Genomic High-Resolution Profiling of Single CKpos/CD45neg Flow-Sorting Purified Circulating Tumor Cells from Patients with Metastatic Breast Cancer

Abstract

BACKGROUND:Circulating tumor cells (CTCs) are promising surrogate markers for systemic disease, and their molecular characterization might be relevant to guide more individualized cancer therapies. To enable fast and efficient purification of individual CTCs, we developed a work flow from CellSearch TM cartridges enabling high-resolution genomic profiling on the singlecell level. METHODS:Single CTCs were sorted from 40 CellSearch samples from patients with metastatic breast cancer using a MoFlo XDP cell sorter. Genomes of sorted single cells were amplified using an adapter-linker PCR. Amplification products were analyzed by array-based comparative genomic hybridization, a gene-specific quantitative PCR (qPCR) assay for cyclin D1 (CCND1) locus amplification, and genomic sequencing to screen for mutations in exons 1, 9, and 20 of the phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) gene and exons 5, 7, and 8 of the tumor protein p53 (TP53) gene. RESULTS:One common flow-sorting protocol was appropriate for 90% of the analyzed CellSearch cartridges, and the detected CTC numbers correlated positively with those originally detected with the CellSearch system (R 2 ϭ 0.9257). Whole genome amplification was successful in 72.9% of the sorted single CTCs. Over 95% of the cells displayed chromosomal aberrations typical for metastatic breast cancers, and amplifications at the CCND1 locus were validated by qPCR. Aberrant CTCs from 2 patients harbored mutations in exon 20 of the PIK3CA gene. CONCLUSIONS:This work flow enabled effective CTC isolation and provided insights into genomic alterations of CTCs in metastatic breast cancer. This approach might facilitate further molecular characterization of rare CTCs to increase understanding of their biology and as a basis for their molecular screening in the clinical setting.

Clinical Applications of Circulating Tumor Cells and Circulating Tumor DNA as Liquid Biopsy

“…Schneck and colleagues analyzed CTCs using the SNaPshot method and found PIK3CA mutations in enriched CTC pools of 15.9% of patients with metastatic breast cancer ( 80 ). Other authors isolated single CTCs and reported strong heterogeneity in the PIK3CA mutational status among CTCs from individual patients (81)(82)(83). These single CTC analyses resulted in higher detection rates of PIK3CA mutations; however, more information about the occurrence of these mutations in the context of therapeutic interventions is urgently needed.…”

Improving the CellSearch® system

“…An inevitable step therefore is to pick and isolate the single CTCs in individual tubes or as a pure CTC fraction for genomic analysis. Methods used to isolate single cell for individual analysis in combination with CellSearch-enriched CTCs: Micromanipulation [113], ALS Cellselector [114], the Silicon Biosystems DEPArray [115][116][117][118], FACS [28,119,120], and the Punch system using self-seeding Microwell Chips [121]. This step seems a minor intermediate part of the whole workflow but is crucial for the end result.…”

Microfluidic Sample Preparation for Single Cell Analysis

“…This system is useful for bulk CTC enrichment but cannot be integrated directly with well plates for single cell molecular analysis. To overcome this, Neves et al 34 developed a protocol for CELLSEARCH® based CTC enrichment, FACS sorting and isolation into PCR tubes followed by single cell molecular analysis via qPCR. This study illustrated the combination of CELLSEARCH® and FACS for molecular analysis of single CTCs.…”