title: Mutation Analysis of Cell-Free DNA and Single Circulating Tumor Cells in Metastatic Breast Cancer Patients with High Circulating Tumor Cell Counts.
abstract: OBJECTIVE
  The purpose of this study was to directly compare mutation profiles in multiple single circulating tumor cells (CTC) and cell-free DNA (cfDNA) isolated from the same blood samples taken from patients with metastatic breast cancer (MBC). We aimed to determine whether cfDNA would reflect the heterogeneity observed in 40 single CTCs.
  
  
  METHODS
  CTCs were enumerated by CELLSEARCH. CTC count was compared with the quantity of matched cfDNA and serum CA15-3 and alkaline phosphatase (ALP) in 112 patients with MBC. In 5 patients with ≥100 CTCs, multiple individual EpCAM-positive CTCs were isolated by DEPArray and compared with matched cfDNA and primary tumor tissue by targeted next-generation sequencing (NGS) of about 2,200 mutations in 50 cancer genes.
  
  
  RESULTS
  In the whole cohort, total cfDNA levels and cell counts (≥5 CTCs) were both significantly associated with overall survival, unlike CA15-3 and ALP. NGS analysis of 40 individual EpCAM-positive CTCs from 5 patients with MBC revealed mutational heterogeneity in PIK3CA, TP53, ESR1, and KRAS genes between individual CTCs. In all 5 patients, cfDNA profiles provided an accurate reflection of mutations seen in individual CTCs. ESR1 and KRAS gene mutations were absent from primary tumor tissue and therefore likely either reflect a minor subclonal mutation or were acquired with disease progression.
  
  
  CONCLUSIONS
  Our results demonstrate that cfDNA reflects persisting EpCAM-positive CTCs in patients with high CTC counts and therefore may enable monitoring of the metastatic burden for clinical decision-making. Clin Cancer Res; 23(1); 88-96. ©2016 AACR.
authors.affiliation: Department of Cancer Studies, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary, Leicester, United Kingdom. c.coombes@imperial.ac.uk js39@le.ac.uk.
authors.forename: Jacqueline A
authors.initials: JA
authors.lastname: Shaw
authors.affiliation: Department of Cancer Studies, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary, Leicester, United Kingdom.
authors.forename: David S
authors.initials: DS
authors.lastname: Guttery
authors.affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith, United Kingdom.
authors.forename: Allison
authors.initials: A
authors.lastname: Hills
authors.affiliation: Department of Cancer Studies, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary, Leicester, United Kingdom.
authors.forename: Daniel
authors.initials: D
authors.lastname: Fernandez-Garcia
authors.affiliation: Department of Cancer Studies, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary, Leicester, United Kingdom.
authors.forename: Karen
authors.initials: K
authors.lastname: Page
authors.affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith, United Kingdom.
authors.forename: Brenda M
authors.initials: BM
authors.lastname: Rosales
authors.affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith, United Kingdom.
authors.forename: Kate S
authors.initials: KS
authors.lastname: Goddard
authors.affiliation: Cancer Research UK Leicester Centre, University of Leicester, Leicester, London, United Kingdom.
authors.forename: Robert K
authors.initials: RK
authors.lastname: Hastings
authors.affiliation: Cancer Research UK Leicester Centre, University of Leicester, Leicester, London, United Kingdom.
authors.forename: Jinli
authors.initials: J
authors.lastname: Luo
authors.affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith, United Kingdom.
authors.forename: Olivia
authors.initials: O
authors.lastname: Ogle
authors.affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith, United Kingdom.
authors.forename: Laura
authors.initials: L
authors.lastname: Woodley
authors.affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith, United Kingdom.
authors.forename: Simak
authors.initials: S
authors.lastname: Ali
authors.affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith, United Kingdom.
authors.forename: Justin
authors.initials: J
authors.lastname: Stebbing
authors.affiliation: Department of Surgery and Cancer, Imperial College London, Hammersmith, United Kingdom. c.coombes@imperial.ac.uk js39@le.ac.uk.
authors.forename: R Charles
authors.initials: RC
authors.lastname: Coombes
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000235:genetics* / Q000378:metabolism; D002452:Cell Count; D000074141:Circulating Tumor DNA; D004252:DNA Mutational Analysis; D005260:Female; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008875:Middle Aged; D009154:Mutation; D009362:Neoplasm Metastasis; D009360:Neoplastic Cells, Circulating / Q000378:metabolism* / Q000473:pathology*; D057188:Workflow
chemical_list: D014408:Biomarkers, Tumor; D000074141:Circulating Tumor DNA