Urinary TERT promoter mutations as non-invasive biomarkers for the comprehensive detection of urothelial cancer

Abstract

Background Recurrent mutations in the promoter of the telomerase reverse transcriptase ( TERT) gene (C228T and C250T) detected in tumours and cells shed into urine of urothelial cancer (UC) patients are putative biomarkers for UC detection and monitoring. However, the possibility of detecting these mutations in cell-free circulating DNA (cfDNA) in blood and urine, or DNA from urinary exfoliated cells (cellDNA) with a single-gene sensitive assay has never been tested in a case-control setting. Methods We developed a single-plex assay (UroMuTERT) for the detection of low-abundance TERT promoter mutations. We tested 93 primary and recurrent UC cases and 94 controls recruited in France (blood, urine samples and tumours for the cases), and 50 primary UC cases and 50 controls recruited in Portugal (urinary exfoliated cell samples). We compared our assay with urine cytology. Findings In the French series, C228T or C250T were detected in urinary cfDNA or cellDNA in 81 cases (87·1%; 95% CI 78·6–93·2), and five controls (Specificity 94·7%; 95%CI 88·0–98·3), with 98·6% (95% CI 92·5–99·96) concordance in matched tumours. Detection rate in plasma cfDNA among cases was 7·1%. The UroMuTERT sensitivity was (i) highest for urinary cfDNA and cellDNA combined, (ii) consistent across primary and recurrent cases, tumour stages and grades, (iii) higher for low-risk non-muscle invasive UC (86·1%) than urine cytology (23·0%) ( P < 0·0001) and (iv) 93·9% when combined with cytology. In the Portuguese series – the sensitivity and specificity for detection of UC with urinary cellDNA was 68·0% (95% CI 53·3–80·5) and 98·0% (95% CI 89·3–100·0). Interpretation TERT promoter mutations detected by the UroMuTERT assay in urinary DNA (cfDNA or cellDNA) show excellent sensitivity and specificity for the detection of UC, significantly outperforming that of urine cytology notably for detection of low-grade early stages UC. Fund French Cancer League; French Foster Research in Molecular Biology and European Commission FP7 Marie Curie COFUND.

TERT Promoter Mutation C228T Increases Risk for Tumor Recurrence and Death in Head and Neck Cancer Patients

“…Studies validating these results should be performed in order to confirm the predictive value of this marker for prognosis. In addition, minimally invasive approaches to detect TERT promoter mutations could be performed in head and neck samples in order to evaluate the feasibility of this marker, and since oral cavity is the most affected site, saliva could possibly be a good source, taking into consideration previous data in other tumors: FNA (Fine-Needle Aspiration) in thyroid tumors (51)(52)(53) and cell-free DNA (cfDNA) from urine in urothelial tumors (54)(55)(56), which were able to detect the mutation in fluids.…”

Practicability of clinical application of bladder cancer molecular classification and additional value of epithelial-to-mesenchymal transition: prognostic value of vimentin expression

“…On the one hand, NMIBC is the most frequent BlCa phenotype [35], and disease recurrence is very frequent. Substantial research efforts have been put towards uncovering non-invasive, liquid biopsy-based biomarkers for accurately diagnosing and following-up these patients [36,37]. One major gap in NMIBC relates to patient prognostication and risk stratification after resection, fundamental for establishing the most appropriate follow-up strategy.…”

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

“…In our previous study, we have established a single-gene assay based on the Ion Torrent Proton system (UroMuTERT) for the quantification of tumor-derived TERT promoter mutations load in urine cellDNA or cell-free DNA and evaluated its clinical performance in the DIAGURO and IPO-PORTO case-control studies [11]. Here, we compared the sensitivity, specificity, and accuracy of urinary TERT promoter mutations ddPCR assays for the detection of UC to that of UroMuTERT assay using the whole set of data (n = 187 in the DIAGURO and n = 100 in IPO-PORTO series) [11] and a restricted set of samples with available data for both techniques (n = 181 in the DIAGURO and n = 99 in IPO-PORTO series) ( Table 1). We also compared the analytical sensitivity and specificity of detecting urinary TERT promoter mutations in corresponding matched tumors of DIAGURO cases (n = 81) using the two evaluated approaches.…”

“…Should these mutations be detectable in the urine of BC patients as a means of liquid biopsy, they would provide an unprecedented opportunity for a simple diagnostic assay of UCs, which could be used for detection as well as monitoring progression and recurrence. We have recently developed a Next-Generation Sequencing (NGS)-based assay, called UroMuTERT, for the comprehensive detection of BC in urine cell pellet and cell-free DNA samples [11]. We specifically showed in case-control studies that urinary TERT promoter mutations have excellent sensitivity and specificity for the detection of BC [11] and that these mutations could also be detected in urinary DNA of asymptomatic individuals years prior to primary diagnosis of BC with high specificity [12], highlighting their potential to be used as simple and inexpensive non-invasive biomarkers for early detection of BC.…”

“…We have recently developed a Next-Generation Sequencing (NGS)-based assay, called UroMuTERT, for the comprehensive detection of BC in urine cell pellet and cell-free DNA samples [11]. We specifically showed in case-control studies that urinary TERT promoter mutations have excellent sensitivity and specificity for the detection of BC [11] and that these mutations could also be detected in urinary DNA of asymptomatic individuals years prior to primary diagnosis of BC with high specificity [12], highlighting their potential to be used as simple and inexpensive non-invasive biomarkers for early detection of BC. While the NGS assay shows great promise in detecting TERT promoter mutations in urine samples, the complex laboratory workflow and requirement of extensive bioinformatics skills for data analysis are still considered as a bottleneck for their large-scale clinical application.…”