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Comparative Study
. 2022 Feb 9;15(1):25.
doi: 10.1186/s12920-022-01175-2.

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

So Young Kang  1 , Deok Geun Kim  2   3 , Hyunjin Kim  1   4 , Yoon Ah Cho  5 , Sang Yun Ha  1 , Ghee Young Kwon  1 , Kee-Taek Jang  6 , Kyoung-Mee Kim  7   8   9
Affiliations
Comparative Study

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

So Young Kang et al. BMC Med Genomics. .

Abstract

Background: 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.

Methods: 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.

Results: 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).

Conclusions: 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.

Keywords: Comparison; Next-generation sequencing; PCR; TERT promoter mutation.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
The average depth of sequencing coverage in the telomerase reverse transcriptase (TERT) promoter region a and other genes b. There was a statistically significant decrease in the sequencing read depth in the TERT promoter region than the other genes c. Sequencing read depth was significantly higher at C250 compared to C228 d
Fig. 2
Fig. 2
Results of iTERT polymerase chain reaction and Sanger sequencing in the representative cases. According to the variant allele frequencies of the TERT promoter mutation, there was good correlation among the heights of the mutant peaks
See this image and copyright information in PMC

References

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