Evaluation of TERT promoter mutations in urinary cell-free DNA and sediment DNA for detection of bladder cancer

Stasik, Sebastian; Salomo, Karsten; Heberling, Ulrike; Froehner, Michael; Sommer, Ulrich; Baretton, Gustavo; Ehninger, Gerhard; Wirth, Manfred P.; Thiede, Christian; Fuessel, Susanne

doi:10.1016/j.clinbiochem.2018.11.009

Cited by 41 publications

(42 citation statements)

References 11 publications

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“…Other workers have also utilised cpDNA and targeted deep sequencing for the identification of genomic alterations in urine samples from patients with UBC [7,9,30]; however, few studies have directly compared cpDNA and cfDNA by targeted deep sequencing in this setting [11,31]. Although cpDNA is conventionally utilised for urinary biomarker studies (principally due to higher yields than cfDNA), we have shown that SM detection in urinary cfDNA works as well as (or marginally better than) SM detection in cpDNA.…”

Section: -Gene Panel For Urinary Ubc Diagnosismentioning

confidence: 77%

Targeted deep sequencing of urothelial bladder cancers and associated urinary DNA: a 23‐gene panel with utility for non‐invasive diagnosis and risk stratification

et al. 2019

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Objectives To develop a focused panel of somatic mutations (SMs) present in the majority of urothelial bladder cancers (UBCs), to investigate the diagnostic and prognostic utility of this panel, and to compare the identification of SMs in urinary cell‐pellet (cp)DNA and cell‐free (cf)DNA as part of the development of a non‐invasive clinical assay. Patients and Methods A panel of SMs was validated by targeted deep‐sequencing of tumour DNA from 956 patients with UBC. In addition, amplicon and capture‐based targeted sequencing measured mutant allele frequencies (MAFs) of SMs in 314 urine cpDNAs and 153 urine cfDNAs. The association of SMs with grade, stage, and clinical outcomes was investigated by univariate and multivariate Cox models. Concordance between SMs detected in tumour tissue and cpDNA and cfDNA was assessed. Results The panel comprised SMs in 23 genes: TERT (promoter), FGFR3, PIK3CA, TP53, ERCC2, RHOB, ERBB2, HRAS, RXRA, ELF3, CDKN1A, KRAS, KDM6A, AKT1, FBXW7, ERBB3, SF3B1, CTNNB1, BRAF, C3orf70, CREBBP, CDKN2A, and NRAS; 93.5–98.3% of UBCs of all grades and stages harboured ≥1 SM (mean: 2.5 SMs/tumour). RAS mutations were associated with better overall survival (P = 0.04). Mutations in RXRA, RHOB and TERT (promoter) were associated with shorter time to recurrence (P < 0.05). MAFs in urinary cfDNA and cpDNA were highly correlated; using a capture‐based approach, >94% of tumour SMs were detected in both cpDNA and cfDNA. Conclusions SMs are reliably detected in urinary cpDNA and cfDNA. The technical capability to identify very low MAFs is essential to reliably detect UBC, regardless of the use of cpDNA or cfDNA. This 23‐gene panel shows promise for the non‐invasive diagnosis and risk stratification of UBC.

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Section: -Gene Panel For Urinary Ubc Diagnosismentioning

confidence: 77%

Targeted deep sequencing of urothelial bladder cancers and associated urinary DNA: a 23‐gene panel with utility for non‐invasive diagnosis and risk stratification

et al. 2019

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“…Although the refinement of molecular technologies enables the analysis of cell-free DNA from urine, molecular approaches employing DNA from the urinary cell pellet display similar or even superior diagnostic value due to a substantial shedding of tumor cells into the urine [9,10]. However, urine does not only accumulate urothelial cells, but also cells from the prostate and kidney [5,11], making it necessary to account for potential cross-specificities of biomarkers.…”

