An Exhaled microRNA Panel Interrogated for Lung Cancer Case-Control Discrimination

Abstract

Background: An exhaled microRNA-based lung cancer case-control discriminant biomarker strategy is reported.Methods: A microRNA-seq discovery effort compared paired tumor to non-tumor tissue, was reconciled with analogous TCGA and published literature-based tissue-discriminant microRNA data, yielding a candidate panel of 24 microRNAs that are upregulated in either adenocarcinomas and/or squamous cell carcinomas. The technical feasibility of microRNA-PCR assays in exhaled breath condensate (EBC) was tested. The airway origin of exhaled microRNAs was then topographically “fingerprinted”, using paired EBC and bronchoscopic samples. For initial EBC testing, a clinic-based case-control set of 351 individuals (166 NSCLC cases, 185 non-cancer controls) was interrogated with the 24-candidate microRNA panel by qualitative RT-PCR, and curated by melt curve analysis. Data were analyzed by both logistic regression (LR), and by random-forest (RF) models, validated by iterative resampling.Results: Both feasibility of exhaled microRNA detection, and its origins in part from lower airway sources, were confirmed. LR models adjusted for age, sex, smoking status, pack years, quit-years, and underlying lung disease identified exhaled miR-21, 33b, 212 (p.adj,=0.019, 0.018, 0.033, resp.) as case-control discriminant. For the RF analysis, the combined clinical + microRNA models showed modest added discrimination capacity (1.1–2.5%) beyond the clinical models alone: by subgroup, all subjects 1.1% (p = 8.7e-04)); former smokers 2.5% (p = 3.6e-05); early stage 1.2% (p = 9.0e-03). Sensitivity, specificity, positive- and negative-predictive values of the clinical + microRNA models for the entire cohort were 71%-76%.Conclusion: This work suggests that exhaled microRNAs are measurable qualitatively; reflect in part lower airway signatures; and if improved/refined, can potentially help distinguish lung cancer cases from controls.

Advances in proteomics methods for the analysis of exhaled breath condensate

“…In this context, metabolomic and proteomic assays are attractive in that they are proximate to the actual cellular activity shifts that are inherent to lung carcinogenesis. We have previously reported exhaled DNA methylation, and more recently, microRNA (Han et al, 2009; Shi et al, 2021), but the crux of the issue is that exhaled specimens carry low levels of analytes, presenting a significant technical challenge. An EBC‐based strategy samples a broad field of injury (Boyle et al, 2010; Sridhar et al, 2008; Zhang et al, 2010), including bilateral lung epithelium undergoing carcinogenesis, as well as the area surrounding the deep, hard‐to‐reach small/early‐stage cancers that are of primary interest for lung cancer screening (Silvestri et al, 2015; Spira et al, 2007).…”

“…Thus, individuals with predisposing lung epithelial features can hypothetically be tested for their risk for lung cancer. There has been a renewed interest in EBC assays because (i) the origins of condensate tend to be alveolar epithelial lining fluid and more proximate airways, rather than the commonly studied gas phase molecules such as small volatile organic compounds (VOCs) that can be derived from blood, and (ii) it is more practical to collect than exhaled gas phase samples, using a handheld disposable (RTube®) device, or small portable condenser (TurboDECCs®) device (Kelly et al, 2017; Keskin et al, 2021; Maniscalco et al, 2019; Shi et al, 2021). As of early 2022, there are no published or ongoing trials of EBC proteomics in lung cancer risk or diagnostics setting, but technologies and standards for EBC assays are evolving.…”