Development of Novel Mutation-Specific Droplet Digital PCR Assays Detecting TERT Promoter Mutations in Tumor and Plasma Samples

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.

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Droplet digital PCR assay for detecting TERT promoter mutations in patients with glioma

Brain Tumor Pathology ◽

10.1007/s10014-021-00403-4 ◽

2021 ◽

Author(s):

Jun-ichi Adachi ◽

Mitsuaki Shirahata ◽

Tomonari Suzuki ◽

Kazuhiko Mishima ◽

Eita Uchida ◽

...

Keyword(s):

Digital Pcr ◽

Droplet Digital Pcr ◽

Pcr Assay ◽

Tert Promoter ◽

Tert Promoter Mutations ◽

Promoter Mutations

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Sensitive droplet digital PCR method for detection of TERT promoter mutations in cell free DNA from patients with metastatic melanoma

Oncotarget ◽

10.18632/oncotarget.20354 ◽

2017 ◽

Vol 8 (45) ◽

pp. 78890-78900 ◽

Cited By ~ 27

Author(s):

Ashleigh C. McEvoy ◽

Leslie Calapre ◽

Michelle R. Pereira ◽

Tindaro Giardina ◽

Cleo Robinson ◽

...

Keyword(s):

Metastatic Melanoma ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Cell Free Dna ◽

Free Dna ◽

Tert Promoter ◽

Tert Promoter Mutations ◽

Pcr Method ◽

Promoter Mutations

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Plasma cell-free circulating tumor DNA (ctDNA) detection in longitudinally followed glioblastoma patients using TERT promoter mutation-specific droplet digital PCR assays.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.2026 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. 2026-2026 ◽

Cited By ~ 3

Author(s):

Christine Cordova ◽

Mahrukh M. Syeda ◽

Broderick Corless ◽

Jennifer M. Wiggins ◽

Amie Patel ◽

...

Keyword(s):

Digital Pcr ◽

Circulating Tumor Dna ◽

Droplet Digital Pcr ◽

Cross Sectional ◽

Tert Promoter ◽

Tert Promoter Mutations ◽

Ffpe Tumor ◽

Pharmacodynamic Biomarkers ◽

Promoter Mutations ◽

Clinical Stability

2026 Background: There is a critical need for more specific and less invasive diagnostic and pharmacodynamic biomarkers in glioblastoma (GBM) patients (pts). Previously, we detected TERT promoter hotspot mutations (C228T and C250T) in the ctDNA of IDH wildtype ( IDHwt) TERT promoter mutant GBM pts with 100% specificity using mutation-specific droplet digital PCR (ddPCR) assays. Here, we explored the dynamics and clinical associations of mutant TERT ctDNA levels in GBM pts undergoing therapy. Methods: We examined 14 pts with suspected IDHwt GBM based on preoperative MRI. Plasma was isolated and frozen from ~15 mL whole blood samples collected pre- and post-op, at end of radiation (RT), and 1, 3, and 6 m after end of RT. TERT promoter mutations were identified in FFPE tumor samples using ddPCR assays for C228T/C250T. Plasma samples were analyzed using ddPCR assays specific for the corresponding tumor mutation. The validated thresholds for positive detection were 1.5 (C228T) and 1.7 copies/mL (C250T). Results: 13/14 (92.9%) IDHwt tumors had TERT mutations (7 C228T and 6 C250T). Six of these 13 (46%) pts had positive plasma TERT ctDNA preop (4 C228T, 2 C250T). The mean cross sectional area of enhancing disease at presentation for positive or negative preop mutant ctDNA was similar. All 4 pts with multiple contrast enhancing lesions had positive preop mutant ctDNA. 2 pts who were negative initially developed detectable mutant ctDNA preceding progression. 3/4 pts with equivocal radiographic pseudoprogression had ctDNA dynamics that correlated with eventual clinical outcome. One patient with unresectable GBM had declining mutant ctDNA in later collections during clinical stability. Conclusions: We detected plasma TERT ctDNA in 46% of TERT mutant GBM pts before surgery, and in 100% of pts with multiple contrast enhancing lesions. TERT mutant ctDNA levels correlated with pseudoprogression or true disease progression and predicted progression before MRI. These data suggest that larger studies to test circulating cell-free TERT mutation as a diagnostic and pharmacodynamic biomarker in GBM are warranted.

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Optimizing Amplification of the GC-Rich TERT Promoter Region Using 7-Deaza-dGTP for Droplet Digital PCR Quantification of TERT Promoter Mutations

Clinical Chemistry ◽

10.1373/clinchem.2017.284257 ◽

2018 ◽

Vol 64 (4) ◽

pp. 745-747 ◽

Cited By ~ 6

Author(s):

Andrew J Colebatch ◽

Tom Witkowski ◽

Paul M Waring ◽

Grant A McArthur ◽

Stephen Q Wong ◽

...

