antecedent injury or associated disease process. iSGS is a fibrotic disease marked histologically by excessive accumulation of fibrous connective tissue components of the extracellular matrix (ECM, i.e., collagen and fibronectin) in inflamed tissue, which leads to airway obstruction and clinical dyspnea. Diverse diseases in divergent organ systems are associated with fibrosis, suggesting common pathogenic pathways. One of the most common is sustained host inflammation. Recent investigations focusing on the inflammatory response associated with iSGS have sought to characterize the immunophenotype and cytokine profile of the airway scar in iSGS. While the role of the immune response as inciting event in iSGS remains unresolved, the centrality of an active immune response to the observed subglottic tissue remodeling is becoming more defined.

Pathologic Fibroblasts in Idiopathic Subglottic Stenosis Amplify Local Inflammatory Signals.

OBJECTIVE: To characterize the phenotype and function of fibroblasts derived from airway scar in idiopathic subglottic stenosis (iSGS) and to explore scar fibroblast response to interleukin 17A (IL-17A). STUDY DESIGN: Basic science. SETTING: Laboratory. SUBJECTS AND METHODS: Primary fibroblast cell lines from iSGS subjects, idiopathic pulmonary fibrosis subjects, and normal control airways were utilized for analysis. Protein, molecular, and flow cytometric techniques were applied in vitro to assess the phenotype and functional response of disease fibroblasts to IL-17A. RESULTS: Mechanistically, IL-17A drives iSGS scar fibroblast proliferation ( P < .01), synergizes with transforming growth factor ß1 to promote extracellular matrix production (collagen and fibronectin; P = .04), and directly stimulates scar fibroblasts to produce chemokines (chemokine ligand 2) and cytokines (IL-6 and granulocyte-macrophage colony-stimulating factor) critical to the recruitment and differentiation of myeloid cells ( P < .01). Glucocorticoids abrogated IL-17A-dependent iSGS scar fibroblast production of granulocyte-macrophage colony-stimulating factor ( P = .02). CONCLUSION: IL-17A directly drives iSGS scar fibroblast proliferation, synergizes with transforming growth factor ß1 to promote extracellular matrix production, and amplifies local inflammatory signaling. Glucocorticoids appear to partially abrogate fibroblast-dependent inflammatory signaling. These results offer mechanistic support for future translational study of clinical reagents for manipulation of the IL-17A pathway in iSGS patients.

The Impact of a Stepwise Approach to Primary Tumor Detection in Squamous Cell Carcinoma of the Neck With Unknown Primary.

OBJECTIVES/HYPOTHESIS: To examine the cumulative effect of diagnostic steps for primary tumor identification in patients with head and neck squamous cell carcinoma of unknown primary (HNSCCUP), including lingual tonsillectomy, and the impact of primary tumor identification on subsequent treatment. STUDY DESIGN: Retrospective analysis. METHODS: We reviewed the records of 110 patients diagnosed with HNSCCUP between 2003 and 2015. Results of diagnostic imaging (fluorodeoxyglucose-positron emission tomography/computed tomography [FDG-PET/CT]), tumor detection with direct laryngoscopy with biopsies, palatine tonsillectomy, and transoral robotic surgery (TORS) lingual tonsillectomy were recorded. Associations between demographic and treatment variables with overall survival (OS) and progression-free survival (PFS) were modeled with Cox proportional hazards models. RESULTS: FDG-PET/CT was suspicious for a primary site in 23/77 (30%) patients. Direct laryngoscopy identified a primary tumor in 34/110 patients (31%). Forty-seven patients underwent palatine tonsillectomy, which identified 17 primaries (36%), yielding a cumulative primary tumor identification of 51/110 (46%). Fourteen patients underwent TORS lingual tonsillectomy, which identified eight primaries (57%), resulting in a cumulative identification of 59/110 (53%). The detection rate increased from 28/63 (44%) to 31/47 (66%) after the addition of TORS lingual tonsillectomy to our institutional approach. Detection rates varied by HPV status. Primary tumor identification altered subsequent radiation planning, as patients with an identified primary tumor received radiation to a smaller volume of tissue than did those without an identified primary tumor. However, there was no significant association between primary tumor identification and OS or PFS. CONCLUSIONS: A stepwise approach to primary tumor identification identifies a primary tumor in a majority of patients. LEVEL OF EVIDENCE: 4 Laryngoscope, 129:1610-1616, 2019.

