Optimized (Pre) Analytical Conditions and Workflow for Droplet Digital PCR Analysis of Cell-Free DNA from Patients with Suspected Lung Carcinoma

Publication date: Available online 21 June 2019Source: The Journal of Molecular DiagnosticsAuthor(s): Remco de Kock, Birgit Deiman, Raisa Kraaijvanger, Volkher ScharnhorstFor patients with suspected lung carcinoma, the analysis of circulating tumor DNA, obtained by liquid biopsy, has the potential to support cancer diagnosis and guide targeted therapy. To ensure sensitive and reproducible detection of circulating tumor DNA in routine clinical practice, a standardized (pre) analytical workflow is required. Plasma was obtained from patients and healthy volunteers. Using the QIAmp Circulating Nucleic Acid Kit (Qiagen Valencia, CA), six different procedures for the isolation of cell-free DNA (cfDNA) were compared. cfDNA was analyzed by droplet digital PCR (ddPCR) for KRAS G12/13 mutations and for EGFR Ex19Del, L858R, and L861Q mutations using an in-house EGFR multiplex assay. A new isolation procedure that yields extracts with significantly higher cfDNA concentrations than described previously was selected (P < 0.001). EGFR and KRAS assay sensitivity of at least 0.2% fractional abundance was guaranteed for approximately 76% of patient samples in one run. A flowchart that includes validity criteria for a standardized analytical workflow of ddPCR analysis was designed. An improved protocol for cfDNA isolation enables a higher cfDNA input for ddPCR. The use of sensitive KRAS and EGFR multiplex assays and accompanying validity criteria allows for controlled and efficient testin...
Source: The Journal of Molecular Diagnostics - Category: Pathology Source Type: research