Mucosal Microbiome Disruption in Acute Laryngeal Injury Following Intubation.

The objective of this study was to characterize mucosal microbial shifts in patients with acute laryngeal injury (ALgI) after intubation. This cross-sectional study included 20 patients with ALgI who underwent early endoscopic intervention with tissue culture, 20 patients with idiopathic subglottic stenosis (iSGS) who underwent tissue culture during the routine endoscopic intervention, and 3 control patients who underwent mucosal swab culture. 70% of the ALgI patients had a positive culture compared to 5% of the iSGS patients and none of the controls. The most identified microbes isolated from ALgI patients included Staphylococcus species in 30% and Streptococcus species in 25%. The high rate of pathologic bacterial infiltration into postintubation laryngeal wounds supports efforts to reduce bacterial colonization of endotracheal tubes and highlights the role of culture-directed antibiotic therapy as a part of early intervention to improve outcomes for patients with ALgI.

Localizing Hormone Receptor Expression to Cellular Compartments in Idiopathic Subglottic Stenosis.

OBJECTIVES: Idiopathic subglottic stenosis (iSGS) is an unexplained progressive fibrosis of the upper airway. iSGS almost exclusively affects women; as a result, female hormones (estrogen and progesterone) have been proposed to participate in the pathogenesis of iSGS. Our aim was to localize cell-specific gene expression of estrogen receptors (ESR1 and ESR2) and progesterone receptor (PGR) using an established iSGS single-cell RNA sequencing (scRNAseq) cell atlas. STUDY DESIGN: Ex vivo molecular study of airway scar and healthy mucosa from iSGS patients. METHODS: An established scRNAseq atlas consisting of 25,974 individually sequenced cells from subglottic scar (n = 7) or matched unaffected mucosa (n = 3) in iSGS patients was interrogated for RNA expression of ESR1, ESR2, and PGR. Results were quantified and compared across cell subsets, then visualized using Uniform Manifold Approximation and Projection (UMAP). Confirmatory protein assessment of endocrine receptors in fibroblasts from iSGS patients (n = 5) was performed via flow cytometry. RESULTS: The proximal airway mucosa in iSGS patients demonstrates differential expression of endocrine receptors (ESR1, ESR2, PGR). Within airway scar, endocrine receptors are primarily expressed by fibroblasts, immune cells, and endothelial cells. Fibroblasts show strong ESR1 and PGR expression, while immune cells possess RNA for both ESR1 and ESR2. Endothelial cells predominantly express ESR2. Epithelial cells in unaffected mucosa express all three receptors, which are all reduced in airway scar. CONCLUSIONS: scRNAseq data localized endocrine receptor expression to specific cell subsets. These results provide the foundation for future work interrogating how hormone-dependent mechanisms promote, sustain, or participate in iSGS disease pathogenesis. LEVEL OF EVIDENCE: NA; Basic science Laryngoscope, 133:3506-3511, 2023.

Interdisciplinary assessment of tracheostomy care knowledge: An opportunity for quality improvement.

PURPOSE: A 2013 AAOHNS consensus statement called for reduced variation in tracheostomy care. Multidisciplinary approaches and standardized protocols have been shown to improve tracheostomy outcomes. This study aims to identify inconsistencies in knowledge in order to design standardized education targeting these areas to improve quality of care. MATERIALS AND METHODS: An online, multiple-choice tracheostomy care knowledge assessment was administered to nurses and respiratory therapists in ICUs, stepdown units, and regular nursing floors, as well as residents in otolaryngology, general surgery, and thoracic surgery. The survey was administered and data were recorded using the Select Survey online platform. RESULTS: 173 nurses, respiratory therapists, and residents participated in this study. Over 75 % of respondents identified correct answers to questions addressing basic tracheostomy care, such as suctioning and humidification. Significant variation was observed in identification and management of tracheostomy emergencies, and appropriate use of speaking valves. Only 47 % of all respondents identified all potential signs of tracheostomy tube displacement. Respiratory therapists with over 20 years of experience (p = 0.001), were more likely to answer correctly than those with less. Nurses were less likely than respiratory therapists to have received standardized tracheostomy education (p = 0.006) and were less likely than others to choose the appropriate scenario for speaking valve use (p = 0.042), highlighting the need for interdisciplinary education. CONCLUSIONS: An interdisciplinary assessment of tracheostomy care knowledge demonstrates variation, especially in identification and management of tracheostomy emergencies and appropriate use of speaking valves. Design of a standardized educational program targeting these areas is underway.