Targeting metabolic abnormalities to reverse fibrosis in iatrogenic laryngotracheal stenosis.

OBJECTIVE: Management of laryngotracheal stenosis (LTS) remains primarily surgical, with a critical need to identify targets for adjuvant therapy. Laryngotracheal stenosis scar fibroblasts exhibit a profibrotic phenotype with distinct metabolic shifts, including an increased glycolysis/oxidative phosphorylation ratio. This study examines the effects of the glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) on collagen production, gene expression, proliferation, and metabolism of human LTS-derived fibroblasts in vitro. METHOD: Paired normal and scar-derived fibroblasts isolated from subglottic and proximal tracheal tissue in patients with iatrogenic laryngotracheal stenosis (iLTS) were cultured. Proliferation rate, gene expression, protein production, and cellular metabolism were assessed in two conditions: 1) fibroblast growth medium, and 2) fibroblast growth medium with 1 × 10-4 M DON. RESULTS: DON treatment reduced cellular proliferation rate (n = 7, P = 0.0150). Expression of genes collagen 1 and collagen 3 both were reduced (n = 7, P = 0.0102, 0.0143, respectively). Soluble collagen production decreased (n = 7, P = 0.0056). As measured by the rate of extracellular acidification, glycolysis and glycolytic capacity decreased (n = 7, P = 0.0082, 0.0003, respectively). adenosine triphosphate (ATP) production and basal respiration decreased (n = 7, P = 0.0045, 0.0258, respectively), determined by measuring the cellular rate of oxygen consumption. CONCLUSION: The glutamine antagonist DON reverses profibrotic changes by inhibiting both glycolysis and oxidative phosphorylation in iLTS scar fibroblasts. In contrast to untreated iLTS scar fibroblasts, collagen gene expression, protein production, metabolic rate, and proliferation were significantly reduced. These results suggest DON and/or its derivatives as strong candidates for adjuvant therapy in the management of iatrogenic laryngotracheal stenosis. Enzymes involved in glutamine metabolism inhibited by DON offer targets for future investigation. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E59-E67, 2018.

Quantification of Inflammatory Markers in Laryngotracheal Stenosis.

Objectives (1) Develop a novel method for serial assessment of gene and protein expression in laryngotracheal stenosis (LTS). (2) Assess cytokine expression and determine an immunophenotype in LTS. Study Design A matched comparison of endolaryngeal brush biopsy samples from laryngotracheal scar and normal airway. Setting Tertiary care hospital, 2015-2016. Methods Brush biopsy specimens of laryngotracheal scar and normal trachea were obtained from 17 patients with LTS at the time of operating room dilation and were used for protein and RNA extraction. Gene expression of the TH1 cytokine interferon γ (INF-γ), TH2 cytokine interleukin 4 (IL-4), transforming growth factor ß, and collagen 1 (Coll1) was quantified with quantitative real-time polymerase chain reaction. Cytokine analysis was performed with flow cytometry with a cytometric bead array. Results LTS specimens demonstrated a 13.68-fold increase in Coll1 gene expression versus normal ( P < .001, N = 17). Additionally, IL-4 gene expression showed a 3.76-fold increase ( P < .001, N = 17) in LTS scar. When stratified into iatrogenic LTS and idiopathic subglottic stenosis cohorts, INF-γ gene expression was significantly increased in idiopathic subglottic stenosis ( P = .011). Soluble cytokine measurements were below the limit of detection for reliable quantification and thus could not be assessed. Conclusions Brush biopsies from LTS samples can be successfully utilized for RNA extraction and demonstrate the expected increase in Coll1 gene expression associated with LTS. Preliminary gene expression suggests that abnormal collagen production may be mediated by the TH2 cytokine IL-4 and that increased INF-γ expression may represent a key difference between iatrogenic LTS and idiopathic subglottic stenosis. Further analysis of soluble cytokines is needed to confirm these findings.

Fibroblasts in Hypoxic Conditions Mimic Laryngotracheal Stenosis.

