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

2018 ◽  
Vol 64 (4) ◽  
pp. 745-747 ◽  
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

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

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3541 ◽  
Author(s):  
Md Ismail Hosen ◽  
Nathalie Forey ◽  
Geoffroy Durand ◽  
Catherine Voegele ◽  
Selin Bilici ◽  
...  
Keyword(s):  
Urothelial Cancer ◽  
Digital Pcr ◽  
Kappa Coefficient ◽  
Droplet Digital Pcr ◽  
Cell Free Dna ◽  
Non Invasive ◽  
Free Dna ◽  
Tert Promoter ◽  
Tert Promoter Mutations ◽  
Promoter Mutations

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.

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

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

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

2019 ◽  
Vol 21 (2) ◽  
pp. 274-285 ◽  
Author(s):  
Broderick C. Corless ◽  
Gregory A. Chang ◽  
Samantha Cooper ◽  
Mahrukh M. Syeda ◽  
Yongzhao Shao ◽  
...  
Keyword(s):  
Novel Mutation ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Plasma Samples ◽  
Tert Promoter ◽  
Tert Promoter Mutations ◽  
Pcr Assays ◽  
Promoter Mutations

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

Oncotarget ◽  
2017 ◽  
Vol 8 (45) ◽  
pp. 78890-78900 ◽  
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

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

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 2026-2026 ◽  
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.

Highly sensitive detection of TERT promoter mutations in recurrent glioblastomas using digital PCR

2020 ◽  
Vol 37 (4) ◽  
pp. 154-158
Author(s):  
Shunichiro Miki ◽  
Kaishi Satomi ◽  
Makoto Ohno ◽  
Yuko Matsushita ◽  
Mai Kitahara ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Sensitive Detection ◽  
Tert Promoter ◽  
Highly Sensitive ◽  
Tert Promoter Mutations ◽  
Highly Sensitive Detection ◽  
Promoter Mutations

Comparison of genomic alterations in bladder urothelial tumors with and without telomerase reverse transcriptase promoter mutation using a next-generation sequencing assay.

2017 ◽  
Vol 35 (6_suppl) ◽  
pp. 310-310 ◽  
Author(s):  
Sumit Isharwal ◽  
Francois Audenet ◽  
Eugene J. Pietzak ◽  
Eugene K. Cha ◽  
Gopa Iyer ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Copy Number ◽  
Promoter Region ◽  
Telomerase Reverse Transcriptase ◽  
Copy Number Alterations ◽  
Tert Promoter ◽  
Tert Promoter Mutations ◽  
Urothelial Tumors ◽  
Altered Gene ◽  
Promoter Mutations

310 Background: Telomerase reverse transcriptase (TERT) is the most frequently altered gene in urothelial cancer (UC), detected across all grades and stages of disease. We sought to characterize TERT alterations within a prospective cohort of UC treated at our institute and compare the frequency of genomic alterations in TERT promoter mutant vs wild-type UC specimens. Methods: Patients diagnosed with bladder urothelial tumors were enrolled onto an institutional review board approved prospective sequencing protocol. Tumor and matched germline DNA were analyzed for somatic point mutations, truncations, copy number alterations, and insertions/deletions using the MSK-IMPACT NGS assay that detects alterations in all exons and select introns of 410 oncogenes and tumor suppressor genes as well as the entire TERT promoter region. Results: 329 UC were sequenced of which 236 (71.7%) harbored TERT mutations, the majority being promoter region hotspots (chr5: 1295228 G > A [81%] and chr5:1295250 G > A [16%]). Patients with TERT promoter mutations were significantly older than those without (69.01±10.70 years vs. 65.44±11.78 years, p = 0.0317). UC with TERT promoter mutations had significantly higher mutation count [median 10 (range: 2-76) vs median 5 (range: 0-119)] as well as copy number alterations [median 0.11 (range: 0-0.68) vs median 0.047 (range: 0-0.65)]. Between UC with and without TERT promoter mutations, there was a very significant difference in mutation frequencies of ARID1A(33% vs 11%), PIK3CA(28% vs 13%), FGFR3(32% vs 16%), CREBBP(19% vs 4%), CDKN1A(17% vs 2%), ERBB2(26% vs 9%), ERCC2(15% vs 3%), TSC1(11% vs 0%), MT2C(17% vs 9%) (all with p < 0.005). Conclusions: TERT is the most frequently altered gene in bladder cancer with the majority of TERT alterations comprised of two hotspot mutations. UC with TERT promoter mutations tends to occur in older patients and is associated with overall higher mutation count and copy number alterations. A number of genes are differentially mutated in UC with and without TERT promoter mutations and may suggest a link between TERT promoter mutations and distinct mutations profiles in UC.

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