Optimization of circulating cell-free DNA recovery for KRAS mutation and HPV detection in plasma.

BACKGROUND: The precise analysis of tumour markers in blood such as circulating cell-free DNA (cfDNA) could have a significant impact in facilitating monitoring of patients after initial therapy. Although high levels of total cfDNA in plasma of cancer patients are consistently demonstrated, a low sensitivity of DNA alterations is reported. OBJECTIVE: The major question regards the recovery of tumour-specific cfDNA such as KRAS mutated DNA and cancer-associated type 16 of human papillomavirus (HPV16). METHODS: TaqMan technology was used for detection of KRAS mutation, HPV16 and to quantify cfDNA in blood plasma. RESULTS: Comparison of four different column-based commercial kits shows that the cfDNA purification carried out by the Genomic Mini AX Body Fluids kit and the QIAamp Circulating Nucleic Acid kit gave us the possibility to improve the sensitivity of detection of KRAS mutation and HPV16. The optimized method was used to follow the reduction in cancer-specific cfDNA after therapy. We found that large volume extractions with low volume of DNA eluate enabled trace amounts of tumour-specific cfDNA from cancer patients to be effectively identified. CONCLUSIONS: Data presented in this study facilitate detection of tumour-specific cfDNA and improve standards needed for the implementation of cfDNA technology into routine clinical practice.