Introduction:

Upper tract urothelial carcinoma (UTUC) is a relatively uncommon malignancy, accounting for 5% of urothelial carcinoma (UC). Patients suspected of having UTUC need to receive invasive procedures such as CT urography, retrograde pyelography, or ureteroscopy for a definitive diagnosis. Many researchers have tried to develop useful urinary markers to detect UTUC, but urine cytology still remains as the only non-invasive diagnostic marker recommended by many guidelines although its sensitivity for detection of UTUC is as low as 40%. Cell-free DNA (cfDNA) in bodily fluids has huge potential in disease diagnosis. Cell-free tumor DNA (ctDNA) is shed into the urine or circulation along with DNA from normal cells. Even though ctDNA constitutes a small fraction of the total DNA, ctDNA is thought to be a promising diagnostic biomarker. Hotspot mutations of Telomerase Reverse Transcriptase (TERT) promoter and fibroblast growth factor receptor (FGFR3) (S249C) are frequently identified in UTUC specimens. To our knowledge, there is no research on the relationship between urinary cfDNA alteration of UTUC and clinical utility. In this study, we developed droplet digital polymerase chain reaction (ddPCR) assays for the detection of hotspot mutations of the TERT promoter region and FGFR3 and analyzed the diagnostic potential of urine supernatant cfDNA collected from patients with localized UTUC. 

Methods:

We investigated voided urine samples from four cohorts of patients: those with localized UTUC (UTUC cohort), those with microscopic or macroscopic hematuria without UC (Hematuria cohort), those who were treated with transurethral resection of bladder tumor (TURBT) or radical nephroureterectomy (RNU) and had no evidence of disease recurrence for at least one year (UC surveillance cohort), and a healthy control cohort (HC cohort) that included kidney transplantation donors, healthy volunteers, benign disease patients and patients with urological carcinoma other than UC. All patients in the UTUC cohort were histologically diagnosed as having UC, and urine samples were collected from patients within four days before RNU or transurethral biopsy. We excluded UTUC patients with concurrent bladder cancer. In 12 of the patients in the UTUC cohort, post-treatment urine samples were also collected about one week after RNU.

Results:

 Fifty-six UTUC patients (UTUC cohort), 50 patients with microscopic or macroscopic hematuria caused by other than UC (Hematuria cohort), 21 patients with no evidence of disease for at least one year after TURBT or RNU (UC surveillance cohort), and 26 healthy controls (HC cohort) were included in this study. The median age was 74.5 years (range 55–92 years) in the UTUC cohort, 68 years (range 33-89 years) in the Hematuria cohort, 70 years (range 47–89 years) in the UC surveillance cohort, and 57 years (range 31–81 years) in the HC cohort. The median follow-up time was 13 months (range 1–60 months). Of the 56 UTUC patients, 54 (96.4%) received RNU, 1 (1.8%) received Bacillus Calmette-Guérin therapy for carcinoma in situ of UTUC, and 1 (1.8%) received platinum-based chemotherapy for clinical T4 UTUC. We detected mutations of TERT C228T in 22/56 (39.3%), TERT C250T in 4/56 (7.1%), and FGFR3 S249C in 9/56 (16.1%) patients. FGFR3 mutation was found only in ≤pT1 tumors (positive predictive value: 100.0%). In combination with cytology results, the sensitivity was 78.6%, and the specificity was 96.0%.

Conclusion:

 We could detect TERT promoter and FGFR3 hotspot mutations in urinary cfDNA from UTUC patients. In combination with cytology results, the sensitivity of our non-invasive urinary test was high enough to apply this assay to the clinical setting. Liquid biopsy analysis of TERT promoter and FGFR3 mutations in urinary cfDNA could be a novel biomarker and a reliable factor for staging UTUC.

Funding: N/A