Concentration and Methylation of Cell-Free DNA from Blood Plasma as Diagnostic Markers of Renal Cancer

Abstract

The critical point for successful treatment of cancer is diagnosis at early stages of tumor development. Cancer cell-specific methylated DNA has been found in the blood of cancer patients, indicating that cell-free DNA (cfDNA) circulating in the blood is a convenient tumor-associated DNA marker. Therefore methylated cfDNA can be used as a minimally invasive diagnostic marker. We analysed the concentration of plasma cfDNA and methylation of six tumor suppressor genes in samples of 27 patients with renal cancer and 15 healthy donors as controls. The cfDNA concentrations in samples from cancer patients and healthy donors was measured using two different methods, the SYBR Green I fluorescence test and quantitative real-time PCR. Both methods revealed a statistically significant increase of cfDNA concentrations in cancer patients. Hypermethylation on cfDNA was detected for the LRRC3B (74.1%), APC (51.9%), FHIT (55.6%), and RASSF1 (62.9%) genes in patients with renal cancer. Promoter methylation of VHL and ITGA9 genes was not found on cfDNA. Our results confirmed that the cfDNA level and methylation of CpG islands of RASSF1A, FHIT, and APC genes in blood plasma can be used as noninvasive diagnostic markers of cancer.

Figure 1

Analysis of cfDNA concentration in plasma of patients with renal carcinoma and controls. cfDNA concentrations were determined by measuring the fluorescence level of intercalated SYBR Green I dye (a) and by qPCR (c). ROC curve analysis of cfDNA concentration in cancer patients compared with the control group ((b) fluorescence test; (d) qPCR).

Figure 2

Receiver-operating characteristics (ROC) curves obtained by using different models for the discrimination between healthy controls (n = 15) and renal carcinoma patients (n = 27). Conc_qPCR: concentration of cfDNA determined by qPCR.