title: Reproducibility of Digital PCR Assays for Circulating Tumor DNA Analysis in Advanced Breast Cancer.
abstract: Circulating tumor DNA (ctDNA) analysis has the potential to allow non-invasive analysis of tumor mutations in advanced cancer. In this study we assessed the reproducibility of digital PCR (dPCR) assays of circulating tumor DNA in a cohort of patients with advanced breast cancer and assessed delayed plasma processing using cell free DNA preservative tubes. We recruited a cohort of 96 paired samples from 71 women with advanced breast cancer who had paired blood samples processed either immediately or delayed in preservative tubes with processing 48-72 hours after collection. Plasma DNA was analysed with multiplex digital PCR (mdPCR) assays for hotspot mutations in PIK3CA, ESR1 and ERBB2, and for AKT1 E17K. There was 94.8% (91/96) agreement in mutation calling between immediate and delayed processed tubes, kappa 0.88 95% CI 0.77-0.98). Discordance in mutation calling resulted from low allele frequency and likely stochastic effects. In concordant samples there was high correlation in mutant copies per ml plasma (r2 = 0.98; p<0.0001). There was elevation of total cell free plasma DNA concentrations in 10.3% of delayed processed tubes, although overall quantification of total cell free plasma DNA had similar prognostic effects in immediate (HR 3.6) and delayed (HR 3.0) tubes. There was moderate agreement in changes in allele fraction between sequential samples in quantitative mutation tracking (r = 0.84, p = 0.0002). Delayed processing of samples using preservative tubes allows for centralized ctDNA digital PCR mutation screening in advanced breast cancer. The potential of preservative tubes in quantitative mutation tracking requires further research.
authors.affiliation: The Breast Cancer Now Research Centre, The Institute of Cancer Research, London, SW3 6JB, United Kingdom.
authors.forename: Sarah
authors.initials: S
authors.lastname: Hrebien
authors.affiliation: The Breast Cancer Now Research Centre, The Institute of Cancer Research, London, SW3 6JB, United Kingdom.|Breast Unit, Royal Marsden Hospital, Fulham Road, London, SW3 6JJ, United Kingdom.
authors.forename: Ben
authors.initials: B
authors.lastname: O'Leary
authors.affiliation: The Breast Cancer Now Research Centre, The Institute of Cancer Research, London, SW3 6JB, United Kingdom.
authors.forename: Matthew
authors.initials: M
authors.lastname: Beaney
authors.affiliation: Translational Science, Oncology iMed, AstraZeneca, Cambridge, CB4 0WG, United Kingdom.
authors.forename: Gaia
authors.initials: G
authors.lastname: Schiavon
authors.affiliation: The Breast Cancer Now Research Centre, The Institute of Cancer Research, London, SW3 6JB, United Kingdom.|Breast Unit, Royal Marsden Hospital, Fulham Road, London, SW3 6JJ, United Kingdom.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Fribbens
authors.affiliation: Breast Unit, Royal Marsden Hospital, Fulham Road, London, SW3 6JJ, United Kingdom.
authors.forename: Amarjit
authors.initials: A
authors.lastname: Bhambra
authors.affiliation: Breast Unit, Royal Marsden Hospital, Fulham Road, London, SW3 6JJ, United Kingdom.
authors.forename: Richard
authors.initials: R
authors.lastname: Johnson
authors.affiliation: The Breast Cancer Now Research Centre, The Institute of Cancer Research, London, SW3 6JB, United Kingdom.
authors.forename: Isaac
authors.identifier: http://orcid.org/0000-0001-8183-7969
authors.initials: I
authors.lastname: Garcia-Murillas
authors.affiliation: The Breast Cancer Now Research Centre, The Institute of Cancer Research, London, SW3 6JB, United Kingdom.|Breast Unit, Royal Marsden Hospital, Fulham Road, London, SW3 6JJ, United Kingdom.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Turner
mesh_terms: D014408:Biomarkers, Tumor / Q000097:blood / Q000235:genetics; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D004273:DNA, Neoplasm / Q000097:blood*; D001330:Electronic Data Processing; D005260:Female; D006801:Humans; D061986:MCF-7 Cells; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D009154:Mutation; D011379:Prognosis; D015203:Reproducibility of Results
chemical_list: D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm