Abstract
We directly compared two different approaches used for Circulating Tumor Cell (CTC) isolation, a size-dependent microfluidic system versus an EpCAM-dependent positive selection for downstream molecular characterization of CTC both at the gene expression and DNA methylation level in Head and Neck Squamous Cell Carcinoma (HNSCC). A size-dependent microfluidic device (Parsortix, ANGLE) and an EpCAM-dependent positive immune-magnetic isolation procedure were applied in parallel, using 10 mL PB from 50 HNSCC patients and 18 healthy donors. Total RNA was isolated from enriched CTCs and RT-qPCR was used to study the expression levels of CK-19, PD-L1, EGFR, TWIST1, CDH2 and B2M (reference gene). Real time methylation specific PCR (MSP) was used to study the methylation status of RASSF1A and MLL3 genes. In identical blood draws, the label-free size-dependent CTC-isolation system was superior in terms of sensitivity when compared to the EpCAM-dependent CTC enrichment, since a significantly higher percentage of identical PB samples was found positive at the gene expression and DNA methylation level, while the specificity was not affected. Our results indicate that future studies focused on the evaluation of clinical utility of CTC molecular characterization in HNSCC should be based on size-dependent enrichment approaches.
Highlights
Liquid biopsy provides a valuable source of biomarkers on prognosis and response to treatment of cancer patients[1] and has recently shown a significant potential even for early cancer diagnosis and screening[2]
We first evaluated in a quantitative way the performance of these two different Circulating Tumor Cell (CTC) enrichment approaches by downstream RNA-based CTC analysis run in parallel, using exactly the same procedure and the same room temperature (RT)-qPCR assays in material extracted from 50 Head and Neck Squamous Cell carcinoma (HNSCC) patients and 18 HD
Our results clearly reveal a significant difference in the expression levels of B2M in CTCs isolated through Parsortix versus EpCAM-dependent CTC enrichment
Summary
Liquid biopsy provides a valuable source of biomarkers on prognosis and response to treatment of cancer patients[1] and has recently shown a significant potential even for early cancer diagnosis and screening[2]. Isolation of circulating tumor cells (CTCs) from peripheral blood (PB) and their further downstream molecular characterization at the DNA, RNA and protein level is very important for reliable liquid biopsy analysis[3]. DNA methylation analysis in CTCs has a high potential to provide novel epigenetic biomarkers for diagnosis, prognosis, risk assessment, and disease monitoring in many types of cancer[4]. It is obvious that in all EpCAM-based CTC-capture systems, EpCAM-negative CTC subpopulations are non-detectable This can have serious implications in cases where CTCs are characterized by a phenotypic plasticity that mainly reflects an epithelial to mesenchymal transition state (EMT)[15,16]. Antibody combinations on immunomagnetic beads when compared to anti-EpCAM antibodies enable capturing of a larger number of CTCs20
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