Development and validation of a multi-marker liquid bead array assay for the simultaneous detection of and hotspot mutations in single circulating tumor cells (CTCs).

BACKGROUND

and mutations are associated with progression and therapy resistance in metastatic breast cancer (MBC). CTCs are highly heterogeneous and their analysis at single cell level can provide unique information for mutational profiling and the existence of different sub-clones related to tumor progression. We have developed a novel multi-marker liquid bead array assay based on combination of an enzymatic mutation enrichment method, multiplex PCR-based assay, and liquid bead array technology for the simultaneous detection of and hotspot mutations in liquid biopsy samples. We focus on single CTCs, however the assay can be used for bulk CTC and ctDNA analysis.

MATERIALS AND METHODS

Single CTCs were isolated from an ER+/HER2+ MBC patient from CellSearch® cartridges using the VyCAP Puncher System and subjected to whole genome amplification followed by nuclease-assisted minor-allele enrichment with probe-overlap (NaME-PrO) enrichment. The assay was validated for analytical sensitivity and specificity for the simultaneous detection of () and (Y537S) mutations in single CTCs, while its clinical performance was evaluated on 22 single CTCs and three single white blood cells (WBCs).

RESULTS

The developed multi-marker liquid bead array assay is novel, highly specific and sensitive for both mutation panels. The assay can reliably detect mutation-allelic-frequencies (MAFs) as low as 0.1 %. The presence of and mutations was detected in 13.6 % and 72.7 % of single CTCs, respectively. The developed assay is sample-saving since it requires only 2 μL of amplified DNA to check for nine hotspot and mutations in a single cell. The developed liquid bead array assay (Luminex, US), based on a 96 microwell plate format, enables the simultaneous analysis of 96 single cells.

CONCLUSIONS

The developed novel multi-marker liquid bead array assay for the simultaneous detection of and hotspot mutations in single CTCs is highly specific, highly sensitive, high-throughput, and sample-, cost-, and time-saving. This multi-marker liquid bead array assay can be extended to detect up to 100 mutations in many genes at once and can be applied for bulk CTC and ctDNA analysis.