Cancer Res Treat.  2022 Jan;54(1):75-83. 10.4143/crt.2021.107.

Detection of TERT Promoter Mutations Using Targeted Next-Generation Sequencing: Overcoming GC Bias through Trial and Error

Affiliations
  • 1Department of Pathology and Translational Genomics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
  • 2Samsung Genome Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
  • 3Department of Digital Health, Samsung Advanced Institute of Health Science and Technology, Sungkyunkwan University School of Medicine, Seoul, Korea
  • 4Department of Clinical Genomic Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
  • 5Department of Pathology, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang, Korea

Abstract

Purpose
Detection of telomerase reverse transcriptase (TERT) promoter mutations is a crucial process in the integrated diagnosis of glioblastomas. However, the TERT promoter region is difficult to amplify because of its high guanine-cytosine (GC) content (> 80%). This study aimed to analyze the capturing of TERT mutations by targeted next-generation sequencing (NGS) using formalin-fixed paraffin-embedded tissues.
Materials and Methods
We compared the detection rate of TERT mutations between targeted NGS and Sanger sequencing in 25 cases of isocitrate dehydrgenase (IDH)-wildtype glioblastomas and 10 cases of non-neoplastic gastric tissues. Our customized panel consisted of 232 essential glioma-associated genes.
Results
Sanger sequencing detected TERT mutations in 17 out of 25 glioblastomas, but all TERT mutations were missed by targeted NGS. After the manual visualization of the NGS data using an integrative genomics viewer, 16 cases showed a TERT mutation with a very low read depth (mean, 21.59; median, 25), which revealed false-negative results using auto-filtering. We optimized our customized panel by extending the length of oligonucleotide baits and increasing the number of baits spanning the coverage of the TERT promoter, which did not amplify well due to the high GC content.
Conclusion
Our study confirmed that it is crucial to consider the recognition of molecular bias and to carefully interpret NGS data.

Keyword

Next-generation sequencing; Glioblastoma; GC-rich sequence

Figure

  • Fig. 1 The result of integrative genomics viewer (IGV). Cases 3 (A) and 8 (B) reveal telomerase reverse transcriptase (TERT) promoter mutation in Chr5: 1,295,228 (C228T). Cases 2 (C) and 10 (D) reveal TERT promoter mutation in Chr5: 1,295,250 (C250T).

  • Fig. 2 The comparison of total read depth between telomerase reverse transcriptase (TERT) promoter region and other region. The total read depth of TERT promoter region is significantly lower than that of other region (paired t test, p < 0.001).

  • Fig. 3 Optimization of sequence of baits around the telomerase reverse transcriptase (TERT) promoter area. After optimization, the number of baits are increased and the length of oligonucleotide sequence of baits are elongated.

  • Fig. 4 The mean total read depth of two hotspots (A, Chr 5:1,295,228; B, Chr 5:1,295,250) of telomerase reverse transcriptase (TERT) promoter area before and after optimization of baits. After optimization, the mean total read depth of two hotspots are significantly increased (Mann-Whitney U test, p < 0.001).

  • Fig. 5 Genetic and histological landscape of glioblastomas. We excluded variants which had been reported as benign or likely benign in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/). CDK4, cyclin-dependent kinase 4; CDKN2A/B, cyclin-dependent kinase inhibitor 2A/B; EGFR, epidermal growth factor receptor; FGFR3, fibroblast growth factor receptor 3; IDH1/2, isocitrate dehydrogenase 1/2; MGMT, O-6-methylguanine-DNA-methyltransferase; NF1, neurofibromin 1; NGS, next-generation sequencing; PDGFRA, platelet-derived growth factor receptor A; PI3K, phosphoinositide 3-kinase; RB, retinoblastoma; RTK, receptor tyrosine kinase; TERT, telomerase reverse transcriptase.


Reference

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