A multicenter, prospective evaluation of a urine-based assay for bladder cancer diagnosis.

Abstract

e16509 Background: At present, bladder cancer is diagnosed mainly through cystoscopy as the "gold standard" method, which is invasive, costly, and painful for patients. Meanwhile, the current non-invasive liquid biopsy approaches, such as FISH or cytology, still do not show adequate performance in the diagnosis of bladder cancer because of the unsatisfactory sensitivity. Therefore, developing a novel method that performs higher sensitivity and specificity, especially negative predictive value(NPV) is an urgent issue to be addressed and will be very helpful for the clinical application. Methods: From October 2019 to October 2020, we designed a multicenter, prospective study, in which 321 hematuria patients were enrolled, including 218 males and 103 females, and their average age was 56. Based on the final pathological diagnosis, 121 patients were confirmed as bladder cancer and 200 samples were benign urinary diseases. 100ml urine samples were collected from each patient before the endoscopy examination. The Genetron Uro-18 assay, which included a 17 gene mutation panel and the methylation test on ONECUT2, was used in this study. The regions in the panel were selected based on the TCGA database, as well as previous publications. 321 urine DNA libraries were generated using multiplex PCR methods and then sequenced on the Ion S5 System. The CpG-sites on the ONECUT2 gene were detected by bisulfite-specific real-time PCR. Results: In these samples, a total of 205 oncogenic mutations were found in 13 genes in the panel. Mutations on TERT, PIK3CA, FGFR3, and TP53 genes showed significantly higher occurrence. 103 samples were found to be mutation-positive, 92 of which were pathologically confirmed as bladder cancers. When only gene mutations were considered, the sensitivity and specificity of this assay were 76.0% and 94.5%, respectively. The methylation of ONECUT2 had a performance in the test with a sensitivity of 74.4% and specificity of 93.0%. Combining the mutation panel with the ONECUT2 methylation test will improve the sensitivity (86.0%) of this assay, but slightly hampered the specificity (91.0%). The positive predictive value(PPV) and negative predictive value(NPV) of the combination were 85.2% and 91.5%. The sensitivity was as high as 91.5% when low-grade bladder cancer samples were excluded. In contrast, the sensitivity of cytology and FISH tests were only 48.4% and 62.0%. Conclusions: The novel urine-based liquid biopsy assay has excellent performances in the diagnosis of bladder cancer, with sensitivity and NPV reaching 86.0% and 91.5%, which improved significantly than cytology and FISH tests. Thus this assay will be more beneficial for patients with urinary diseases which significantly reduces the current burden of repeat cystoscopies and cytology tests.