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.

Voice Quality and Laryngeal Findings in Patients With Suspected Lung Cancer.

OBJECTIVE: To describe laryngeal findings and voice quality in patients with suspected lung cancer, relative to voice quality and possible laryngeal pathology. STUDY DESIGN: Prospective cohort study. SETTING: Tertiary care center. METHODS: Patients with known or suspected lung cancer were approached before planned thoracic surgery, and they completed acoustic analysis, the Voice-Related Quality of Life (V-RQOL) questionnaire, and stroboscopy. The prevalence of dysphonia, V-RQOL and Cepstral Spectral Index of Dysphonia (CSID) scores, and laryngeal findings were examined and compared between patients ultimately found to have lung cancer and those without cancer. RESULTS: Sixty-one patients (45 cancer, 16 noncancer) were analyzed. Patients with cancer were older than those without (mean ± SD, 72.3 ± 9.94 vs 62.6 ± 9.30 years; P = .001). Otherwise, the distribution of stroboscopy findings, acoustic measures, and self-reported voice handicap were similar between the cancer and noncancer cohorts. Prior to surgery, no patients had vocal cord paralysis or obvious neoplasm, though 4 (6.56%) had leukoplakia and 28 (45.9%) had vocal fold movement asymmetry on stroboscopy. Overall, 21 patients (35.0%) had average CSID scores >19, and 13 (21.7%) had CSID scores >24; however, only 4 self-described their voice as not working as it should, and only 2 had a V-RQOL score <85. CONCLUSION: Patients with suspected lung cancer have moderate dysphonia on acoustic measures, though self-reported impact on quality of life is low. While leukoplakia was seen in 4 patients, obvious neoplasm and occult paralysis were not seen in this cohort. Together, these findings suggest that patients with suspected lung cancer should be assessed for subjective voice dysfunction, but routine laryngeal screening may otherwise be unnecessary.

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.

Endoscopic Resection and Mucosal Reconstitution With Epidermal Grafting: A Pilot Study in Idiopathic Subglottic Stenosis.

OBJECTIVE: To describe technical aspects and surgical outcomes of endoscopic resection and mucosal reconstitution with epidermal grafting (ie, the Maddern procedure) in the treatment of idiopathic subglottic stenosis. STUDY DESIGN: Medical record abstraction. SETTING: Johns Hopkins Hospital. METHODS: Retrospective series of 9 adults with idiopathic subglottic stenosis who underwent the Maddern procedure by a single surgeon over a 5-year period. Prespecified outcomes included (1) perioperative outcomes (Clavien-Dindo grade 4/5 complications, need for staged tracheostomy, hospital length of stay), (2) postoperative outcomes (peak expiratory flow rate [PEFR], need for subsequent airway surgery, tracheostomy at follow-up), and (3) patient-reported quality-of-life outcomes (Clinical COPD Questionnaire, Voice Handicap Index-10, Eating Assessment Tool-10, and 12-Item Short Form Version 2). Wilcoxon matched-pairs signed rank test and Kaplan-Meier analysis were performed. RESULTS: There were no Clavien-Dindo grade 4/5 complications; 2 patients required unplanned staged tracheostomy; and the median length of stay was 3 days. Following endoscopic resection and stent removal, a median of 2 laser resurfacing procedures were required. Two patients developed recurrent stenosis requiring cricotracheal resection (CTR). There were significant improvements in PEFR, Clinical COPD Questionnaire, and Voice Handicap Index-10, without significant difference in Eating Assessment Tool-10. The 12-Item Short Form Version 2 approximated the population norm. Kaplan-Meier analysis demonstrated significant improvement in time to surgery after the final laser resurfacing. CONCLUSION: The Maddern procedure has a low complication rate and offers durable physiologic improvement in PEFR, limiting need for additional procedures. Risks included need for CTR salvage, temporary tracheostomy, phlegm accumulation, and laryngospasm. It is a surgical option for patients with short dilation intervals who prefer to avoid the risks of CTR.

