Development of Novel Mutation-Specific Droplet Digital PCR Assays Detecting TERT Promoter Mutations in Tumor and Plasma Samples.

Development of Novel Mutation-Specific Droplet Digital PCR Assays Detecting TERT Promoter Mutations in Tumor and Plasma Samples.

Publication date: Mar 01, 2019

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 DNA was also identified in 21 of 27 plasma samples (78%) from patients with TERT 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 DNA in tumors and plasma of cancer patients.

Corless, B.C., Chang, G.A., Cooper, S., Syeda, M.M., Shao, Y., , Osman, Karlin-Neumann, G., and Polsky, D. Development of Novel Mutation-Specific Droplet Digital PCR Assays Detecting TERT Promoter Mutations in Tumor and Plasma Samples. 21854. 2019 J Mol Diagn (21):2.

Concepts Keywords
Allele COLD-PCR
Cancer Melanoma
Droplet Assay
Formalin Cancer
Melanoma Biotechnology
Mutant Molecular biology
Mutation Polymerase chain reaction
Orthogonal Laboratory techniques
Paraffin Physical sciences
Plasma Branches of biology
Sequencing Natural sciences
TERT Cancer
Tumor Tumor plasma melanoma
TERT tumors

Semantics

Type Source Name
drug DRUGBANK Formaldehyde
pathway BSID Melanoma
disease DOID melanoma
disease MESH melanoma
disease MESH multiple
disease DOID cancer
disease MESH Tumor
gene UNIPROT TERT
disease MESH Development

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