Join Dr. Cristian Ionescu-Zanetti from Fluxion Biosciences for an IsoFlux NGS Webinar.
Introduction: Next generation sequencing (NGS) of blood-derived nucleic acids is an emerging paradigm for determining the mutational status of cancer patients over time. Here we present data from a CTC enrichment modality that results in tumor cell purities of >10% and a high sensitivity NGS data analysis workflow that enables the use of standard amplicon panels typically used for primary tissue.
Methods: Blood samples were enriched for CTCs using the IsoFluxTM System, from a starting blood volume of 7.5-14ml. Matched samples were enumerated to determine the CTC load, where CTCs were defined as CK+, CD45- nucleated cells (DAPI+). Cells were lysed and DNA was amplified using the IsoFlux NGS Kit (Fluxion Biosciences. Targeted libraries were sequenced using both the MiSeq (Illumina) and PGM (ThermoFisher) sequencing instruments; data was analyzed using a customized variant calling/filtering pipeline.
Results: Multisite analytical validation data, based on spiking of cells into whole blood, demonstrated a detection limit down to 2 cells/ml blood, with a very low false positive rate. Clinical data from two different clinical pilot studies demonstrates the detection of somatic variants for a majority of samples, and significant overlap between detected mutations and known somatic mutation sites. This assay makes possible the detection of somatic variants from urological cancer patients without the need for a tissue biopsy.