Extracellular DNA in patients with urothelial carcinoma of the bladder.

Abstract

834 Background: Higher concentration of extracellular DNA (ecDNA) in plasma of subjects with various cancers is associated with worse prognosis. The association of ecDNA and its subcellular origin – nuclear DNA (ncDNA) and mitochondrial DNA (mtDNA) with urothelial carcinoma of the bladder (BC) is unexplored. Deoxyribonuclease (DNase) can cleave ecDNA and modulate the observed association. The objective of this study was to identify the potential differences in ecDNA, ncDNA, mtDNA and DNase between patients with non-muscle-infiltrating BC (NMIBC) and muscle-infiltrating BC (MIBC) compared to controls. Methods: We analyzed a population of 62 individuals with BC (44 males, median age 67.5 years), 20 had Ta stage, 26 T1 stage (NMIBC) and 16 T2–T3 stage (MIBC). The control group consisted of 15 males and 6 females without history of cancer, median age 67.3 years. EcDNA was quantified fluorometrically after isolation from double centrifuged plasma. The subcellular origin of ecDNA was analyzed using quantitative real time PCR targeting specific nuclear and mitochondrial sequences. DNase activity was assessed using the single radial enzyme diffusion method. For statistical analysis, basic nonparametric tests were used. Results: In patients with BC compared to controls, we identified significantly higher ecDNA (median 15.0 v 11.7 ng/ml, P=.021) and ncDNA (median 4707 v 2136 GE/ml, P=.0046), but not mtDNA and DNase. Moreover, we determined a higher activity of DNase (median 2.3 vs 1.9 ng/ml, P=.0084) in subjects with NMIBC (Ta+T1) v MIBC. By mutual comparisons of all subgroups, following significant differences were recognized: ecDNA and ncDNA in MIBC v controls (P=.0065 and P=.0127, respectively) and DNase for Ta v MIBC (P=.0177). In addition, we detected a significant correlation on a stage of BC and rising ecDNA concentrations (P=.0076). Conclusions: In this study, we showed higher plasma ecDNA and ncDNA in patients with MIBC than controls and lower DNase activity in MIBC compared to NMIBC. The ecDNA concentrations increased with rising tumor stage. These results suggest a possible diagnostic and a likely prognostic value of ecDNA and DNase in BC. Further studies will focus on a potential association between plasma ecDNA and urinary ecDNA, which are assessed non-invasively and more frequently. This study was supported by the Ministry of Education, Science, Research and Sport of the Slovak Republic (grant number : VEGA 1/0090/22), the Slovak Research and Development Agency (grant number: APVV-22-0231) and OncoReSearch. Key Words: Bladder Cancer. Extracellular DNA. Nuclear DNA. Mitochondrial DNA. Nuclease.