Comparative Liquid Biopsy Testing for KRAS Mutations From Plasma Cell-Free DNA (cfDNA) and Extracellular Vesicles in
Caeli J Zahra1, Tian Mun Chee1, Edward K H Stephens1
1UQ Thoracic Research Centre, Faculty of Health, Medicine and Behavioural Sciences, The University of Queensland, Brisbane, Australia.
Background:
Liquid biopsy has emerged as a promising, minimally invasive tool to detect cancer biomarkers. Extracellular vesicles (EVs) are secreted into biofluids to transport cargo such as DNA and are a potential biosource for liquid biopsy biomarkers. To determine the optimal use of liquid biopsy for diagnosing mutations in lung adenocarcinoma (LUAD), we compared the KRAS mutation status in DNA isolated from four different plasma fractions, including EVs.
Methods:
Plasma was collected from 58 participants diagnosed with LUAD (early-stage (I, II), n = 30; late-stage (IIIB, IV), n = 28) with known KRAS mutation (KRASmt) or wild-type KRAS (KRASwt). Three distinct plasma-derived fractions were prepared by sequential differential ultracentrifugation aiming to isolate EVs (pellets 1, 2, 3 (P1-P3)). These were tested together with the corresponding plasma supernatant (SUP) for the presence of KRAS G12/G13 mutations by droplet digital PCR (ddPCR).
Results:
In early-stage KRASmt LUAD, mutations were detected by ddPCR in only 1 of 15 processed plasma supernatant samples and 1 of 15 P3 samples, but not in any P1 or P2 pellets. In late-stage KRASmt LUAD, mutations were detected in 13/14 of SUP samples, but only in a small fraction of the pellet preparations: P1 (0/14), P2 (2/14) and P3 (2/13) samples.
Conclusion:
We conclude that ddPCR testing of plasma supernatant achieved high overall agreement with tumour KRASmt status. There was varying abundance of KRAS detected in the plasma fractions.
