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Page 178 Pavan et al. J Transl Genet Genom 2021;5:173-81 https://dx.doi.org/10.20517/jtgg.2021.18

[9]
Silva et al. , designed a prospective study to investigate the role of blood and urine in capturing the PCa
methylome. They selected a cohort of 4 patients with de novo metastatic PCa (mPCa) and a post-DRE and
FV sample of urine were analyzed. Detection of tumor DNA methylation probes in urine ranged from
6.98% to 39.40%. Authors demonstrated, through a DNA methylation analysis, highly correlated patterns
between the different liquid types (ρ = 0.93, P < 0.0001), with large contributions from non-tumor sources.

[10]
Finally, a promising Danish study investigated the role of novel aberrant promoter hypermethylation of
specific genes to improve the diagnosis and prognosis of PCa. Methylation in the promoter region of genes
was analyzed in prostate tissue and liquid biopsy. In particular, ST6GALNAC3 encoding a member of the
sialyltransferases for the modification of glycoproteins, ZNF660 encoding a transcriptional regulator,
T6GALNAC3 encoding a protein identified in neutrophil granules, ZNF660 encoding a transcription factor,
CCDC181 encoding a microtubule binding protein, and HAPLN3 encoding a membrane protein. 815
samples (705 PCa and 110 non-cancer) were processed by methylation-specific qPCR or methylation array.
The AUC of the ROC analysis demonstrated the role of hypermethylation of ST6GALNAC3 and ZNF660 in
the diagnosis of PCa (0.917-0.995 vs. 0.846-0.903 in cancer vs. non-cancer samples, respectively). Moreover,
ZNF660 hypermethylation was tested in two radical prostatectomy cohorts of 158 and 392 patients.

ZNF660 hypermethylation was also significantly associated with poor overall and PCa-specific survival in a
different cohort of radical prostatectomy (n = 158) with long clinical follow-up available showing a potential
prognostic role. In the same study, a panel of hypermethylated circulating tumor DNA (ctDNA) for
ST6GALNAC3, ZNF660, HAPLN3, and CCDC181 was proposed for liquid biopsy. A final ctDNA
hypermethylation model of 3 genes (ST6GAL- NAC3/CCDC181/HAPLN3) was developed with 100% of
specificity and 67% of sensitivity in the detection of PCa.

METHYLATED BIOMARKERS IN CIRCULATING CELL-FREE DNA FROM BLOOD
The cfDNA, containing ctDNA, has been collected from plasma or from serum
In 2015, Reis et al. studied, in serum, cfDNA the methylation of GADD45a gene, which they previously
[11]
found to be methylated at different sites in tissue PCa tissue. The authors found a statistically significant
difference between the methylation of GADD45a in Pca with respect to Benign Prostatic Hyperplasia (BPH)
patients’ serum. The PCa samples were more methylated than BPH controls, although in PCa patients a
higher methylation variability than BPH controls was found. No correlation between GADD45a
methylation and Gleason score was evidenced. Interestingly, the methylation status of GADD45a and the
PSA level better define PCa versus BPH patients than GADD45a methylation alone.

The role of aberrant DNA promoter methylation was also studied as a possible tool for simultaneous
[12]
detection of several types of cancers . In a large multicenter study, this hypothesis was tested for lung,
prostate, and colorectal cancers. More deeply, cfDNA was extracted from 121 PCa patients and the level of
methylation of different promoters was assessed. The authors proposed a “Pan-Cancer” panel (FOXA1me,
RARβ2me and RASSF1Ame) able to simultaneously detect PCa and lung cancer (SP 70% and SS 64%). The
panel was also able to discriminate between intermediate and high-risk PCa with a sensitivity of 71% and a
specificity of 65%. These results can be interesting when future studies will apply this panel in an AS setting
or in the decision-making process for a diagnostic biopsy in the suspected cases of PCa.

[9]
As previously described, the study published by Silva et al. analyzed the role of blood in capturing DNA
methylation. Utilizing the Infinium® MethylationEPIC BeadChip (Illumina) they were able to detect DNA
methylation probes from 7.19% to 64.14% in plasma. Matching liquid and prostate biopsies controls authors
prevented the effect of unwanted variables and reduced the inter-individual variability. Despite the small