A study of pre-analytical variables and optimization of extraction method for circulating tumor DNA measurements by digital droplet PCR

Circulating free DNA (cfDNA) is an exciting novel method to diagnose, monitor and predict resistance and response to cancer therapies, with the potential to radically alter the management of cancer patients. In order to fulfill its potential, greater knowledge about pre-analytical variables is required to optimize and standardize the collection process, and maximize the yield and utility of the small quantities of cfDNA extracted.In this study, we have compared the cfDNA extraction efficiency of three different protocols, including a protocol developed in house (JGH). We evaluated the impact on cfDNA levels of pre-analytical variables including speed and timing of the second centrifugation and the use of k-EDTA and CTAD blood collection tubes. Finally, we analyzed the impact on fractional abundance of targeted pre-amplification and whole genome amplification on tumor and circulating tumor DNA (ctDNA) from breast cancer patients.