Next Generation Sequencing of Circulating Tumor Cells Isolated from the Peripheral Blood of Patients with Gastrointestinal Cancer. Circle-1 Trial

Abstract

ABSTRACT Aim: Monitoring of tumor biology provides pivotal information for suitable therapy. Circulating tumor cells (CTCs) released into the blood stream from primary tumor and metastases may reflect current tumor status. The ClearCell FX system (Clearbridge Biomedics, Singapore), which uses a label-free inertial microfluidics approach based on biomechanical properties, is able to capture CTCs independent of EpCAM expression. We investigated the tumor biology by performing genomic profiling of CTCs using next-generation sequencing (NGS). Methods: Patients with advanced gastric and colorectal cancer were enrolled in this study. We collected 10mL of blood from each patient. CTCs were extracted by using the Clearcell FX system. DNA was prepared immediately after isolation, after a week's culture with RepCellTM (CellSeed) which was a temperature-responsive cell cultureware. Whole genome amplification (WGA) was performed by using REPLI-g Single Cell Kit (Qiagen), and NGS was performed by using Ion Ampliseq (LifeTechnology). Cells were enumerated using cytokeratin staining and immunocytochemical analysis. At the same time, we prepared DNA not only from peripheral blood mononuclear cells but also from plasma (cell-free DNA). Results: 8 patients were enrolled; 6 had colorectal cancer and 2 had gastric cancer. Median of CTC count was 6/mL (ranges 3 to 14). On the other hand, the number of CTCs collected by CellSearch ranged from 0 to 1/7.5mL. In these 8 cases, either right after isolation or after 1 week of culture, DNA derived from CTCs was successfully prepared by WGA. NGS was successfully performed with WGA treated DNA from CTCs and related materials. Conclusions: By using ClearCell FX System's label-free technology, we were able to capture a larger number of CTCs from patients with gastric and colorectal cancer than with using the CellSearch system. NGS from CTCs and related materials can be performed successfully. Disclosure: A. Kakimoto: I am a employee of SRL Inc.; T. Sakuma: I am a employee of Mitsui Knowledge Industry Co and Ltd.; S. Fang: I am a employee of Clearbridge Accelarator; A. Wu: I am a employee of Clearbridge Bio Medics; C.T. Lim: I have stock of Clearbridge Biomedics Pte Ltd. All other authors have declared no conflicts of interest.