The enzyme Telomerase maintains telomeres at the ends of chromosomes. The Telomerase Reverse Transcriptase (TERT) gene codes for the enzyme’s catalytic domain and is not expressed in normal somatic cells. As a consequence, normal cells acquire senescence by shortening of their telomeres during cell division and eventually undergo apoptosis. In contrast to normal somatic cells, expression of TERT is reinstated in cancer cells causing escape from senescence and apoptosis by maintaining the telomeres. It has recently been shown that mutations in the TERT promoter region play a key role in regulating and reinstating TERT expression. 90% of cancers carry a mutation in the TERT promoter region. Mutations like C228T and C250T create new binding site for the E26 transformation-specific (ETS) transcription factor that regulates TERT expression. Experimental evidence showed that the ETS factor GA-binding protein, alpha subunit (GABPA) binds to the de novo ETS motif and activates TERT transcription in cancer cells.

We undertook a project designing TaqMan Rare Mutation assays addressing mutations in the TERT promoter. These assays are TaqMan SNP Genotyping assays that are optimized for use in digital PCR with the Applied Biosystems QuantStudio 3D. In digital PCR, partitioning the sample into many individual reaction wells facilitates detection and quantification of rare mutant alleles. TaqMan SNP Genotyping Assays ensure reliable discrimination of mutant and wild-type allele. This will enable easy and sensitive detection of TERT promoter mutations in cancer research samples. These assays are suitable for detection in liquid biopsy applications with cell free DNA (cfDNA).

Assay design proved to be challenging due to high GC content and repetitive elements in this region of the TERT gene and required varying both the assay design and experimental cycling conditions. Template for wet-lab testing was synthetic plasmid carrying the mutation spiked into wild-type genomic DNA and wild-type genomic DNA control.

The result was a TaqMan Rare Mutation assay detecting the C228T mutation in the TERT promoter region. We showed that designing TaqMan Rare Mutation Assays for digital PCR is feasible for the challenging TERT promoter region. Work addressing additional mutations in this region is ongoing.

For Research Use Only. Not for use in diagnostic procedures.

Citation Format: Marion Laig, Kamini Varma, Vidya Venkatesh. TaqMan Rare Mutation Assays targeting the TERT promoter region [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 746. doi:10.1158/1538-7445.AM2017-746