Refined Procedure to Purify and Sequence Circulating Cell-Free DNA in Prostate Cancer

Cell-free DNA (cfDNA), a fragmented DNA circulating in blood, is a promising biomarker for cancer diagnosis and monitoring. Standardization of cfDNA isolation to enhance the sensitivity of molecular analyses in prostate cancer (PCa) is required. Towards this goal, we optimized existing methods to obtain a high quantity and quality of cfDNA from low volumes of plasma. The protocol was applied to samples from healthy males and three patient categories: radical prostatectomy (RP), disease-free (>6 years post-RP), and metastatic castration-resistant PCa (mCRPC). The yield was significantly higher in mCRPC cases, and the size of fragments was shorter. We compared for the first time library preparation using two cfDNA inputs and low vs. high sequencing depth. Clonal events were observed irrespective of input and depth, but lower input showed more subclonal events. The clinical application of the refined protocols to cfDNA samples from an mCRPC patient showed no tumor fraction before RP, while it increased to 25% at the advanced stage. Among chromosomal changes and mutations, the androgen receptor gene amplification was detected. Altogether, this comprehensive study on improved cfDNA procedures is highly promising to enhance the quality of liquid biopsy-based research for discoveries and much-needed clinical applications.