The Utility of Cell-free DNA in Exhaled Breath Condensate in the Detection of Lung Cancer Somatic Mutations

Background: Exhaled breath condensate (EBC) is an acellular, organ specific biofluid that is known to harbour cell free DNA (cfDNA). CfDNA in plasma is increasingly used in lung cancer as a minimally invasive method to detect targetable mutations such as EGFR (‘liquid biopsy’). Plasma based testing, while representing the industry standard for liquid biopsy in advanced stage disease has shown mixed results in detecting tumour specific mutations in early-stage NSCLC. Furthermore, studies of liquid biopsies in advanced EGFR mutated lung cancer have shown differences in detection rates between M1a and M1b disease. The phenomenon of spatial heterogeneity is also a current concern in NSCLC given its association with treatment resistance, disease recurrence and sampling bias in diagnostic biopsies. Given EBC is a cellular and lung specific, we sought to perform an investigation into its ability to detect both lung cancer specific mutations and spatial heterogeneity and compare these results to plasma-based analysis using tumour tissue as the gold standard.

Methods: In 2 separate proof of concept pilot studies using a combination of highly sensitive detection methods (UltraSEEKTM and ddPCR) we explored the ability of EBC to detect lung cancer mutations. Study 1 sought to detect the EGFR-T790M resistance mutation in the EBC-DNA of a cohort of 19 patients with advanced EGFR mutated lung cancer (ten cases and nine controls) . Study 2 investigated spatial genomic heterogeneity in 8 patients with early-stage NSCLC using multi-region whole exome sequencing in FFPE tissue from the resected specimens. A patient specific bespoke panel of 5 mutations was chosen to best represent genomic heterogeneity and include important oncogenic driver mutations. We then sought to identify these mutations in EBC and plasma both pre and postoperatively at 6 and 12 week intervals. We then compared performance of EBC to that of plasma in detecting these mutations at the various timepoints.

Results: Using UltraSEEK(TM) EBC successfully detected EGFR-T790M in 9/10 cases compared to 7/10 in plasma. None of the 9 control patients were found to have the EGFR-T790M mutation. Using a combination of EBC and plasma all T790M positive patients were correctly genotyped. In our second project of 8 patients with early-stage NSCLC, tumours were genomically heterogeneous with a median of 57% (IQR 38.5-84.5) homogenously found throughout the tumour. EBC detected 39/40 mutations compared to 40/40 in plasma. CfDNA in EBC and plasma both outperformed the original diagnostic biopsy sample.

Conclusion: Both studies suggested that EBC is comparable to plasma in the detection of lung cancer specific mutations from cfDNA. Our preliminary proof of concept data supports the further investigation into the utility of EBC in lung cancer genotyping and its potential adjunctive role to plasma-based analysis.