title: Genomic high-resolution profiling of single CKpos/CD45neg flow-sorting purified circulating tumor cells from patients with metastatic breast cancer.
abstract: BACKGROUND
  Circulating tumor cells (CTCs) are promising surrogate markers for systemic disease, and their molecular characterization might be relevant to guide more individualized cancer therapies. To enable fast and efficient purification of individual CTCs, we developed a work flow from CellSearch(TM) cartridges enabling high-resolution genomic profiling on the single-cell level.
  
  
  METHODS
  Single CTCs were sorted from 40 CellSearch samples from patients with metastatic breast cancer using a MoFlo XDP cell sorter. Genomes of sorted single cells were amplified using an adapter-linker PCR. Amplification products were analyzed by array-based comparative genomic hybridization, a gene-specific quantitative PCR (qPCR) assay for cyclin D1 (CCND1) locus amplification, and genomic sequencing to screen for mutations in exons 1, 9, and 20 of the phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) gene and exons 5, 7, and 8 of the tumor protein p53 (TP53) gene.
  
  
  RESULTS
  One common flow-sorting protocol was appropriate for 90% of the analyzed CellSearch cartridges, and the detected CTC numbers correlated positively with those originally detected with the CellSearch system (R(2) = 0.9257). Whole genome amplification was successful in 72.9% of the sorted single CTCs. Over 95% of the cells displayed chromosomal aberrations typical for metastatic breast cancers, and amplifications at the CCND1 locus were validated by qPCR. Aberrant CTCs from 2 patients harbored mutations in exon 20 of the PIK3CA gene.
  
  
  CONCLUSIONS
  This work flow enabled effective CTC isolation and provided insights into genomic alterations of CTCs in metastatic breast cancer. This approach might facilitate further molecular characterization of rare CTCs to increase understanding of their biology and as a basis for their molecular screening in the clinical setting.
authors.affiliation: Department of General, Visceral and Pediatric Surgery.
authors.forename: Rui P L
authors.initials: RP
authors.lastname: Neves
authors.affiliation: Institute for Transplantation Diagnostics and Cell Therapeutics.
authors.forename: Katharina
authors.initials: K
authors.lastname: Raba
authors.affiliation: Chair of Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany;
authors.forename: Oliver
authors.initials: O
authors.lastname: Schmidt
authors.affiliation: Department of Obstetrics and Gynecology, and.
authors.forename: Ellen
authors.initials: E
authors.lastname: Honisch
authors.affiliation: Department of Obstetrics and Gynecology, and.
authors.forename: Franziska
authors.initials: F
authors.lastname: Meier-Stiegen
authors.affiliation: Department of General, Visceral and Pediatric Surgery.
authors.forename: Bianca
authors.initials: B
authors.lastname: Behrens
authors.affiliation: Department of General, Visceral and Pediatric Surgery.
authors.forename: Birte
authors.initials: B
authors.lastname: Möhlendick
authors.affiliation: Department of Obstetrics and Gynecology, and.
authors.forename: Tanja
authors.initials: T
authors.lastname: Fehm
authors.affiliation: Department of Obstetrics and Gynecology, and.
authors.forename: Hans
authors.initials: H
authors.lastname: Neubauer
authors.affiliation: Chair of Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany; Fraunhofer Institute for Toxicology and Experimental Medicine, Regensburg, Germany.
authors.forename: Christoph A
authors.initials: CA
authors.lastname: Klein
authors.affiliation: Fraunhofer Institute for Toxicology and Experimental Medicine, Regensburg, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Polzer
authors.affiliation: Department of Maxillo- and Facial Plastic Surgery, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany;
authors.forename: Christoph
authors.initials: C
authors.lastname: Sproll
authors.affiliation: Institute for Transplantation Diagnostics and Cell Therapeutics.
authors.forename: Johannes C
authors.initials: JC
authors.lastname: Fischer
authors.affiliation: Department of Obstetrics and Gynecology, and.
authors.forename: Dieter
authors.initials: D
authors.lastname: Niederacher
authors.affiliation: Department of General, Visceral and Pediatric Surgery, nikolas.stoecklein@uni-duesseldorf.de.
authors.forename: Nikolas H
authors.initials: NH
authors.lastname: Stoecklein
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics*; D001943:Breast Neoplasms / Q000097:blood* / Q000235:genetics / Q000473:pathology; D058534:Class I Phosphatidylinositol 3-Kinases; D055028:Comparative Genomic Hybridization / Q000379:methods*; D019938:Cyclin D1 / Q000235:genetics; D056915:DNA Copy Number Variations; D005091:Exons; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D017493:Leukocyte Common Antigens / Q000235:genetics; D009154:Mutation; D009362:Neoplasm Metastasis; D009360:Neoplastic Cells, Circulating / Q000378:metabolism* / Q000473:pathology; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics*; D010798:Phycocyanin / Q000235:genetics; D010799:Phycoerythrin / Q000235:genetics; D059010:Single-Cell Analysis; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
chemical_list: D014408:Biomarkers, Tumor; C530104:CCND1 protein, human; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C002497:allophycocyanin; D010798:Phycocyanin; D010799:Phycoerythrin; D019938:Cyclin D1; D019869:Phosphatidylinositol 3-Kinases; D058534:Class I Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human