Feasibility of a workflow for the molecular characterization of single Circulating Tumor Cells by Next Generation Sequencing

Francesca Salvianti1, Giada Rotunno2, Francesca Galardi3, Francesca De Luca3, Marta Pestrin3, Alessandro Maria Vannucchi2, Angelo Di Leo3, Mario Pazzagli1, Pamela Pinzani1
1Department of Clinical, Experimental and Biomedical Sciences, University of Florence, Florence Italy.; 
2Department of Clinical and Experimental Medicine, University of Florence, Florence Italy; 
3Sandro Pitigliani Medical Oncology Department, Hospital of Prato, Istituto Toscano Tumori, Prato, Italy

Circulating Tumor Cells (CTCs) represent a “liquid biopsy of the tumor” which might allow real-time monitoring of cancer biology and therapies in individual patients. CTCs are extremely rare in the blood stream and their analysis is technically challenging. The purpose of the study was to explore the feasibility of a protocol for the molecular characterization of single CTCs. CTCs were enriched and enumerated by CellSearch in blood samples collected from four metastatic breast cancer patients and subsequently isolated by DEPArray to obtain single CTCs to be submitted to Whole Genome Amplification. Samples (3-5 single CTC per patient) were analysed by NGS on the Ion Torrent system using the Ion AmpliSeq™ Cancer Hotspot Panel (Thermo Scientific) able to investigate genomic “hot spot” regions of 50 oncogenes and tumor suppressor genes. We found 53 sequence variants in 26 genes: 36 variants with possible deleterious consequences and 17 supposed benign variants on the basis of the Polyphen software. Twenty-two mutations were already reported in the COSMIC database. The gene with the highest number of sequence variants is TP53 (with 10 variants) followed by PDGFRA (5 variants) and KIT (4 variants). We observed inter- and intra-patient heterogeneity in the mutational status of CTCs. In 3 patients we could compare the NGS results from CTC with those from the primary tissue evidencing few mutations common to the two different compartments. The discordance between the mutational status of the primary tumor and CTCs suggests that CTCs in advanced stages may reflect the disease status better than the primary tumor. This study demonstrates the feasibility of a non-invasive approach based on the liquid biopsy in metastatic breast cancer patients.

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