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Pavan et al. J Transl Genet Genom 2021;5:173-81 https://dx.doi.org/10.20517/jtgg.2021.18 Page 179

number of patients, the authors have shown that both plasma and urine serve as excellent surrogates for
detecting tumoral epigenomic alterations.

[13]
Menschikowski et al. developed a novel amplification system based on digital-droplet PCR (ddPCR),
named optimized bias-based pre-amplification ddPCR (OBBPA-ddPCR), for early detection of rare DNA
methylation targets. They demonstrated that this technique can specifically detect PLA2R1 gene methylation
in serum of PCa patients with a very high sensitivity. PLA2R1 encodes a phospholipase A2 receptor. If this
novel assay could be usable for the early identification of PCa patients remains to be demonstrated in a large
sample cohort.

Bjerre et al. proved from a panel of 24 candidate biomarkers that three of them, DOCKK2, HAPLN3,
[14]
encoding an important protein that binds hyaluronic acid involved in many cellular function and cell
adhesion, and FBXO30, encoding a member of F-box protein family involved in protein degradation, were
strongly related to the progression of hormone-naïve mPCa to castration-resistant mPCa. They used plasma
samples that were analyzed by MS-ddPCR. Interestingly, these markers did not result in methylation in
healthy controls, BPH, or localized PCa patients. The authors noted that plasma cfDNA quantity did not
differ between healthy donors, BPH, localized PCa, or de novo mPCa patients. However, a higher level of
cfDNA was found to be related to cases in a more advanced clinical stage. It is important to underline that
in BPH or localized PCa samples, the cfDNA methylation in the biomarkers was rarely found; on the
contrary, in corresponding tissue samples, the methylation of all markers was present. The markers were
highly sensitive and specific to identify high tumor volume, de novo mPCa. From the clinical point of view,
the methylation of any of the three biomarkers was related with shorter OS in these patients, as an
independent predictor.

[15]
Beltran et al. studied cfDNA in castration-resistant neuroendocrine prostate cancer (CRPC-NE). A
significant proportion of PCa with this phenotype are linked with a poor prognosis. They performed whole-
exome sequencing in cfDNA from plasma to identify any aberration in the expression of important tumor
suppressor genes such as TP53 and RB1. The same analysis was applied also in genes involved in DNA
repair such as BRCA1, BRCA2, FANCA, or an important checkpoint signaling regulators such as Ataxia-
telangiectasia mutated gene (ATM) gene. The authors also performed whole-exome genome bisulfite
sequencing in a small sample of patients harboring CRPC-adeno or CRPC-NE and compared the results
with the methylation pattern in the tissues. A concordance of the methylation status of the targets between
cfDNA and tissue biopsy was found. In CRPC-NE samples hypo or hypermethylation status of 20 different
sites marked this tumor phenotype in cfDNA.

CONCLUSION
The ctDNA can be easily identified from the quote of cfDNA release from cancer cells in bloodstream or in
urine. It is important to note that the total level of cfDNA in plasma did not relate with presence of PCA,
although higher values of cfDNA have been found in advanced disease patients . However, the measure of
[14]
cfDNA level alone remains a poor predictor of the disease. Methylation of specific biomarkers and, in
particular, the number of methylated biomarkers in a panel can provide useful tools for clinicians either to
manage the risk of asymptomatic patients or to predict the progression.

Molecular tests are more expensive than PSA, however the reduction of improper prostate biopsies, the
precise identification of patients with risk should fully balance the initial screening test costs, and
prospectively save costs and suffering. Reducing the number of unnecessary biopsies will be one of the best
targets that we can achieve in PCa. The finding of low-grade PCa and an increase demand of AS need to be