Analysis of TERT promoter hotspot mutations using droplet digital PCR in hepatoblastoma and hepatocellular carcinoma.

Somatic mutations in the telomerase reverse transcriptase promoter (TERTp) region are common in many cancers, including in liver cancers. Detection of TERTp mutations in tumor tissue DNAs and cell-free tumor DNAs is useful for diagnosing and monitoring cancers. Since the most common TERTp hotspot mutations, C228T and C250T, are difficult to identify using Sanger sequencing, we tested an easy and highly sensitive alternative method that targets these two sites using droplet digital PCR. Using this method, both the sensitivity and specificity for detecting these two mutations were 100 % in DNA samples derived from cell lines and liver cancer tissues, including hepatocellular carcinoma (HCC) and hepatoblastoma (HB). The detection limit for the allele frequencies of these mutations was approximately 0.1 %. This method is also widely applicable; for instance, it can be applied to DNA derived from FFPE (formalin-fixed paraffin embedded) samples. In addition, we applied this method to detecting TERTp mutations in cell-free DNA samples of patients with TERTp-mutated tumors. Finally, we found that outcomes for HB patients with TERTp mutations were significantly worse than in those without mutations, indicating the importance of this method for improving patient outcomes. In light of this, we discuss the advantages of this method for clinical implementation in the detection and monitoring of cancers.