Increased Expression of PD-1 and PD-L1 in Patients With Laryngotracheal Stenosis.

OBJECTIVES: Laryngotracheal stenosis (LTS) is a fibrotic condition of the upper airway. Recent evidence suggests dysregulated host immunity plays a role in LTS development and progression. The programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) axis, targeted by paradigm-shifting immunotherapies for cancer treatment, has also recently been implicated in the pathogenesis of fibrotic pulmonary disease. However, a role for the PD-1/PD-L1 axis in the proximal airway fibrosis seen in LTS patients has not been explored. STUDY DESIGN: Controlled ex vivo study. METHODS: Expression of PD-1, PD-L1, CD4, and CD8 were evaluated using immunohistochemical staining of cricotracheal resection specimens from postintubation iatrogenic laryngotracheal stenosis (iLTS), idiopathic subglottic stenosis (iSGS) patients, and normal controls derived from rapid autopsy (n = 8 per group). Fibroblasts derived from iLTS scar were also treated with transforming growth factor beta 1 (TGFß1) and analyzed for PD-L1 expression by quantitative real-time polymerase chain reaction (n = 6). RESULTS: iLTS specimens exhibited increased expression of PD-1, PD-L1, and CD4 (all P < .0167) compared to controls, whereas iSGS specimens exhibited increased expression of PD-1 and CD4 (P < .0167) compared to controls. PD-1, PD-L1, and CD4 showed periepithelial patterns of expression in both disease cohorts. TGFß1 treatment of iLTS fibroblasts increased expression of PD-L1 (the cognate ligand for PD-1). CONCLUSION: Expression of both PD-1 and its ligand PD-L1 are significantly greater in patients with iLTS compared to controls, and PD-1 expression is also elevated in patients with iSGS. Given published evidence implicating the PD-1/PD-L1 axis in pulmonary fibrosis, this suggests a possible role for checkpoint inhibitors targeting the PD-1/PD-L1 axis for the treatment of LTS. LEVEL OF EVIDENCE: N/A Laryngoscope, 131:967-974, 2021.

Quantitative Assessment of the Immune Microenvironment in Patients With Iatrogenic Laryngotracheal Stenosis.

OBJECTIVE: Iatrogenic laryngotracheal stenosis (iLTS) is characterized by fibroinflammatory narrowing of the upper airway and is most commonly caused by intubation injury. Evidence suggests a key role for CD4 T cells in its pathogenesis. The objective of this study is to validate emerging multiplex immunofluorescence (mIF) technology for use in the larynx and trachea while quantitatively characterizing the immune cell infiltrate in iLTS. In addition to analyzing previously unstudied immune cell subsets, this study aims to validate previously observed elevations in the immune checkpoint PD-1 and its ligand PD-L1 while exploring their spatial and cellular distributions in the iLTS microenvironment. STUDY DESIGN: Controlled ex vivo cohort study. SETTING: Tertiary care center. METHODS: mIF staining was performed with formalin-fixed, paraffin-embedded slides from 10 patients with iLTS who underwent cricotracheal resection and 10 control specimens derived from rapid autopsy for CD4, CD8, CD20, FoxP3, PD-1, PD-L1, and cytokeratin. RESULTS: There was greater infiltration of CD4+ T cells, CD8+ T cells, CD20+ B cells, FoxP3+CD4+ Tregs, and FoxP3+CD8+ early effector T cells in the submucosa of iLTS specimens as compared with controls (P < .05 for all). PD-1 was primarily expressed on T cells and PD-L1 predominantly on CD4+ cells and "other" cells. CONCLUSION: This study leverages the power of mIF to quantify the iLTS immune infiltrate in greater detail. It confirms the highly inflammatory nature of iLTS, with CD4+ cells dominating the immune cell infiltrate; it further characterizes the cellular and spatial distribution of PD-1 and PD-L1; and it identifies novel immunologic targets in iLTS.

