title: A novel approach for next-generation sequencing of circulating tumor cells.
abstract: BACKGROUND
  Next-generation sequencing (NGS) of surgically resected solid tumor samples has become integral to personalized medicine approaches for cancer treatment and monitoring. Liquid biopsies, or the enrichment and characterization of circulating tumor cells (CTCs) from blood, can provide noninvasive detection of evolving tumor mutations to improve cancer patient care. However, the application of solid tumor NGS approaches to circulating tumor samples has been hampered by the low-input DNA available from rare CTCs. Moreover, whole genome amplification (WGA) approaches used to generate sufficient input DNA are often incompatible with blood collection tube preservatives used to facilitate clinical sample batching.
  
  
  METHODS
  To address this, we have developed a novel approach combining tumor cell isolation from preserved blood with Repli-G WGA and Illumina TruSeq Amplicon Cancer Panel-based NGS. We purified cell pools ranging from 10 to 1000 cells from three different cell lines, and quantitatively demonstrate comparable quality of DNA extracted from preserved versus unpreserved samples.
  
  
  RESULTS
  Preservation and WGA were compatible with the generation of high-quality libraries. Known point mutations and gene amplification were detected for libraries that had been prepared from amplified DNA from preserved blood.
  
  
  CONCLUSIONS
  These spiking experiments provide proof of concept of a clinically applicable workflow for real-time monitoring of patient tumor using noninvasive liquid biopsies.
authors.affiliation: Department of Medicine University of Pennsylvania Perelman School of Medicine Philadelphia Pennsylvania.
authors.forename: Stephanie S
authors.initials: SS
authors.lastname: Yee
authors.affiliation: Department of Pathology and Laboratory Medicine Hospital of the University of Pennsylvania Philadelphia Pennsylvania.
authors.forename: David B
authors.initials: DB
authors.lastname: Lieberman
authors.affiliation: Department of Cancer BiologyUniversity of Pennsylvania Perelman School of MedicinePhiladelphiaPennsylvania; Abramson Family Cancer Research InstituteUniversity of Pennsylvania Perelman School of MedicinePhiladelphiaPennsylvania.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Blanchard
authors.affiliation: Division of Oncology Center for Childhood Cancer Research Children's Hospital of Philadelphia Philadelphia Pennsylvania.
authors.forename: JulieAnn
authors.initials: J
authors.lastname: Rader
authors.affiliation: Department of Pathology and Laboratory Medicine Hospital of the University of Pennsylvania Philadelphia Pennsylvania.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Center for Clinical Epidemiology and BiostatisticsUniversity of PennsylvaniaPhiladelphiaPennsylvania; Abramson Cancer CenterUniversity of Pennsylvania Perelman School of MedicinePhiladelphiaPennsylvania.
authors.forename: Andrea B
authors.initials: AB
authors.lastname: Troxel
authors.affiliation: Department of Pathology and Laboratory Medicine Hospital of the University of Pennsylvania Philadelphia Pennsylvania.
authors.forename: Daniel
authors.initials: D
authors.lastname: DeSloover
authors.affiliation: Department of Pathology and Laboratory Medicine Hospital of the University of Pennsylvania Philadelphia Pennsylvania.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Fox
authors.affiliation: Department of Pathology and Laboratory Medicine Hospital of the University of Pennsylvania Philadelphia Pennsylvania.
authors.forename: Robert D
authors.initials: RD
authors.lastname: Daber
authors.affiliation: Department of Medicine University of Pennsylvania Perelman School of Medicine Philadelphia Pennsylvania.
authors.forename: Bijal
authors.initials: B
authors.lastname: Kakrecha
authors.affiliation: Department of Pathology and Laboratory Medicine Hospital of the University of Pennsylvania Philadelphia Pennsylvania.
authors.forename: Shrey
authors.initials: S
authors.lastname: Sukhadia
authors.affiliation: Department of Cancer BiologyUniversity of Pennsylvania Perelman School of MedicinePhiladelphiaPennsylvania; Abramson Family Cancer Research InstituteUniversity of Pennsylvania Perelman School of MedicinePhiladelphiaPennsylvania.
authors.forename: George K
authors.initials: GK
authors.lastname: Belka
authors.affiliation: Department of MedicineUniversity of Pennsylvania Perelman School of MedicinePhiladelphiaPennsylvania; Abramson Cancer CenterUniversity of Pennsylvania Perelman School of MedicinePhiladelphiaPennsylvania.
authors.forename: Angela M
authors.initials: AM
authors.lastname: DeMichele
authors.affiliation: Department of MedicineUniversity of Pennsylvania Perelman School of MedicinePhiladelphiaPennsylvania; Department of Cancer BiologyUniversity of Pennsylvania Perelman School of MedicinePhiladelphiaPennsylvania; Abramson Family Cancer Research InstituteUniversity of Pennsylvania Perelman School of MedicinePhiladelphiaPennsylvania.
authors.forename: Lewis A
authors.initials: LA
authors.lastname: Chodosh
authors.affiliation: Department of Pathology and Laboratory Medicine Hospital of the University of Pennsylvania Philadelphia Pennsylvania.
authors.forename: Jennifer J D
authors.initials: JJ
authors.lastname: Morrissette
authors.affiliation: Department of MedicineUniversity of Pennsylvania Perelman School of MedicinePhiladelphiaPennsylvania; Abramson Cancer CenterUniversity of Pennsylvania Perelman School of MedicinePhiladelphiaPennsylvania.
authors.forename: Erica L
authors.initials: EL
authors.lastname: Carpenter
keywords: Breast cancer; circulating tumor cell; liquid biopsy; next‐generation sequencing; personalized medicine; whole genome amplification