Objective To elucidate the role of hypoxia and inflammatory pathways in the pathogenesis of iatrogenic laryngotracheal stenosis (iLTS). Study Design (1) Examination of mucosal surface gene expression in human iLTS. (2) In vitro comparison of normal and scar laryngotracheal fibroblasts under normoxic and hypoxic conditions. Setting Tertiary care hospital in a research university (2012-2016). Subjects and Methods Brush biopsies were obtained from normal laryngotracheal tissue and scar in iLTS patients; gene expression was compared. Fibroblasts were isolated from normal and scarred trachea and grown in vitro in either a 1% O2 or normoxic environment. Cell growth and gene and protein expression were compared. Statistical analysis utilized a multilevel mixed effects model. Results Expression of IL-6 (fold change = 2.8, P < .01), myofibroblast marker αSMA (fold change = 3.0, P = .01), and MMP13 (fold change = 5.4, P = .02) was significantly increased in scar biopsy samples as compared to normal. Under hypoxic conditions in vitro, normal laryngotracheal fibroblasts proliferated significantly faster (n = 8, P < .01 each day). Expression of IL-6 (n = 8, fold change = 2.6, P < .01) increased significantly after 12 hours under hypoxia. Expression of αSMA (n = 8, fold change= 2.0, P = .03), COL1 (n = 8, fold change = 1.1, P = .03), and MMP13 (n = 8, fold change = 1.6, P = .01) increased significantly after 48 hours under hypoxia. Scar fibroblasts also proliferated significantly faster under hypoxic conditions but did not display the same expression profile. Conclusion Human iLTS scar has a myofibroblast phenotype. Under hypoxic conditions in vitro, normal laryngotracheal fibroblasts can transdifferentiate into a similar phenotype. These changes may be mediated by IL-6, a fibrosis-related cytokine.

Laryngotracheal Stenosis: Risk Factors for Tracheostomy Dependence and Dilation Interval

Objective Laryngotracheal stenosis (LTS) is a fibrotic process that narrows the upper airway and has a significant impact on breathing and phonation. Iatrogenic injury from endotracheal and/or tracheostomy tubes is the most common etiology. This study investigates differences in LTS etiologies as they relate to tracheostomy dependence and dilation interval. Study Design Case series with chart review. Setting Single-center tertiary care facility. Subjects and Methods Review of adult patients with LTS was performed between 2004 and 2015. The association of patient demographics, comorbidities, disease etiology, and treatment modalities with patient outcomes was assessed. Multiple logistic regression analysis and Kaplan-Meier analysis were performed to determine factors associated with tracheostomy dependence and time to second procedure, respectively. Results A total of 262 patients met inclusion criteria. Iatrogenic patients presented with greater stenosis ( P = .023), greater length of stenosis ( P = .004), and stenosis farther from the vocal folds ( P < .001) as compared with other etiologies. Iatrogenic patients were more likely to be African American, use tobacco, and have obstructive sleep apnea, type II diabetes, hypertension, chronic obstructive pulmonary disease, or a history of stroke. Iatrogenic LTS (odds ratio [OR] = 3.1, 95% confidence interval [95% CI] = 1.2-8.2), Cotton-Myer grade 3-4 (OR = 2.6, 95% CI = 1.1-6.4), and lack of intraoperative steroids (OR = 2.9, 95% CI = 1.2-6.9) were associated with tracheostomy dependence. Nonsmokers, patients without tracheostomy, and idiopathic LTS patients had a significantly longer time to second dilation procedure. Conclusion Iatrogenic LTS presents with a greater disease burden and higher risk of tracheostomy dependence when compared with other etiologies of LTS. Comorbid conditions promoting microvascular injury-including smoking, COPD, and diabetes-were prevalent in the iatrogenic cohort. Changes in hospital practice patterns to promote earlier tracheostomy in high-risk patients could reduce the incidence of LTS.

Office-based Management of Recurrent Respiratory Papilloma.

This review will highlight the indications and benefits of office-based therapy for recurrent respiratory papillomatosis (RRP) and discuss the utilization of photo-dynamic lasers and adjuvant medical therapy in office-based settings. Office-based management of RRP allows for more timely interventions, is preferred by the majority of patients, and negates the risk of general anesthesia. Current literature argues for the utilization of KTP laser over CO2 laser for office-based treatment of RRP. Medical therapies for RRP are limited, but agents such as bevacizumab are promising and have been shown to reduce disease burden. Medical therapies that can induce disease remission are still needed. Office-based procedures save time and healthcare expenses compared to like procedures in the operating room. However, the increased frequency for office-based procedures predicts similar overall healthcare costs for office-based and OR laser excision of RRP. Office-based management of RRP is a feasible and well-tolerated strategy in appropriately selected patients with adequate local anesthesia.