Recent advances in cancer therapeutics allow the targeting of specific molecular pathways. In particular, PD-1/PD-L1 checkpoint blockers and BRAF kinase inhibitors have improved progression-free survival in patients with advanced melanoma. However, therapeutic efficacy is difficult to predict due to the lack of molecular biomarkers and heterogeneity in primary and metastatic tumor deposits. Circulating tumor cells (CTCs) are cells shed from primary or metastatic tumor deposits and have entered peripheral blood circulation. The VERSA platform (Versatile Exclusion-based Rare Sample Analysis), is capable of simultaneously analyzing proteins and extracting nucleic acids from CTCs down to the single cell level. Using this platform, we have developed a method to isolate circulating tumor cells in patients with advanced melanoma. We have optimized immunomagnetic-based cell capture with extracellular targets of CD146, GD2 and NG2. Individually, these targets yield captures of 52%, 54% and 45% in the SK-Mel-28 cell line, respectively. We developed a multi-marker, immunomagnetic cell capture methodology with these targets which improved cell recovery to 92%, with comparable results occurred in the M21 and 624 melanoma cell lines. After cell capture, sequential addition of lysis buffer and paramagnetic particles (PMPs) targeting mRNA then DNA were added for paired genomic/transcriptomic analyses from the same cell population. PMPs were eluted in nuclease-free water and analyzed via quantitative polymerase chain reaction (qPCR). Molecular targets included the BRAF-V600E oncogene in DNA, PD-L1 mRNA and transcription products known to upregulate PD-L1 expression including phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. We are now collecting patient samples to validate these targets as potential predictive biomarkers for these novel agents.

Development of microfluidic extraction and qPCR analysis of nucleic acids in circulating melanoma cells

248th National Meeting (2014)

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