title: Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA) from Patients with Non-Small Cell Lung Cancer (NSCLC).
abstract: BACKGROUND
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Personalised Healthcare & Biomarkers, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Darwin Building, 310 Cambridge Science Park, Milton Road, Cambridge, CB4 0WG, United Kingdom.
authors.forename: James L
authors.initials: JL
authors.lastname: Sherwood
authors.affiliation: Personalised Healthcare & Biomarkers, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Darwin Building, 310 Cambridge Science Park, Milton Road, Cambridge, CB4 0WG, United Kingdom.
authors.forename: Claire
authors.initials: C
authors.lastname: Corcoran
authors.affiliation: Personalised Healthcare & Biomarkers, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Darwin Building, 310 Cambridge Science Park, Milton Road, Cambridge, CB4 0WG, United Kingdom.
authors.forename: Helen
authors.initials: H
authors.lastname: Brown
authors.affiliation: Personalised Healthcare & Biomarkers, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Darwin Building, 310 Cambridge Science Park, Milton Road, Cambridge, CB4 0WG, United Kingdom.
authors.forename: Alan D
authors.initials: AD
authors.lastname: Sharpe
authors.affiliation: Personalised Healthcare & Biomarkers, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Darwin Building, 310 Cambridge Science Park, Milton Road, Cambridge, CB4 0WG, United Kingdom.
authors.forename: Milena
authors.initials: M
authors.lastname: Musilova
authors.affiliation: Personalised Healthcare & Biomarkers, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Darwin Building, 310 Cambridge Science Park, Milton Road, Cambridge, CB4 0WG, United Kingdom.
authors.forename: Alexander
authors.initials: A
authors.lastname: Kohlmann
mesh_terms: D000230:Adenocarcinoma / Q000097:blood / Q000737:chemistry / Q000235:genetics*; D001331:Automation; D001800:Blood Specimen Collection / Q000295:instrumentation / Q000379:methods*; D002289:Carcinoma, Non-Small-Cell Lung / Q000097:blood / Q000737:chemistry / Q000235:genetics*; D002498:Centrifugation / Q000379:methods; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification; D011905:Genes, ras; D006801:Humans; D008175:Lung Neoplasms / Q000097:blood / Q000737:chemistry / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D011933:Reagent Kits, Diagnostic; D012996:Solutions; D013048:Specimen Handling / Q000379:methods; D013696:Temperature; D013997:Time Factors
chemical_list: D004273:DNA, Neoplasm; C117307:KRAS protein, human; D009363:Neoplasm Proteins; D011933:Reagent Kits, Diagnostic; D012996:Solutions; D016283:Proto-Oncogene Proteins p21(ras)