Keyword(s):

Promoter Region ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Tert Promoter ◽

Tert Promoter Mutations ◽

Promoter Mutations

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Cell-free tumor DNA and TERT promoter mutations in bladder cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.6_suppl.353 ◽

2017 ◽

Vol 35 (6_suppl) ◽

pp. 353-353 ◽

Cited By ~ 1

Author(s):

Franklin W. Huang ◽

Mikael L. Rinne ◽

Kevin T. Lundgren ◽

Stephanie Anne Mullane ◽

Irene Moreno ◽

...

Keyword(s):

Urothelial Carcinoma ◽

Metastatic Disease ◽

Tert Promoter Mutation ◽

Plasma Samples ◽

Promoter Mutation ◽

Library Construction ◽

Non Invasive ◽

Tert Promoter ◽

Tert Promoter Mutations ◽

Promoter Mutations

353 Background: Currently, there are no FDA-approved blood biomarkers for the prognosis or prediction of outcomes in urothelial carcinoma (UC). The telomerase reverse transcriptase ( TERT) promoter is recurrently mutated at high frequency in UC (50%). These mutations have been correlated with tumor recurrence and survival. Tumor cell-free DNA (cfDNA) with somatic genomic alterations can be found in the plasma of cancer patients and has the potential for use as a non-invasive cancer biomarker. Detection of TERT promoter mutations in cfDNA might be used as a prognostic tool to monitor disease outcome in UC patients. We set out to detect tumor cfDNA and TERT promoter mutations in cfDNA from patients with UC at different stages. Methods: UC patients receiving chemotherapy in the neoadjuvant, first or second-line metastatic setting had blood collected either before or during therapy. cfDNA was isolated from ~1ml plasma samples using the QIAmp (Qiagen) kit. Samples underwent ultra-low pass whole genome sequencing (ULP-WGS) to determine whether tumor cfDNA could be detected in these samples. TERT promoter mutations were detected using a sensitive qPCR assay. Results: 40 plasma samples from a total of 32 patients with urothelial carcinoma were analyzed. Sufficient amounts of plasma cfDNA were obtained for library construction and ULP-WGS in 11 patients. 6 of these 11 patients were determined to be positive for detectable tumor cfDNA and of these, all were metastatic and 50% (3/6) were positive for a TERT promoter mutation. In total, 8 out of 40 samples (20%) were positive for a TERT promoter mutation, including samples from two patients where total cfDNA yield was insufficient for library construction. A total of ~20% of patients with metastatic disease were positive for TERT promoter mutations in cfDNA. The low percentage of samples having sufficient cfDNA most likely reflects the low volume of plasma used. Conclusions: TERT promoter mutations were identified in cfDNA of UC patients. ULP-WGS showed tumor cfDNA in patients with a high tumor burden and metastatic disease. TERTpromoter mutations in cfDNA could potentially be used as a non-invasive method for detection of disease. These results have implications for the use of cfDNA in the evaluation of advanced UC.

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Limited Clinical and Diagnostic Utility of Circulating Tumor DNA Detection in Patients with Early-Stage Well-Differentiated Thyroid Cancer: Comparison with Benign Thyroid Nodules and Healthy Individuals

Healthcare ◽

10.3390/healthcare9040386 ◽

2021 ◽

Vol 9 (4) ◽

pp. 386

Author(s):

Yong Joon Suh ◽

Mi Jung Kwon ◽

Hye-Mi Noh ◽

Hong Kyu Lee ◽

Yong Joon Ra ◽

...

Keyword(s):

Kras Mutation ◽

Early Stage ◽

Circulating Tumor Dna ◽

Diagnostic Utility ◽

Plasma Samples ◽

Healthy Individuals ◽

Thyroid Cancers ◽

Tert Promoter ◽

Tert Promoter Mutations ◽

Promoter Mutations

Limited data are available on the diagnostic utility of circulating tumor DNA (ctDNA) in early-stage thyroid cancers for BRAF, KRAS, NRAS, and TERT promoter mutations, which are known detectable markers for thyroid cancers. Here, we analyzed the above driver mutations in ctDNA and matched neoplastic tissues from patients with early-stage thyroid cancers in order to investigate diagnostic utility of circulating markers in distinguishing from other mimicking thyroid lesions and healthy individuals. In total, 73 matched neoplastic tissue and plasma samples [thyroid cancers (n = 62), benign thyroid disorders (n = 8), and parathyroid lesions (n = 3)] and 54 plasma samples from healthy individuals (as controls) were analyzed for BRAF, KRAS, NRAS, and TERT promoter mutations using peptide nucleic acid clamp real-time PCR. Although only one patient with an indeterminate lesion on thyroid cytology showed KRAS mutation (codon 146) in the preoperative plasma, that KRAS mutation was not identified in the stage I papillary thyroid carcinoma tissue. In the remaining 72 plasma samples, no other mutations were identified in BRAF, NRAS, and TERT promoter genes. The concordance rates of mutational results between the plasma and tumor tissue or metastatic lymph node were very low. One (1.9%) of the 54 healthy individuals harbored a KRAS mutation in the plasma samples. The ctDNA results did not represent the mutational profile of primary or metastatic thyroid cancers, warranting a caution for interpretation. The clinical utility of BRAF, KRAS, NRAS, and TERT promoter mutation analysis on ctDNA appears to be limited to early-stage thyroid cancers.

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