INTRODUCTION AND OBJECTIVE:
The analysis of circulating cell-free DNA (ccfDNA) and its tumoral fraction, the circulating tumor DNA (ctDNA), represents an innovative strategy for cancer management. It can provide information on the molecular profile of patients, useful for early cancer diagnosis and monitoring. However, considering localized prostate cancer (PCa), the presence of a pseudo-capsule surrounding the organ limits the release of ccfDNA in the circulation and its tumoral fraction is under the threshold for detection. We hypothesized that during the biopsy the needle punctures performed on the organ are likely to cause a release of prostate-derived ccfDNA, suitable for further molecular tests.
We collected needle biopsies and a blood sample (3 mL) before the start of the biopsy procedure from 38 patients undergoing transperineal prostate biopsy. A second blood sample was collected at the end of the procedure, at different sampling times: for 13 patients after 60 minutes, for 19 after 120 minutes and for 6 patients 4 post-biopsy blood samples were taken (after 10, 30, 60 and 120 minutes). ccfDNA was extracted from 1 mL of plasma using the Maxwell RSC ccfDNA Plasma Kit (Promega) and quantified with a Qubit fluorometer (Thermo Fisher). The size distribution analysis was performed using a TapeStation (Agilent). Patients’ specific somatic mutations were identified with the TruSight RNA PanCancer panel (Illumina) on bioptic material and were searched in ccfDNA of the corresponding patient using a targeted sequencing (NEBNext Ultra II DNA Library Prep kit, NEB) of selected amplicons.
We demonstrated a significant increase in the amount of ccfDNA after the biopsy procedure, both after 60 and 120 minutes (P≤0.0024). The size distribution analysis revealed the presence of longer DNA molecules, up to 1 kb, in the post-biopsy samples compared to the pre-biopsy condition. Longer molecules are probably prostate-derived and freshly released after the end of the biopsy, not yet being degraded by nucleases in the circulation. Five patient-specific somatic variants were detected with an experiment of targeted RNA-sequencing performed on needle biopsy samples. The NGS analysis of amplicons targeting the selected variants in pre- and post-biopsy ccfDNA in the corresponding patients revealed an enrichment in ctDNA (from 2.2 to 164 fold) in the post-biopsy sample
This study opens the possibility of exploiting the enrichment of ctDNA in the post-biopsy condition for the analysis of somatic mutations/epigenetic modifications in ccfDNA for the first time in the context of localized PCa.
Source of Funding: