Urine Cell-Free DNA Integrity as a Marker for Early Bladder and Prostate Cancer Diagnosis: Preliminary studies

Roberta Gunelli1, Valentina Casadio2, Samanta Salvi2, Daniele Calistri 2, Massimo Fiori1, Cristiano Salaris1, Umberto Salomone1, Marco Urbinati1, Ferdinando Di Giacomo1, Teo Zenico1, Wainer Zoli2
  • 1 Ospedale Morgagni-Pierantoni, U.O. Urologia (Forlì)
  • 2 IRCCS Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (Meldola)

Objective

Early diagnosis plays an important role in increasing disease-free survival and reducing mortality in patients with various tumor types.
Urological cancers are such tumor types which could benefit of an early diagnostic approach; moreover, regarding prostate and bladder cancer, the possibility to find tumor specific markers in urine samples pave the way for the research of new early-non invasive approaches.
Many studies have demonstrated the role of important urine markers in the diagnosis of prostate and bladder cancer (1) but there is still the need for new more accurate approaches able to implement those actually used in clinical practice (PSA and urine cytology) (2,3).
Many studies have been published on various markers detected in urine cells, but only few studies have considered the role of cell free DNA obtained from urine supernatant (4,5,6).
Taking into account that normal apoptotic cells produce highly fragmented DNA while cancer cells release longer DNA (7), the objective of our preliminary study is to evaluate the potential role of UCF DNA integrity as an early diagnostic marker, able to distinguish cancer patients from healthy individuals.

Methods and results

A total of 112 individuals were enrolled: 51 with bladder cancer, 29 with prostate cancer and 32 without malignant urological diseases. Urine samples were collected before surgery and DNA was isolated from 1 ml of urine supernatant and quantified by spectrophotometry.
DNA sequences longer than 250 bp, corresponding to three oncogenes (c-Myc, BCAS1, and HER2), were quantified by real-time PCR to verify UCF DNA integrity. In parallel, it was analyzed a short sequence, called STOX1 to exclude PCR inhibitors.
Urine cell free DNA was quantifiable for all samples and showed a median value of 6ng/µl.
To verify diagnostic accuracy, we performed ROC curve analyses, showing the following AUCs: 0.7959 (95% CI 0.6729–0.9188) for prostate cancer versus healthy individuals, 0.8346 (95% CI 0.7391-0.9300) for bladder cancers versus healthy individuals.
UCF DNA integrity demonstrated a sensitivity of 0.73 (95% CI 0.61– 0.85) and a specificity of 0.84 (95% CI 0.71– 0.97) for bladder cancer at the best cut off value of 0,1ng/µl. For prostate cancer it has shown a sensitivity of 0.79 (95% CI 0.62–0.90) and a specificity of 0.84 (95% CI 0.65–0.94) at the best cut-off value of 0.04 ng/µL.

Discussion

These preliminary findings demonstrated the potential utility of urine cell free DNA as a source of biomarkers for urologic malignancies and open the way for further studies on urine cell free DNA characterization.
UCF DNA integrity approach showed a higher accuracy with the respect to conventionally used approaches for bladder and prostate diagnosis (PSA and urine cytology).
Regarding prostate cancer, UCF DNA integrity has a high specificity (0.84), suggesting its potential usefulness in addition to PSA to reduce false positive results.
Regarding bladder cancer, UCF DNA integrity demonstrated a high sensitivity even in low grade and stage tumors, suggesting that it could be used in addition to urine cytology to unmask false negative results.
UCF DNA integrity approach has the advantage to be non invasive, rapid, easy to perform test and only few milliliters of urine samples are necessary to perform the analysis.
The main limitation of this preliminary study is the small number of individuals recruited, however the results are promising and pave the way for larger, confirmatory studies.

References

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