From presumed benign neck masses to delayed recognition of human papillomavirus-positive oropharyngeal cancer.

OBJECTIVES/HYPOTHESIS: To describe patients with delayed diagnosis of human papillomavirus-positive oropharyngeal squamous cell carcinoma (HPV-OPC) after initial incorrect diagnosis of branchial cleft cyst or nondiagnostic workup of unilateral neck mass. STUDY DESIGN: Retrospective case series. METHODS: Patients with delayed diagnosis of HPV-OPC after initial nondiagnostic workup for unilateral neck mass were eligible. Medical record abstraction was performed to describe clinical characteristics at initial presentation and later diagnosis of HPV-OPC. To estimate nodal growth rates, the short axis diameter of the lymph nodes was determined from imaging reports. RESULTS: Six patients met eligibility criteria. After a median interval of 42 months (range, 3 months-7 years) from initial presentation with unilateral neck mass, patients were diagnosed with HPV-OPC. At the time of HPV-OPC diagnosis, five were AJCC eighth edition overall stage I, and one was stage II. Primary tumors were T0 or T1 in the majority (83.3%, n = 5). Among five patients with available serial imaging, despite diagnostic delay, three of five still had a single lymph node without involvement of additional nodes, whereas the remaining two developed additional suspicious nodes (ipsilateral and contralateral). Two of five developed evidence of extranodal extension. Median lymph node growth was 9.5% per year (range, -6% to 32%). CONCLUSIONS: Although the natural history of HPV-OPC is not well understood, this case series suggests that it can be slow growing and mimic benign processes, leading to diagnostic delays. Adults presenting with neck masses should undergo complete diagnostic evaluation. LEVEL OF EVIDENCE: 4 Laryngoscope, 130:392-397, 2020.

Inflammatory pathways in the pathogenesis of iatrogenic laryngotracheal stenosis: what do we know?

Iatrogenic laryngotracheal stenosis (LTS) is pathologic airway narrowing due to scar formation following endotracheal intubation or tracheostomy. It is a chronic and recurrent disease that results in significant morbidity or even mortality. Current data suggests that inflammatory pathways play a significant role in mediating the deposition of collagen and extracellular matrix to generate this scar. In particular, the T-helper 2 cell and M2 macrophage axis appears to be activated in both animal models and patients with iatrogenic LTS. Interleukin-6 production in response to hypoxia has also been implicated. In this paper, we review the data supporting the roles of various inflammatory pathways in the pathogenesis of iatrogenic LTS, discuss potential therapeutic approaches targeting these mechanisms, and outline areas for future study.

Resistance to CTLA-4 checkpoint inhibition reversed through selective elimination of granulocytic myeloid cells.

