title: Clinical validation of an ultra high-throughput spiral microfluidics for the detection and enrichment of viable circulating tumor cells.
abstract: BACKGROUND
  Circulating tumor cells (CTCs) are cancer cells that can be isolated via liquid biopsy from blood and can be phenotypically and genetically characterized to provide critical information for guiding cancer treatment. Current analysis of CTCs is hindered by the throughput, selectivity and specificity of devices or assays used in CTC detection and isolation.
  
  
  RESULTS
  Here, we enriched and characterized putative CTCs from blood samples of patients with both advanced stage metastatic breast and lung cancers using a novel multiplexed spiral microfluidic chip. This system detected putative CTCs under high sensitivity (100%, n = 56) (Breast cancer samples: 12-1275 CTCs/ml; Lung cancer samples: 10-1535 CTCs/ml) rapidly from clinically relevant blood volumes (7.5 ml under 5 min). Blood samples were completely separated into plasma, CTCs and PBMCs components and each fraction were characterized with immunophenotyping (Pan-cytokeratin/CD45, CD44/CD24, EpCAM), fluorescence in-situ hybridization (FISH) (EML4-ALK) or targeted somatic mutation analysis. We used an ultra-sensitive mass spectrometry based system to highlight the presence of an EGFR-activating mutation in both isolated CTCs and plasma cell-free DNA (cf-DNA), and demonstrate concordance with the original tumor-biopsy samples.
  
  
  CONCLUSIONS
  We have clinically validated our multiplexed microfluidic chip for the ultra high-throughput, low-cost and label-free enrichment of CTCs. Retrieved cells were unlabeled and viable, enabling potential propagation and real-time downstream analysis using next generation sequencing (NGS) or proteomic analysis.
authors.affiliation: Mechanobiology Institute, National University of Singapore, Singapore, Singapore.
authors.forename: Bee Luan
authors.initials: BL
authors.lastname: Khoo
authors.affiliation: BioSystems and Micromechanics (BioSyM) IRG, Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore, Singapore.
authors.forename: Majid Ebrahimi
authors.initials: ME
authors.lastname: Warkiani
authors.affiliation: Department of Medical Oncology, National Cancer Centre Singapore, Singapore, Singapore; Cancer Therapeutics Research Laboratory, National Cancer Centre Singapore, Singapore, Singapore.
authors.forename: Daniel Shao-Weng
authors.initials: DS
authors.lastname: Tan
authors.affiliation: Clearbridge BioMedics Pte Ltd, Singapore, Singapore.
authors.forename: Ali Asgar S
authors.initials: AA
authors.lastname: Bhagat
authors.affiliation: Sequenom Inc, San Diego, California, United States of America.
authors.forename: Darryl
authors.initials: D
authors.lastname: Irwin
authors.affiliation: Department of Medical Oncology, National Cancer Centre Singapore, Singapore, Singapore; Cancer Therapeutics Research Laboratory, National Cancer Centre Singapore, Singapore, Singapore.
authors.forename: Dawn Pingxi
authors.initials: DP
authors.lastname: Lau
authors.affiliation: Department of Pathology, Singapore General Hospital, Singapore, Singapore.
authors.forename: Alvin S T
authors.initials: AS
authors.lastname: Lim
authors.affiliation: Department of Pathology, Singapore General Hospital, Singapore, Singapore.
authors.forename: Kiat Hon
authors.initials: KH
authors.lastname: Lim
authors.affiliation: Department of Medical Oncology, National Cancer Centre Singapore, Singapore, Singapore; Cancer Therapeutics Research Laboratory, National Cancer Centre Singapore, Singapore, Singapore.
authors.forename: Sai Sakktee
authors.initials: SS
authors.lastname: Krisna
authors.affiliation: Department of Medical Oncology, National Cancer Centre Singapore, Singapore, Singapore.
authors.forename: Wan-Teck
authors.initials: WT
authors.lastname: Lim
authors.affiliation: Department of Medical Oncology, National Cancer Centre Singapore, Singapore, Singapore.
authors.forename: Yoon Sim
authors.initials: YS
authors.lastname: Yap
authors.affiliation: Department of Hematology-Oncology, National University Hospital, Singapore, Singapore.
authors.forename: Soo Chin
authors.initials: SC
authors.lastname: Lee
authors.affiliation: Department of Hematology-Oncology, National University Hospital, Singapore, Singapore.
authors.forename: Ross A
authors.initials: RA
authors.lastname: Soo
authors.affiliation: BioSystems and Micromechanics (BioSyM) IRG, Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore, Singapore; Department of Electrical Engineering and Computer Science, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
authors.forename: Jongyoon
authors.initials: J
authors.lastname: Han
authors.affiliation: Mechanobiology Institute, National University of Singapore, Singapore, Singapore; BioSystems and Micromechanics (BioSyM) IRG, Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore, Singapore; Department of Biomedical Engineering, National University of Singapore, Singapore, Singapore; Clearbridge BioMedics Pte Ltd, Singapore, Singapore.
authors.forename: Chwee Teck
authors.initials: CT
authors.lastname: Lim
mesh_terms: D001943:Breast Neoplasms / Q000097:blood / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D002469:Cell Separation / Q000295:instrumentation*; D002474:Cell-Free System / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics; D006801:Humans; D008175:Lung Neoplasms / Q000097:blood / Q000235:genetics / Q000473:pathology; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D009154:Mutation; D009362:Neoplasm Metastasis; D009360:Neoplastic Cells, Circulating / Q000378:metabolism / Q000473:pathology*
chemical_list: C512478:EGFR protein, human; D066246:ErbB Receptors