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

Area of Science:

• Molecular Biology
• Oncology
• Biochemistry

Background:

• Cell-free DNA (cfDNA) is a valuable biomarker for cancer detection and monitoring.
• Standardized cfDNA isolation is crucial for sensitive molecular analyses in prostate cancer (PCa).

Purpose of the Study:

• To optimize cfDNA isolation methods for high yield and quality from low plasma volumes.
• To evaluate cfDNA characteristics and library preparation in different PCa patient cohorts.
• To assess the clinical utility of refined cfDNA protocols for PCa liquid biopsy.

Main Methods:

• Optimization of cfDNA isolation protocols from plasma.
• Application of the protocol to samples from healthy males and PCa patients (RP, disease-free, mCRPC).
• Comparison of library preparation with varying cfDNA input and sequencing depths.
• Clinical application in an mCRPC patient for longitudinal monitoring.

Main Results:

• Significantly higher cfDNA yield and shorter fragment sizes were observed in metastatic castration-resistant PCa (mCRPC) cases.
• Clonal and subclonal events were detected, with lower cfDNA input revealing more subclonal events.
• Refined protocols detected a 25% tumor fraction in an mCRPC patient post-radical prostatectomy and identified androgen receptor gene amplification.

Conclusions:

• The optimized cfDNA isolation protocol enhances the quantity and quality of cfDNA for sensitive molecular analysis.
• This improved methodology supports the clinical application of liquid biopsy in prostate cancer research and diagnostics.
• The study highlights the potential of cfDNA analysis for detecting tumor burden and genetic alterations in PCa.