Direct comparison of the next-generation sequencing and iTERT PCR methods for the diagnosis of TERT hotspot mutations in advanced solid cancers

• Published in: BMC medical genomics Vol. 15; no. 1; p. 25
• Main Authors: Kang, So Young, Kim, Deok Geun, Kim, Hyunjin, Cho, Yoon Ah, Ha, Sang Yun, Kwon, Ghee Young, Jang, Kee-Taek, Kim, Kyoung-Mee
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 09-02-2022; BioMed Central; BMC
• Subjects: Analysis; Bladder cancer; Cancer; Comparison; Data analysis; Diagnosis; DNA sequencing; Gene mutations; Genomes; Health aspects; High-Throughput Nucleotide Sequencing; Humans; Liver cancer; Melanoma; Mutation; Mutation hot spots; Next-generation sequencing; PCR; Polymerase Chain Reaction; RNA-directed DNA polymerase; Skin cancer; Software; Solid tumors; Telomerase; Telomerase - genetics; Telomerase reverse transcriptase; TERT promoter mutation; Testing; Thyroid cancer; Transcription activation; Tumors; Urinary bladder; South Korea; United States > US; TERT promoter mutation; Next-generation sequencing; Comparison; PCR
• ISSN: 1755-8794; 1755-8794
• DOI: 10.1186/s12920-022-01175-2

Mutations in the telomerase reverse transcriptase (TERT) promoter region have been proposed as novel mechanisms for the transcriptional activation of telomerase. Two recurrent mutations in the TERT promoter, C228T and C250T, are prognostic biomarkers. Herein, we directly compared the commercially available iTERT PCR kit with NGS-based deep sequencing to validate the NGS results and determine the analytical sensitivity of the PCR kit.
Of the 2032 advanced solid tumors diagnosed using the TruSight Oncology 500 NGS test, mutations in the TERT promoter region were detected in 103 cases, with 79 cases of C228T, 22 cases of C250T, and 2 cases of C228A hotspot mutations. TERT promoter mutations were detected from 31 urinary bladder, 19 pancreato-biliary, 22 hepatic, 12 malignant melanoma, and 12 other tumor samples.
In all 103 TERT-mutated cases detected using NGS, the same DNA samples were also tested with the iTERT PCR/Sanger sequencing. PCR successfully verified the presence of the same mutations in all cases with 100% agreement. The average read depth of the TERT promoter region was 320.4, which was significantly lower than that of the other genes (mean, 743.5). Interestingly, NGS read depth was significantly higher at C250 compared to C228 (p < 0.001).
The NGS test results were validated by a PCR test and iTERT PCR/Sanger sequencing is sensitive for the identification of the TERT promoter mutations.