title: Genetic profiling of tumours using both circulating free DNA and circulating tumour cells isolated from the same preserved whole blood sample.
abstract: Molecular information obtained from cancer patients' blood is an emerging and powerful research tool with immense potential as a companion diagnostic for patient stratification and monitoring. Blood, which can be sampled routinely, provides a means of inferring the current genetic status of patients' tumours via analysis of circulating tumour cells (CTCs) or circulating tumour DNA (ctDNA). However, accurate assessment of both CTCs and ctDNA requires all blood cells to be maintained intact until samples are processed. This dictates for ctDNA analysis EDTA blood samples must be processed with 4 h of draw, severely limiting the use of ctDNA in multi-site trials. Here we describe a blood collection protocol that is amenable for analysis of both CTCs and ctDNA up to four days after blood collection. We demonstrate that yields of circulating free DNA (cfDNA) obtained from whole blood CellSave samples are equivalent to those obtained from conventional EDTA plasma processed within 4 h of blood draw. Targeted and genome-wide NGS revealed comparable DNA quality and resultant sequence information from cfDNA within CellSave and EDTA samples. We also demonstrate that CTCs and ctDNA can be isolated from the same patient blood sample, and give the same patterns of CNA enabling direct analysis of the genetic status of patients' tumours. In summary, our results demonstrate the utility of a simple approach that enabling robust molecular analysis of CTCs and cfDNA for genotype-directed therapies in multi-site clinical trials and represent a significant methodological improvement for clinical benefit.
authors.affiliation: Nucleic Acid Biomarker Laboratory, Clinical Experimental Pharmacology Group, CR-UK Manchester Institute, University of Manchester, M20 4BX, UK. Electronic address: dominic.rothwell@cruk.mancehster.ac.uk.
authors.forename: Dominic G
authors.initials: DG
authors.lastname: Rothwell
authors.affiliation: Nucleic Acid Biomarker Laboratory, Clinical Experimental Pharmacology Group, CR-UK Manchester Institute, University of Manchester, M20 4BX, UK.
authors.forename: Nigel
authors.initials: N
authors.lastname: Smith
authors.affiliation: Nucleic Acid Biomarker Laboratory, Clinical Experimental Pharmacology Group, CR-UK Manchester Institute, University of Manchester, M20 4BX, UK.
authors.forename: Daniel
authors.initials: D
authors.lastname: Morris
authors.affiliation: Computational Biology Support, CR-UK Manchester Institute, University of Manchester, M20 4BX, UK.
authors.forename: Hui Sun
authors.initials: HS
authors.lastname: Leong
authors.affiliation: Computational Biology Support, CR-UK Manchester Institute, University of Manchester, M20 4BX, UK; Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK.
authors.forename: Yaoyong
authors.initials: Y
authors.lastname: Li
authors.affiliation: Nucleic Acid Biomarker Laboratory, Clinical Experimental Pharmacology Group, CR-UK Manchester Institute, University of Manchester, M20 4BX, UK.
authors.forename: Antoine
authors.initials: A
authors.lastname: Hollebecque
authors.affiliation: Nucleic Acid Biomarker Laboratory, Clinical Experimental Pharmacology Group, CR-UK Manchester Institute, University of Manchester, M20 4BX, UK.
authors.forename: Mahmood
authors.initials: M
authors.lastname: Ayub
authors.affiliation: Nucleic Acid Biomarker Laboratory, Clinical Experimental Pharmacology Group, CR-UK Manchester Institute, University of Manchester, M20 4BX, UK.
authors.forename: Louise
authors.initials: L
authors.lastname: Carter
authors.affiliation: Nucleic Acid Biomarker Laboratory, Clinical Experimental Pharmacology Group, CR-UK Manchester Institute, University of Manchester, M20 4BX, UK.
authors.forename: Jenny
authors.initials: J
authors.lastname: Antonello
authors.affiliation: Nucleic Acid Biomarker Laboratory, Clinical Experimental Pharmacology Group, CR-UK Manchester Institute, University of Manchester, M20 4BX, UK.
authors.forename: Lynsey
authors.initials: L
authors.lastname: Franklin
authors.affiliation: Computational Biology Support, CR-UK Manchester Institute, University of Manchester, M20 4BX, UK; RNA Biology Group, CR-UK Manchester Institute, University of Manchester, M20 4BX, UK.
authors.forename: Crispin
authors.initials: C
authors.lastname: Miller
authors.affiliation: Christie NHS Foundation Trust, Institute of Cancer Sciences, University of Manchester, M20 4BX, UK.
authors.forename: Fiona
authors.initials: F
authors.lastname: Blackhall
authors.affiliation: Nucleic Acid Biomarker Laboratory, Clinical Experimental Pharmacology Group, CR-UK Manchester Institute, University of Manchester, M20 4BX, UK.
authors.forename: Caroline
authors.initials: C
authors.lastname: Dive
authors.affiliation: Nucleic Acid Biomarker Laboratory, Clinical Experimental Pharmacology Group, CR-UK Manchester Institute, University of Manchester, M20 4BX, UK.
authors.forename: Ged
authors.initials: G
authors.lastname: Brady
mesh_terms: D001793:Blood Preservation; D004273:DNA, Neoplasm / Q000097:blood* / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D009360:Neoplastic Cells, Circulating / Q000378:metabolism*; D013997:Time Factors
chemical_list: D004273:DNA, Neoplasm
keywords: Biomarker; Blood; CTC; NGS; ctDNA