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

Ismail Hosen ,Olesia Lole,Selin Bilici,Patrice Hodonou Avogbe,Nathalie Forey,Florence Le Calvez-Kelm,Gilles Polo,Berengere De Tilly,Catherine Voegele,Antoine Boureille,Rui Henrique,Eduard Pisarev,Matthieu Foll,Graham Byrnes,Sonia Meziani,Emmanuel Vian,Anne‐Marie Manel ,Andrei Ronaldo Oliveira Silva Escartin Salas ,Geoffroy Durand ,Tiffany M Delhomme ,Carmen Jerónimo ,P François ,Ghislaine Scélo ,Sara Monteiro‐Reis ,James Mckay ,Maria I Zvereva

doi:10.3390/cancers12123541

Abstract

Simple SummaryThe gold standard method for the diagnosis of bladder cancer (BC) is the invasive and expensive cystoscopy. Telomerase reverse transcriptase (TERT) promoter mutations occur frequently (60–90%) in BC. In this study, we developed highly sensitive droplet digital PCR (ddPCR) assays for detecting low-allelic fraction TERT promoter mutations (C228T, C228A, CC242-243TT and C250T) in urinary cell-free and/or cell pellet DNA of BC patients and compared their performance with our previously established NGS-based assay (UroMuTERT) in two independent case-control studies: DIAGURO (n = 89 cases and n = 92 controls) and IPO-PORTO (n = 49 cases and n = 50 controls). The sensitivity and specificity of the ddPCR assays in detecting TERT promoter mutations in BC cases and controls were very high and comparable to the UroMuTERT assay. However, the technical and analytical simplicity of the ddPCR assays make them suitable candidates for clinical implementation.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.

Highlights

Bladder cancer (BC), accounting for 90% of urothelial cancer (UC), is the 10th most common cancer worldwide [1]
The GC content of the telomerase reverse transcriptase (TERT) promoter region is approximately 80% which causes the formation of G-quadruplex structures [13,14]
The two most common somatic mutations of the TERT promoter (C228T and C250T) occur within the terminal G-tracts of the second putative quadruplex sequence [14]. These higher-level organization at the TERT promoter mutation sites poses a significant threat for the droplet digital PCR (ddPCR) assays to show distinct droplet clusters

Summary

Introduction

Bladder cancer (BC), accounting for 90% of urothelial cancer (UC), is the 10th most common cancer worldwide [1]. Owing to the high recurrence rate of the non-muscle-invasive BC and expensive cystoscopy as a gold-standard diagnostic method, BC is one of the most expensive cancer to treat and manage [3]. The current guidelines for routine diagnosis and monitoring of BC rely on a sensitive but invasive cystoscopy as the gold standard and urine cytology, which is relatively less sensitive than cystoscopy, especially for low-grade non-muscle-invasive BC [4]. Individuals exposed to BC carcinogens at their workplace and under regular medical surveillance may benefit from a BC screening strategy using urine biomarkers with higher diagnostic predictive values than the common non-invasive tests (hematuria and urine cytology). Detecting molecular alterations of the bladder tumor in the urine promises to be an important addition in the liquid biopsy of BC [7,8]

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