Early Dynamics of Quantitative SEPT9 and SHOX2 Methylation in Circulating Cell-Free Plasma DNA during Prostate Biopsy for Prostate Cancer Diagnosis

The diagnosis and risk stratification of prostate cancer (PCa) is associated with difficulties for both the examiner and the patient: the tumor burden and the aggressiveness of the tumor should be determined as accurately as possible without putting too much psychological and physical strain on the patient. The aim of the present proof-of-concept study was to investigate the potential and dynamics of quantitative SEPT9 and SHOX2 methylation, two promising validated non-invasive pan-cancer biomarkers, in PCa patient tissue and circulating cell-free DNA (ccfDNA) during prostate biopsy as a diagnostic tool. SEPT9 and SHOX2 were hypermethylated in PCa tissue and allowed discrimination of disease status, tumor stage and grade. In ccfDNA, methylation of both biomarkers allowed discrimination of localized and metastatic disease and increased shortly after prostate biopsy only in PCa patients. Hence, mSEPT9 and mSHOX2 are promising non-invasive measure in PCa evaluation. Background: The methylation status of Septin 9 (SEPT9) and short stature homeobox 2 (SHOX2) in circulating cell-free DNA (ccfDNA) are validated pan-cancer biomarkers. The present proof-of-concept study aimed to investigate the potential and dynamics of quantitative SEPT9 and SHOX2 methylation in prostate cancer (PCa) patient tissue and ccfDNA during prostate biopsy as a diagnostic tool. Methods: The methylation patterns of SEPT9 and SHOX2 in prostate tissue were analyzed using The Cancer Genome Atlas data set (n = 498 PCa and n = 50 normal adjacent prostate tissue (NAT)). Next, dynamic changes of ccfDNA methylation were quantified in prospectively enrolled patients undergoing prostate biopsy (n = 72), local treatment for PCa (n = 7; radical prostatectomy and radiotherapy) as well as systemic treatment for PCa (n = 6; chemotherapy and 177-Lu-PSMA-therapy). Biomarker levels were correlated with clinicopathological parameters. Results: SEPT9 and SHOX2 were hypermethylated in PCa tissue (p < 0.001) and allowed discrimination of PCa and non-tumor prostate tissue (mSEPT9: AUC 0.87, 95%CI [0.82-0.92]; mSHOX2: AUC 0.89, 95%CI 0.84-0.94). SHOX2 methylation and mRNA levels were significantly higher in PCa tissue and increased with tumor stage and grade, as well as in patients suffering from biochemical recurrence following radical prostatectomy. SEPT9 and SHOX2 ccfDNA methylation allowed distinguishing patients with localized and metastatic disease (p < 0.001 for both). In addition, methylation levels increased shortly after prostate biopsy only in patients with PCa (Delta mSEPT9: p < 0.001 and Delta mSHOX2: p = 0.001). Conclusions: The early dynamics of methylated SEPT9 and SHOX2 in ccfDNA allow differentiation between PCa patients and patients without PCa and is a promising marker for tumor monitoring in the metastatic stage to determine tumor burden under systemic therapy.