PURPOSE: Local immunosuppression remains a critical problem that limits clinically meaningful response to checkpoint inhibition in patients with head and neck cancer. Here, we assessed the impact of MDSC elimination on responses to CTLA-4 checkpoint inhibition. EXPERIMENTAL DESIGN: Murine syngeneic carcinoma immune infiltrates were characterized by flow cytometry. Granulocytic MDSCs (gMDSCs) were depleted and T-lymphocyte antigen-specific responses were measured. Tumor-bearing mice were treated with MDSC depletion and CTLA-4 checkpoint blockade. Immune signatures within the human HNSCC datasets from The Cancer Genome Atlas (TCGA) were analyzed and differentially expressed genes from sorted human peripheral MDSCs were examined. RESULTS: gMDSCs accumulated with tumor progression and correlated with depletion of effector immune cells. Selective depletion of gMDSC restored tumor and draining lymph node antigen-specific T-lymphocyte responses lost with tumor progression. A subset of T-cell inflamed tumors responded to CTLA-4 mAb alone, but the addition of gMDSC depletion induced CD8 T-lymphocyte-dependent rejection of established tumors in all treated mice that resulted in immunologic memory. MDSCs differentially expressed chemokine receptors. Analysis of the head and neck cancer TCGA cohort revealed high CTLA-4 and MDSC-related chemokine and an MDSC-rich gene expression profile with a T-cell inflamed phenotype in > 60% of patients. CXCR2 and CSF1R expression was validated on sorted peripheral blood MDSCs from HNSCC patients. CONCLUSIONS: MDSCs are a major contributor to local immunosuppression that limits responses to checkpoint inhibition in head and neck cancer. Limitation of MDSC recruitment or function represents a rational strategy to enhance responses to CTLA-4-based checkpoint inhibition in these patients.

Anti-PD-L1 Efficacy Can Be Enhanced by Inhibition of Myeloid-Derived Suppressor Cells with a Selective Inhibitor of PI3Kδ/γ.

Checkpoint inhibitors are relatively inefficacious in head and neck cancers, despite an abundance of genetic alterations and a T-cell-inflamed phenotype. One significant barrier to efficacy may be the recruitment of myeloid-derived suppressor cells (MDSC) into the tumor microenvironment. Here we demonstrate functional inhibition of MDSC with IPI-145, an inhibitor of PI3Kδ and PI3Kγ isoforms, which enhances responses to PD-L1 blockade. Combination therapy induced CD8+ T lymphocyte-dependent primary tumor growth delay and prolonged survival only in T-cell-inflamed tumor models of head and neck cancers. However, higher doses of IPI-145 reversed the observed enhancement of anti-PD-L1 efficacy due to off-target suppression of the activity of tumor-infiltrating T lymphocytes. Together, our results offer a preclinical proof of concept for the low-dose use of isoform-specific PI3Kδ/γ inhibitors to suppress MDSC to enhance responses to immune checkpoint blockade. Cancer Res; 77(10); 2607-19. ©2017 AACR.

Avoiding phagocytosis-related artifact in myeloid derived suppressor cell T-lymphocyte suppression assays.

Myeloid-derived suppressor cells (MDSCs) have garnered much attention in recent years as a potential target for altering the immunosuppressive tumor microenvironment in a variety of solid tumor types. The ability to accurately assess the immunosuppressive capacity of MDSCs is fundamental to the development of therapeutic approaches aimed at disabling these immunosuppressive functions. In this article we provide evidence that the use of CD3/28 coated microbeads leads to artefactual T-lymphocyte suppression due to sequestration of beads by MDSCs isolated from the spleens of wild-type mice bearing subcutaneous syngeneic, carcinogen-induced oral cavity carcinomas. Mechanisms of this finding may include early MDSC death and acquisition of phagocytic capacity. These artefactual findings were avoided by eliminating the use of microbeads and instead using plate bound CD3/28 antibody as the T-lymphocyte stimulus. We propose model-specific validation of microbead-based MDSC assays, or use of an alternative stimulation approach such as plate bound CD3/28 antibodies.

Overcoming barriers to effective immunotherapy: MDSCs, TAMs, and Tregs as mediators of the immunosuppressive microenvironment in head and neck cancer.

A significant subset of head and neck cancers display a T-cell inflamed phenotype, suggesting that patients with these tumors should respond to therapeutic approaches aimed at strengthening anti-tumor immune responses. A major barrier to the development of an effective anti-tumor immune response, at baseline or in response to immunotherapy, is the development of an immunosuppressive tumor microenvironment. Several well described mechanisms of effector immune cell suppression in the head and neck cancer microenvironment are discussed here, along with updates on current trials designed to translate what we have learned from pre-clinical and correlative clinical studies into improved responses in patients with head and neck cancer following immune activating therapies.