File(s) under embargo

8

month(s)

11

day(s)

until file(s) become available

The use of Exhaled Breath Condensate (EBC) in the Identification of Driver Mutations in Lung Cancer and SARS-CoV-2 viral RNA in Covid-19

thesis
posted on 23.03.2022, 16:31 authored by Daniel Ryan
Molecular testing for targetable driver mutations is fundamental in advanced stage lung cancer. Small diagnostic tissue samples can miss driver mutations and fail to reflect intratumour heterogeneity (ITGH). Liquid biopsies are non-invasive and may better reflect ITGH. Most liquid biopsies are performed in the context of circulating tumour DNA (ctDNA) in plasma but Exhaled Breath Condensate (EBC) shows significant promise as a lung specific liquid biopsy. In this prospective, proof on concept study we carried out targeted Next Generation Sequencing (NGS) on diagnostic tissue samples of 150 patients with a potential diagnosis of lung cancer and compared the results to plasma and EBC for 5 lung cancer associated driver mutations (EGFR, KRAS, PIK3CA, ERBB2, BRAF) using an ultrasensitive PCR technique (UltraSEEK™ Lung Panel on the MassARRAY® System, Agena Bioscience, San Diego, CA, USA). 125/150 (83.3%) patients had tissue confirmed lung cancer. There was a significantly higher failure rate due to unamplifiable DNA in tissue NGS samples (57/125 ,45.6%) compared to plasma (27/125, 21.6%, p=3.212e-05) and EBC (26/125,20.8%, p=1.115e-05). Consequently, NGS and plasma only showed significant correlation for PIK3CA (X2 p=0.032) and BRAF (X2 p=0.021) and there was no significant correlation between tissue NGS and EBC.However, there was significant correlation between plasma and EBC for EGFR (X2p=0.002), KRAS (X2P=0.0004), PIK3CA (X2P=0.0004) and BRAF (X2P=0.0004) as well as the identification of mutations unique to both modalities. The results suggest that EBC is effective in identifying lung cancer mutations using ultrasensitive PCR and has a potential role as an adjunct to plasma-based testing.
The research coincided with the outbreak of the COVID-19 pandemic. False negatives from nasopharyngeal swabs (NPS) using reverse transcriptase PCR (RT-PCR) in SARS-CoV-2 are high. Exhaled Breath Condensate (EBC) contains lower respiratory droplets that may improve detection. We performed EBC RT-PCR for SARS-CoV-2 genes ( E,S,N,ORF1ab) on NPS positive (n=16) and NPS negative/clinically positive COVID-19 patients (n=15) using 2 commercial assays. EBC detected SARS-CoV-2 in 93.5% (29/31) of cases using 4 genes. Pre SARS-CoV-2 era controls (n=14) were all negative. EBC was positive in NPS negative/clinically positive patients in 66.6% (10/15) using the identical E/S assay used for NPS, 73.3% (11/15) using the N/ORF1ab assay and 14/15 (93.3%) combined.

History

First Supervisor

Prof. Bryan Hennessy

Second Supervisor

Prof. Ross Morgan

Comments

Submitted for the degree of Doctor of Medicine from the Royal College of Surgeons in Ireland in 2021

Published Citation

Ryan D. The use of Exhaled Breath Condensate (EBC) in the Identification of Driver Mutations in Lung Cancer and SARS-CoV-2 viral RNA in Covid-19 [MD Thesis] Dublin: Royal College of Surgeons in Ireland; 2021

Degree Name

Doctor of Medicine (MD)

Date of award

30/11/2021

Programme

  • Doctor of Medicine (MD)