Urine cell free DNA integrity as a marker for early diagnosis of non invasive bladder cancer

Background: Urine cell free DNA (UF DNA) has recently been proposed as a potential template for bladder cancer characterization and diagnosis. It is known that the origin of extracellular DNA can be established on the basis of its fragmentation; non cancer apoptotic cells produce highly fragmented DNA whereas necrotic cancer cells release longer DNA. The aim of our study was to verify the accuracy of a new non invasive approach in identifying bladder cancers. Attention was focused on three regions frequently amplified in bladder cancer corresponding to the genes c-MYC, BCAS1, HER 2. We also tested the integrity of cell free DNA in urine. Materials and Methods: The study was conducted on a series of 132 individuals: 51 cancer patients, 46 symptomatic patients with benign urogenital diseases, and 32 healthy volunteers. After urine samples were collected, extracellular DNA was isolated from urine supernatant and free DNA integrity was determined blindly by three quantitative Real Time PCRs on three sequences longer than 250 bp: C-MYC, BCAS1 and HER2. A short fragment called STOX 1 was analyzed to exclude the presence of PCR inhibitors. Results: UF DNA integrity analysis highlighted 0.1 ng/ mu l as the best cut-off value with 0.73 (95% CI 0.61-0.85) sensitivity, 0.84 specificity (95% CI 0.71-0.97) in healthy individuals, and 0.83 (95% CI 0.72-0.94) in symptomatic patients. The areas under the ROC curves were 0.8346 (95% CI 0.7391-0.9300) for healthy individuals and 0.7962 (95% CI 0.7070-0.8855) for symptomatic patients. In our case series UF DNA integrity showed higher sensitivity compared to cytology (0.73 versus 0.53) with the highest advantage for low-grade tumors (0.72 vs 0.15). The combination of cytology and UF-DNA analysis increased sensitivity to 0.81 (95% CI 0.69-0.93). Conclusion: Our preliminary data suggest that urine cell free DNA integrity has the potential to be a good marker for the diagnosis of early, non invasive bladder cancer. The diagnostic performance of the test did not vary significantly even when symptomatic individuals instead of healthy individuals were considered as reference group. Furthermore, the DNA analysis showed higher sensitivity with respect to cytology in detecting low-grade tumors, an essential element for early diagnosis. Research is ongoing in a larger case series to confirm these results.