title: Feasibility of a workflow for the molecular characterization of single cells by next generation sequencing.
abstract: The purpose of the study was to explore the feasibility of a protocol for the isolation and molecular characterization of single circulating tumor cells (CTCs) from cancer patients using a single-cell next generation sequencing (NGS) approach. To reach this goal we used as a model an artificial sample obtained by spiking a breast cancer cell line (MDA-MB-231) into the blood of a healthy donor. Tumor cells were enriched and enumerated by CellSearch(®) and subsequently isolated by DEPArray™ to obtain single or pooled pure samples to be submitted to the analysis of the mutational status of multiple genes involved in cancer. Upon whole genome amplification, samples were analysed by NGS on the Ion Torrent PGM™ system (Life Technologies) using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Life Technologies), designed to investigate genomic "hot spot" regions of 50 oncogenes and tumor suppressor genes. We successfully sequenced five single cells, a pool of 5 cells and DNA from a cellular pellet of the same cell line with a mean depth of the sequencing reaction ranging from 1581 to 3479 reads. We found 27 sequence variants in 18 genes, 15 of which already reported in the COSMIC or dbSNP databases. We confirmed the presence of two somatic mutations, in the BRAF and TP53 gene, which had been already reported for this cells line, but also found new mutations and single nucleotide polymorphisms. Three variants were common to all the analysed samples, while 18 were present only in a single cell suggesting a high heterogeneity within the same cell line. This paper presents an optimized workflow for the molecular characterization of multiple genes in single cells by NGS. The described pipeline can be easily transferred to the study of single CTCs from oncologic patients.
authors.affiliation: Department of Clinical, Experimental and Biomedical Sciences, University of Florence, Florence, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Salvianti
authors.affiliation: Department of Clinical and Experimental Medicine, University of Florence, Florence, Italy.
authors.forename: Giada
authors.initials: G
authors.lastname: Rotunno
authors.affiliation: Sandro Pitigliani Medical Oncology Department, Hospital of Prato, Istituto Toscano Tumori, Prato, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Galardi
authors.affiliation: Sandro Pitigliani Medical Oncology Department, Hospital of Prato, Istituto Toscano Tumori, Prato, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: De Luca
authors.affiliation: Sandro Pitigliani Medical Oncology Department, Hospital of Prato, Istituto Toscano Tumori, Prato, Italy.
authors.forename: Marta
authors.initials: M
authors.lastname: Pestrin
authors.affiliation: Department of Clinical and Experimental Medicine, University of Florence, Florence, Italy.
authors.forename: Alessandro Maria
authors.initials: AM
authors.lastname: Vannucchi
authors.affiliation: Sandro Pitigliani Medical Oncology Department, Hospital of Prato, Istituto Toscano Tumori, Prato, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Di Leo
authors.affiliation: Department of Clinical, Experimental and Biomedical Sciences, University of Florence, Florence, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Pazzagli
authors.affiliation: Department of Clinical, Experimental and Biomedical Sciences, University of Florence, Florence, Italy.
authors.forename: Pamela
authors.initials: P
authors.lastname: Pinzani
keywords: Circulating tumor cells; Microgenomics; NGS; Single cell; Somatic mutations