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Page 174 Pavan et al. J Transl Genet Genom 2021;5:173-81 https://dx.doi.org/10.20517/jtgg.2021.18

largely proposed as the first-line test. Moreover, PSA is elevated as well in benign prostate pathologies, and
about 15% of asymptomatic PCa patients do not present elevated PSA levels. Nowadays, prostate biopsy
remains the procedure necessary for the diagnosis of PCa despite the invasiveness, possible side effects and
cost of the procedure.

More specific PCa biomarkers are urgently needed not only to anticipate cancer diagnosis, especially the
identification of the more aggressive forms, but also for the best management of therapeutic interventions
and the surveillance of cancer progression. In particular, for patients in active surveillance (AS), a more
specific test and less invasive procedure are needed to improve the adherence to the protocols and maintain
a good quality of life for patients.

At the genomic level, tumor onset and progression are importantly modulated at the epigenetic level by the
DNA methylation changes in specific regions, mainly at gene promoter sites. The epigenetic modulation of
DNA is a field of intense research to find novel biomarkers for diagnosis/prognosis or targets for innovative
therapeutic strategies. Methylation of the CpG islands is a very frequent aberration in cancer that occurs
also in PCa . Thus, a part of the scientific interest is directed to the “methylome” analysis of the DNA to
[2]
better define the onset and the phenotype evolution of cancer.

To analyze tumor DNA, liquid biopsy can offer a non-invasive tool to monitor specific PCa biomarkers in
different biological fluids. Nowadays, it is possible to detect, with high sensitivity and specificity, circulating
tumor nucleic acids (DNA and RNA) derived from cancer cells that have died. For example, the search of
DNA aberrations of the androgen receptor gene has been found useful at a prognostic and predictive level
[3]
and they are strongly correlated with patients’ outcomes .
This mini-review summarizes the major and recent discoveries of aberration in the methylation pattern of
circulating tumor DNA. We mainly focused on the biomarkers that have been demonstrated to be clinically
useful or promising.

A literature review was performed using PubMed and three main key terms (DNA methylation AND
prostate cancer AND circulating cell-free DNA AND liquid biopsy) We selected research articles from 2015
to 2021. Articles included in this review are summarized in Table 1.

METHYLATED BIOMARKERS IN CIRCULATING CELL-FREE DNA FROM URINE
The cfDNA containing ctDNA has been collected after digital rectal examen or at first void
One of the most interesting translational research comes from Brikun et al. . Using cell-free DNA
[4,5]
(cfDNA) from urine after digital rectal examen (DRE) or at first void (FV), they demonstrated, using a
panel of 19 targets that the number of methylated markers was statistically higher in PCa cases compared to
controls, 10 of 19 vs. 3 of 19, respectively. Six of nineteen methylated markers (6 of 19) were shown to be the
threshold to predict PCa with a negative predictive value (NPV) ≥ 90% for both DRE and FV urine cfDNA.
In addition, authors proved a significative association between the number of methylated markers and the
PCa diagnosis with the tissue biopsies. Finally, in post-DRE urine samples, a higher rate of biomarkers was
reported when compared to urine at FV. In particular, AOX1, coding an aldehyde oxidase that regulates
reactive oxygen species homeostasis, GRFA2, coding a neurotrophic factor involved in cell survival and
differentiation, and NEUROG3, coding a transcriptional regulator, cannot be found in FV samples .
[5]

Furthermore, in a subsequent study the authors proved that other 13 markers can be used to predict early
PCa or to stratify the disease. Using the same sample of cfDNA from urine after DRE or at FV, a panel of 32