Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA) from Patients with Non-Small Cell Lung Cancer (NSCLC)

Abstract

Introduction: Non-invasive mutation testing using circulating tumour DNA (ctDNA) is an attractive premise. This could enable patients without available tumour sample to access more treatment options.

Materials & methods: Peripheral blood and matched tumours were analysed from 45 NSCLC patients. We investigated the impact of pre-analytical variables on DNA yield and/or KRAS mutation detection: sample collection tube type, incubation time, centrifugation steps, plasma input volume and DNA extraction kits.

Results: 2 hr incubation time and double plasma centrifugation (2000 x g) reduced overall DNA yield resulting in lowered levels of contaminating genomic DNA (gDNA). Reduced "contamination" and increased KRAS mutation detection was observed using cell-free DNA Blood Collection Tubes (cfDNA BCT) (Streck), after 72 hrs following blood draw compared to EDTA tubes. Plasma input volume and use of different DNA extraction kits impacted DNA yield.

Conclusion: This study demonstrated that successful ctDNA recovery for mutation detection in NSCLC is dependent on pre-analytical steps. Development of standardised methods for the detection of KRAS mutations from ctDNA specimens is recommended to minimise the impact of pre-analytical steps on mutation detection rates. Where rapid sample processing is not possible the use of cfDNA BCT tubes would be advantageous.

Fig 1. Sample workflow schematic.

Diagram showing the experimental workflow for the different sample cohorts. Panel A. Comparison of Streck Cell Free (BCT) and EDTA tubes in addition to single and double centrifugation on DNA yield and mutation detection. B. Comparison of different plasma volumes and ctDNA extraction kits on DNA yield and mutation detection.

Fig 2. Plasma processing method comparison.

Whole blood was collected from 20 NSCLC patients and processed using various tube types, incubation times and centrifugation steps. A subset of these patients (n = 5) had whole blood processed to assess a single vs. double centrifugation step. DNA was measured using the ABI TaqMan® RNase P Detection Reagent Kit A: Blood was collected using EDTA blood collection tube (EDTA) and processed after 2 hr or 72 hr. B: Blood was collected using cell free DNA blood collection tube (cfDNA BCT) and processed after 2 hr or 72 hr. C: Blood was collected using EDTA or cfDNA BCT tubes and processed after 2 hr. D: Blood was collected using EDTA or cfDNA BCT tubes and processed after 72 hr. E: Blood collected in EDTA tubes after 2 hr incubation and processed using either single or double centrifugation. F: Blood collected in EDTA tubes after 72 hr incubation and processed using either single or double centrifugation. Results are displayed for each patient. Statistical analysis was performed using a paired Student’s t-test where; **p<0.01.

Fig 3. Plasma input volume comparison.

Three volumes of plasma (1 mL, 2 mL and 3 mL) were processed from each sample in a cohort of 15 NSCLC patients. DNA was extracted using the QIAamp CNA Kit and was measured by qPCR using the ABI TaqMan® RNase P Detection Reagent Kit. Results are displayed for each patient. Statistical analysis was performed using a paired Student’s t-test where; **p<0.01.

Fig 4. DNA extraction kit comparison.

Equal volumes of plasma (2 mL) from 10 NSCLC patients were processed using three different DNA extraction methods: QIAamp Circulating Nucleic Acid Kit (Qiagen CNA Kit); PME free-circulating DNA Extraction Kit (Analytik Jena) and the DSP Virus/Pathogen Midi Kit performed on QIAsymphony (QIAsymphony). DNA was measured by qPCR using the ABI TaqMan® RNase P Detection Reagent Kit. Results are displayed for each patient. Statistical analysis was performed using a paired Student’s t-test where; **p<0.01, ***p<0.001.