Cohort-level clinical trajectory and molecular landscape of idiopathic subglottic stenosis for precision laryngology-a study of the Canadian Airways Research (CARE) group

Abstract

Background: First described in 1972, idiopathic subglottic stenosis (iSGS) is a serious chronic orphan disease characterised by recurrent scarring of the subglottis. Although the cause is unknown, iSGS is almost exclusively restricted to Caucasian females typically in their fourth to sixth decade. However, given its rare incidence (1:400,000), understanding the clinical trajectory and molecular factors associated with iSGS disease development and prognosis has been difficult. In the current study we sought to unravel the pathogenesis of iSGS at the clinical, transcriptional, and genetic level in a prospective cohort.

Methods: We prospectively enrolled 126 patients with iSGS, 104 controls, and 13 patients with traumatic SGS. Within this cohort, we profiled 114 human epiglottis and 121 human subglottis biopsies across three different conditions: control, iSGS, and intubation-related traumatic stenosis using bulk and single nucleus RNA-sequencing. Whole exome sequencing for germline variants was performed for 70 controls and 75 patients with iSGS.

Findings: Patients with iSGS received a median number of five (range 0-18) surgical dilations at a rate of 1.031 dilations (range: 0.12-6.2) per year. Older age at diagnosis and higher Cotton-Myers grade were associated with increased number of surgical dilations over time. Cohort-level bulk transcriptomics found that iSGS pathology was restricted within the subglottis and did not affect anatomically adjacent epiglottis, opposite to previous hypotheses. We further identified cellular subsets associated with iSGS prognosis and severity. Finally, patients with iSGS exhibit lower testosterone predicted using a polygenic score.

Interpretation: Together, our data refines our understanding of laryngeal biology and provides insights into the clinical trajectory of subglottic stenoses. Future research should explore the role of testosterone in the development of iSGS.

Funding: This study was funded by a grant from the American Laryngology Association (#1082), an Academic Medical Organization of Southwestern Ontario innovation fund grant (INN21-016), grant support from the Departments of Otolaryngology-Head and Neck Surgery at University of Toronto and Western University. ACN was supported by the Wolfe Surgical Research Professorship in the Biology of Head and Neck Cancers Fund. PYFZ was supported by a Vanier Canada Graduate Scholarship and PSI foundation fellowship.

Keywords: Biomarkers; Genomics; Idiopathic subglottic stenosis; Larynx; Transcriptomics; Traumatic subglottic stenosis.

Fig. 1

The Canadian Airways Research (CARE) group prospective study of subglottic stenoses. The Canadian Airways Research (CARE) group is an ongoing Canada-wide prospective cohort study of clinical, molecular, cellular, and genetic factors underlying the factors determining the pathogenesis and treatment response of subglottic stenosis (SGS). In the first phase of the study, we report detailed characterisation of 104 control, 126 idiopathic SGS (iSGS), and 13 long-term iatrogenic intubation-related trauma patients with SGS. Bulk RNA-sequencing, single-nucleus RNA-sequencing, and whole exome sequencing of germline DNA was performed on epiglottis and subglottis samples as displayed in the figure.

Fig. 2

Clinical trajectory of patients with iSGS. a) Clinical trajectory in follow-up years of all patients with iSGS (left) and those with at least one molecular data (right). Clinical procedures are shown as colour denoted in the left panel. Timing of epiglottis or subglottis biopsy is shown by dark or light grey. b) The total number of surgical dilations patients with iSGS in the clinical cohort (n = 126). c) Average number of surgical dilations per year a patient experienced. Only patients with a minimum of one year follow-up are shown (n = 82). d) Cumulative hazard for surgical dilation of patients (n = 126) according to the index Cotton-Myers grade (first recorded Cotton-Myers grade within the study). e) Forrest plot of multivariate Poisson regression for the number of dilations using follow-up time as an offset (n = 126). In the left box blue and red shows terms with estimates less or greater than 0 and p less than 0.05. Grey shows terms with p values greater than 0.05. Red line in the right box shows p value of 0.05.

Fig. 3

Transcriptomic landscape of healthy and diseased epiglottis and subglottis. a) Scatter plot comparing transcript abundance difference between the epiglottis of patients with iSGS (n = 84) to that of controls (n = 16), and the subglottis of patients with iSGS (n = 83) to controls (n = 18) using a linear mixed model. Log 2-fold change in epiglottis or subglottis is represented on X and Y-axis, respectively. Note the difference in scale on X and Y-axis. p value from negative binomial linear-mixed effects model. Genes in green show significant change (FDR p-value < 0.05) according to disease status. Those in blue/red show differential change in transcript abundance in the tissue subsite (epiglottis or subglottis) between the disease status (iSGS vs control) on significant interaction term disease status x tissue subsite, with greater fold change in the epiglottis or subglottis shown in blue or red, respectively. b) Scatter plots for selected genes shown in a in each disease status and tissue subsite, with black point showing the mean while error bars showing the 95% CI. FDR from the interaction term disease status x tissue subsite. c) Pathway analysis results of downregulated (top panel) or upregulated in iSGS subglottis, compared to control subglottis using GProfiler. X-axis are pathway groups GO:MF (gene ontology, molecular function), GO:CC (GO, cellular component), GO:BP (GO, biological process), KEGG, REAC (reactome), TF (transcription factor), miRNA, HPA (human protein atlas), CORUM (CORUM protein complex database), and HP (human phenotype ontology).

Fig. 4

Connection between molecular and clinical heterogeneity of iSGS. a) Association between each cell subset in iSGS and Cotton-Myers grade (n = 83). FDR value from Benjamini-Hochberg corrected Wilcoxon rank sum test and coloured by log 2-fold change. b) Association between estimated cell abundance and the rate of surgical dilation alone, and rate of surgical dilation or steroid injection (n = 72). Only patients with at least one follow-up after the biopsy were considered. For patients with multiple samples only the first sample was included. Estimate and FDR value from Benjamini-Hochberg corrected Poisson regression. c) Association between estimated cell abundance and time to the next surgical dilation, and time to the next surgical dilation or steroid injection. Only patients with at least one follow-up after the biopsy were considered (n = 72). For patients with multiple samples only the first sample was included. Estimate and p-values from Cox proportional hazard model.

Fig. 5

Patients with iSGS exhibit lower serum testosterone polygenic score compared to controls. p value (0.043) from linear regression analysis of controls (n = 68) and iSGS patients (n = 75), adjusted for the top 5 principal components.