Atsushi Morimoto

High-Density Dielectrophoretic Microwell Array for Detection, Capture, and Single-Cell Analysis of Rare Tumor Cells in Peripheral Blood.

Development of a reliable platform and workflow to detect and capture a small number of mutation-bearing circulating tumor cells (CTCs) from a blood sample is necessary for the development of noninvasive cancer diagnosis. In this preclinical study, we aimed to develop a capture system for molecular characterization of single CTCs based on high-density dielectrophoretic microwell array technology. Spike-in experiments using lung cancer cell lines were conducted. The microwell array was used to capture spiked cancer cells, and captured single cells were subjected to whole genome amplification followed by sequencing. A high detection rate (70.2%–90.0%) and excellent linear performance (R2 = 0.8189–0.9999) were noted between the observed and expected numbers of tumor cells. The detection rate was markedly higher than that obtained using the CellSearch system in a blinded manner, suggesting the superior sensitivity of our system in detecting EpCAM− tumor cells. Isolation of single captured tumor cells, followed by detection of EGFR mutations, was achieved using Sanger sequencing. Using a microwell array, we established an efficient and convenient platform for the capture and characterization of single CTCs. The results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients.

Diversity of circulating tumor cells in peripheral blood: Detection of heterogeneous BRAF mutations in a patient with advanced melanoma by single-cell analysis

The advent of therapeutics targeting the MAPK signal pathway has led to great advances in the treatment of metastatic melanoma. However, a significant number of patients experience a short response and exhibit recurrence within several months after therapy, potentially related to mutation heterogeneity arising due to genetic and epigenetic instability of melanoma cells. Circulating tumor cells (CTCs) released from either primary or metastatic tumors are a non-invasive tool that could be used to characterise changes in tumors over time. However, the low concentrations of circulating tumor cells have significantly impeded the research necessary to evaluate their clinical utility. In this study, we demonstrated genetic heterogeneity in the BRAF V600 region in CTCs using a high-density dielectrophoretic microwell array, a novel rare-cell capture system. In a 77-year-old Japanese male, exon 15 of BRAF was wild-type in the primary lesion, BRAFK601E was detected in a metastatic lymph node. CTCs were defined as MART-1/gp100-positive and CD45-negative cells, and isolated by micromanipulation. Single-cell sequencing was performed in 33 CTCs: three contained BRAFK601E (one heterozygous and two homozygous mutations); two contained BRAFV600E (heterozygous); one contained BRAFV600A (heterozygous); and the remaining 27 CTCs were BRAFwild-type. This study indicates that CTCs are an important indicator of the potential for poor prognosis, treatment response, and disease recurrence.