TERTmonitor-qPCR Detection of TERTp Mutations in Glioma

Abstract

Telomerase promoter (TERTp) mutations are frequently observed in various types of tumours and commonly characterised by two specific hotspots located at positions -124 and -146 upstream of the start codon. They enhance TERTp activity, resulting in increased TERT expression. In central nervous system (CNS) tumours, they are integrated as biomarkers, aiding in the diagnosis and with a role in prognosis, where, in some settings, they are associated with aggressive behaviour. In this study, we evaluated the performance of TERTmonitor for TERTp genotyping in a series of 185 gliomas in comparison to the traditional method, Sanger sequencing. Against the gold-standard Sanger method, TERTmonitor performed with a 97.8% accuracy. Inaccuracy was mainly due to the over-detection of variants in negative cases (by Sanger) and the presence of variants that can modify the chemistry of the probe detection. The distribution of the mutations was comparable to other series, with the -124 being the most represented (38.92% for Sanger and TERTmonitor) and more prevalent in the higher-grade tumours, gliosarcoma (50.00%) and glioblastoma (52.6%). The non-matched cases are debatable, as we may be dealing with the reduced sensitivity of Sanger in detecting rare alleles, which strengthens the use of the TERTmonitor. With this study, we present a reliable and rapid potential tool for TERTp genotyping in gliomas.

Keywords: CNS tumours; TERTmonitor; genotyping; gliomas; qPCR; telomerase promoter.

Figure 1

Comparison of Sanger sequencing and TERTmonitor—frequencies and type of TERTp mutations in the different histological subtypes of the gliomas. § detection of two additional mutated cases (−124 G>A and −146 G>A, respectively) and failure to detect one mutation (−124 G>A) in a case with TERTmonitor in diffuse astrocytoma; §§ detection of an additional mutated case (−146 G>A) of glioblastoma.

Figure 2

Sequencing chromatograms and qPCR multicomponent plots of glioma mutated cases with non-concordant results by Sanger method and TERTmonitor. (A) A non-concordant diffuse astrocytoma case with a hotspot −124 G>A heterozygous mutation detected in the sequencing chromatogram, as well as a rare variant, the −104 A>G (A, top). The corresponding TERTmonitor qPCR multicomponent plot (A, bottom left) for the −124 position fails to detect the −124 G>A mutation ((A) bottom left plots (blue, FAM)). An assay poorer performance is also perceivable in the −124 wt plots (bottom left, green (HEX)) where the late amplification of the wt sequences may be indicative of probe interference; and (B) a glioblastoma case with a −146 G>A mutation detected by TERTmonitor that is not present in the Sanger sequencing chromatograms. An additional alteration is present in the sequencing chromatogram, a −132 G>A rare variant in homozygosity (B, top chromatogram) that generates a novel motif in the opposite strand in reverse complement (CCGGCT) and partially complementary to the well-described TERTp mutations motifs. The bottom component plots (B) correspond to two independent runs for the −146-hotspot evaluation; for each run, in one of the replicates there is false detection of the −146 mutation (mut, blue, FAM), and late (or failed) amplifications in both plots, and in the different runs, indicative of probe interference.

Figure 3

Sequencing chromatograms and qPCR multicomponent plots of mutated diffuse astrocytoma cases only detected by TERTmonitor. (A) A diffuse astrocytoma with a −124 G>A mutation not detected by Sanger sequencing (A, left) and the corresponding two independent qPCR TERTmonitor runs (with 2 replicates) (A, middle and right); whereas in both analysis the assays for the −124 mutation were not concordant for the mutation detection; (B) a −146 G>A diffuse astrocytoma mutated case detected by TERTmonitor that after re-evaluation of sequencing chromatogram, a small peak at the −146 hotspot was considered (initially considered an artifact) (B, left); in one of the qPCR TERTmonitor runs, the presence of the mutation was more clear (B, right). The initial qPCR run failed to detect the mutation (B, middle).