1. Development of Novel Mutation-Specific Droplet Digital PCR Assays Detecting TERT Promoter Mutations in Tumor and Plasma Samples
- Author
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Broderick Corless, David Polsky, Yongzhao Shao, Iman Osman, Mahrukh M. Syeda, Gregory Chang, Samantha Cooper, and George Karlin-Neumann
- Subjects
0301 basic medicine ,Mutant ,medicine.disease_cause ,Polymerase Chain Reaction ,Article ,Pathology and Forensic Medicine ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,medicine ,Humans ,Digital polymerase chain reaction ,Promoter Regions, Genetic ,Telomerase ,Mutation ,Plasma samples ,Melanoma ,Cancer ,medicine.disease ,Molecular biology ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,Molecular Medicine ,GC-content ,DNA - Abstract
Detecting mutations in the plasma of patients with solid tumors is becoming a valuable method of diagnosing and monitoring cancer. The TERT promoter is mutated at high frequencies in multiple cancer types, most commonly at positions -124 and -146 (designated C228T and C250T, respectively). Detection of these mutations has been challenging because of the high GC content of this region (approximately 80%). We describe development of novel probe-based droplet digital PCR assays that specifically detect and quantify these two mutations, along with the less common 242-243 CC>TT mutation, and demonstrate their application using human tumor and plasma samples from melanoma patients. Assay designs and running conditions were optimized using cancer cell line genomic DNAs with the C228T or C250T mutations. The limits of detection were 0.062% and 0.051% mutant allele fraction for the C228T and C250T assays, respectively. Concordance of 100% was observed between droplet digital PCR and sequencing-based orthogonal methods in the detection of TERT mutant DNA in 32 formalin-fixed, paraffin-embedded melanoma tumors. TERT(mutant) DNA was also identified in 21 of 27 plasma samples (78%) from patients with TERT(mutant) tumors, with plasma mutant allele fractions ranging from 0.06% to 15.3%. There were no false positives in plasma. These data demonstrate the potential of these assays to specifically detect and quantify TERT(mutant) DNA in tumors and plasma of cancer patients.
- Published
- 2019