Section: Of 14mentioning

confidence: 99%

In-Vitro Identification and In-Vivo Confirmation of DNA Methylation Biomarkers for Urothelial Cancer

et al. 2020

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We identified DNA methylation targets specific for urothelial cancer (UC) by genome-wide methylation difference analysis of human urothelial (RT4, J82, 5637), prostate (LNCAP, DU-145, PC3) and renal (RCC-KP, CAKI-2, CAL-54) cancer cell lines with their respective primary epithelial cells. A large overlap of differentially methylated targets between all organs was observed and 40 Cytosine-phosphate-Guanine motifs (CpGs) were only specific for UC cells. Of those sites, two also showed high methylation differences (≥47%) in vivo when we further compared our data to those previously obtained in our array-based analyses of urine samples in 12 UC patients and 12 controls. Using mass spectrometry, we finally assessed seven CpG sites in this “bladder-specific” region of interest in urine samples of patients with urothelial (n = 293), prostate (n = 75) and renal (n = 23) cancer, and 143 controls. DNA methylation was significantly increased in UC compared to non-UC individuals. The differences were more pronounced for males rather than females. Male UC cases could be distinguished from non-UC individuals with >30% sensitivity at 95% specificity (Area under the curve (AUC) 0.85). In summary, methylation sites highly specific in UC cell lines were also specific in urine samples of UC patients showing that in-vitro data can be successfully used to identify biomarker candidates of in-vivo relevance.

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“…To improve the predictive value of all three tested biomarkers an increased number of samples and observation time for individual patients is necessary. FGFR3 and TERT promoter mutations were used previously as urine biomarkers for monitoring BC separately and in combination [11,15,[28][29][30]]. An addition of STAG2 as a predictor for recurrence will substantially increase the analytical potential of the assay.…”

Section: Discussionmentioning

confidence: 99%

“…Mutation in cfDNA very tightly matched somatic mutation in the original tumor. High concordance rates of mutation allele frequencies in the FFPE tumor tissue with urinary cfDNA was shown [11]. Another group showed that 80.7% of somatic mutations detected in tumors were found in cfDNA [31].…”

Section: Discussionmentioning

confidence: 99%

“…It was shown that urinary cfDNA of BC patients had a higher tumor genomic burden and greater detection potential as a genomic biomarker (90%) than urinary pellet DNA (61%) [10]. However, data about the diagnostic implications of cfDNA in urine for the detection of BC is still sparse [11]. Therefore, for the current study, we decided to use for cfDNA NMIBC from a patient's urine.…”

Section: Introductionmentioning

confidence: 99%

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Liquid Biopsy Analysis of FGFR3, TERT Promoter and STAG2 Hotspot Mutations for Disease Surveillance in Bladder Cancer

Gnatenko¹,

Forsyth²,

Xiaohui³

et al. 2020

COR

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Patients with non-muscle invasive bladder cancer (NMIBC) are followed by frequent cystoscopies. Innovative approaches partly replacing cystoscopy (uncomfortable, expensive, low sensitive procedure) are demanded. The current study aims to establish a fast, reliable, non-invasive, and inexpensive procedure for NMIBC patient surveillance. Liquid biopsy is a reliable source of biomarkers for cancer patient monitoring. Urine is the most suitable biological liquid to search for bladder cancer biomarkers. Cell-free DNA in urine represents tumor-related mutations for several cancers, including the bladder. We investigated mutations in FGFR3, TERT promoter, and STAG2 as markers for diagnostics and follow-up in NMIBC. Digital PCR was used to detect mutations in urine-derived cell-free DNA. The sensitivity and specificity of the markers in relation to clinical outcomes served as criteria of the assay efficiency. The sensitivity with a single marker (TERT) reached 87%, with a specificity of 77%. Combining two biomarkers (TERT+FGFR3) increased the specificity of the assay to 100% with a sensitivity of 72%. Different mutational status of STAG2 can indicate NMIBC presence or recurrence. Therefore, applying the suggested combination of biomarkers with simple detection procedures to larger patient cohorts will allow developing procedures for BC detection and surveillance with optimal sensitivity and specificity. Based on the results of this proof-in-concept study, we conclude that this simple, fast and inexpensive assay can add diagnostic and prognostic value to cystoscopy/cytology analysis of NMIBC patients.

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Evaluation of TERT promoter mutations in urinary cell-free DNA and sediment DNA for detection of bladder cancer

Cited by 41 publications

References 11 publications

Targeted deep sequencing of urothelial bladder cancers and associated urinary DNA: a 23‐gene panel with utility for non‐invasive diagnosis and risk stratification

Targeted deep sequencing of urothelial bladder cancers and associated urinary DNA: a 23‐gene panel with utility for non‐invasive diagnosis and risk stratification

In-Vitro Identification and In-Vivo Confirmation of DNA Methylation Biomarkers for Urothelial Cancer

Liquid Biopsy Analysis of FGFR3, TERT Promoter and STAG2 Hotspot Mutations for Disease Surveillance in Bladder Cancer

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