Development of Sensitive Droplet Digital PCR Assays for Detecting Urinary TERT Promoter Mutations as Non-Invasive Biomarkers for Detection of Urothelial Cancer

Abstract

Somatic mutations in the telomerase reverse transcriptase (TERT) promoter regions are frequent events in urothelial cancer (UC) and their detection in urine (supernatant cell-free DNA or DNA from exfoliated cells) could serve as putative non-invasive biomarkers for UC detection and monitoring. However, detecting these tumor-borne mutations in urine requires highly sensitive methods, capable of measuring low-level mutations. In this study, we developed sensitive droplet digital PCR (ddPCR) assays for detecting TERT promoter mutations (C228T, C228A, CC242-243TT, and C250T). We tested the C228T and C250T ddPCR assays on all samples with sufficient quantity of urinary DNA (urine supernatant cell-free DNA (US cfDNA) or urine pellet cellular DNA (UP cellDNA)) from the DIAGURO (n = 89/93 cases and n = 92/94 controls) and from the IPO-PORTO (n = 49/50 cases and n = 50/50 controls) series that were previously screened with the UroMuTERT assay and compared the performance of the two approaches. In the DIAGURO series, the sensitivity and specificity of the ddPCR assays for detecting UC using either US cfDNA or UP cellDNA were 86.8% and 92.4%. The sensitivity was slightly higher than that of the UroMuTERT assay in the IPO-PORTO series (67.4% vs. 65.3%, respectively), but not in the DIAGURO series (86.8% vs. 90.7%). The specificity was 100% in the IPO-PORTO controls for both the UroMuTERT and ddPCR assays, whereas in the DIAGURO series, the specificity dropped for ddPCR (92.4% versus 95.6%). Overall, an almost perfect agreement between the two methods was observed for both US cfDNA (n = 164; kappa coefficient of 0.91) and UP cellDNA (n = 280; kappa coefficient of 0.94). In a large independent series of serial urine samples from DIAGURO follow-up BC cases (n = 394), the agreement between ddPCR and UroMuTERT was (i) strong (kappa coefficient of 0.87), regardless of urine DNA types (kappa coefficient 0.89 for US cfDNA and 0.85 for UP cellDNA), (ii) the highest for samples with mutant allelic fractions (MAFs) > 2% (kappa coefficient of 0.99) and (iii) only minimal for the samples with the lowest MAFs (< 0.5%; kappa coefficient 0.32). Altogether, our results indicate that the two methods (ddPCR and UroMuTERT) for detecting urinary TERT promoter mutations are comparable and that the discrepancies relate to the detection of low-allelic fraction mutations. The simplicity of the ddPCR assays makes them suitable for implementation in clinical settings.

Clinical Utility of Plasma KRAS, NRAS and BRAF Mutational Analysis with Real Time PCR in Metastatic Colorectal Cancer Patients—The Importance of Tissue/Plasma Discordant Cases

“…The most probable explanation of this finding is the higher false negativity rate of liquid biopsy testing observed when a specific mutation present in the tumor tissue is analysed in the plasma of patients with low tumor burden. Modern techniques with improved sensitivity would probably detect circulating mutations also in case of low mutant allele fraction (MAF) [ 21 , 22 , 23 ].…”

Liquid Biopsy Biomarkers in Urine: A Route towards Molecular Diagnosis and Personalized Medicine of Bladder Cancer

“…The authors demonstrated that the detection of TERT promoter mutations in urine have high diagnostic accuracy and excellent diagnostic specificity for BC [ 40 , 41 ]. More recently, Hosen et al reported the development of test based on droplet digital PCR able to detect TERT promoter mutations in urine with an easy, inexpensive, non-invasive procedure [ 42 ]. Such an assay showed comparable performance with previously developed NGS-based test [ 40 ] but has the potential to be implemented in large-scale clinical application.…”

Urine TERT promoter mutations‑based tumor DNA detection in patients with bladder cancer: A pilot study

“…Droplet Digital PCR (ddPCR) was the analytical method of choice due to its ability to precisely quantify target DNA, even in case of low allelic frequency (34)(35)(36)(37). This approach to detecting TERT promoter mutations was previously proven to be comparable to the next-generation sequencing (NGS)-based validated system 'UroMuTERT' in terms of diagnostic accuracy (38).…”