Initial development and testing of an exhaled microRNA detection strategy for lung cancer case–control discrimination

Abstract

For detecting field carcinogenesis non-invasively, early technical development and case–control testing of exhaled breath condensate microRNAs was performed. In design, human lung tissue microRNA-seq discovery was reconciled with TCGA and published tumor-discriminant microRNAs, yielding a panel of 24 upregulated microRNAs. The airway origin of exhaled microRNAs was topographically “fingerprinted”, using paired EBC, upper and lower airway donor sample sets. A clinic-based case–control study (166 NSCLC cases, 185 controls) was interrogated with the microRNA panel by qualitative RT-PCR. Data were analyzed by logistic regression (LR), and by random-forest (RF) models. Feasibility testing of exhaled microRNA detection, including optimized whole EBC extraction, and RT and qualitative PCR method evaluation, was performed. For sensitivity in this low template setting, intercalating dye-based URT-PCR was superior to fluorescent probe-based PCR (TaqMan). In application, adjusted logistic regression models identified exhaled miR-21, 33b, 212 as overall case–control discriminant. RF analysis of combined clinical + microRNA models showed modest added discrimination capacity (1.1–2.5%) beyond clinical models alone: all subjects 1.1% (p = 8.7e−04)); former smokers 2.5% (p = 3.6e−05); early stage 1.2% (p = 9.0e−03), yielding combined ROC AUC ranging from 0.74 to 0.83. We conclude that exhaled microRNAs are qualitatively measureable, reflect in part lower airway signatures; and when further refined/quantitated, can potentially help to improve lung cancer risk assessment.

Preliminary results from the EMoLung clinical study showing early lung cancer detection by the LC score

“…The use of isoform ratios as building blocks of the LC score make it resilient to variations that may occur at different steps of the procedure, including RNA isolation, cDNA synthesis or PCR amplification. In addition, the utility of EBCs for expression analysis has been underlined by recent studies comparing non-coding transcripts in NSCLC patients versus control donors [ 10 , 31 , 32 ]. Among the limitations of the present study, the LC score does not allow the distinction of LC stages (Table S2 in Supplementary Material) or NSCLC subtypes (Fig.…”

Developing technologies and areas of interest in lung cancer screening adjuncts

“…They also found three miRNAs that were highly sensitive to detection and highly specific to lung cancer; several other miRNAs were highly specific to either lung adenocarcinoma or squamous cell carcinoma in particular ( 28 ). A recent study using a panel of 24 miRNAs upregulated in lung cancer demonstrated a modest increase in case-control discrimination—between 1.1% to 2.5%—compared to using only clinical models ( 29 ).…”

Non-invasive detection of orthotopic human lung tumors by microRNA expression profiling of mouse exhaled breath condensates and exhaled extracellular vesicles

“…[ 41 , 42 ] . Studies that have been conducted on EBC have revealed that it contains miRNAs, including those deregulated and released by tumors, with unique expression ratios that may be quantifiable and, in turn, allow detection of lung tumors [ 43 – 47 ] . Interestingly, recent investigations have also revealed that EBC contains extracellular vesicles (EVs), whose stably packaged miRNA cargoes can be analyzed and that may also help improve the detection of lung diseases [ 48 – 50 ] .…”