Abstract
Simple SummaryActivating mutations in the promoter region of TERT (TERTp) gene are frequently observed in low- and high-grade dysplastic nodules and defined as early events in hepatocellular carcinoma development. This study shows that the nucleotide change G>A at position −124 in the TERTp region is very common in hepatocellular carcinoma. The concordance rate between droplet digital PCR (ddPCR) (63.6%) and Sanger sequencing (52.1%) detection methods is good (83.5%). HCC patients carrying the TERTp mutation had lower levels of the tumour biomarker Ca19-9 but showed reduced survival. The presence of TERTp mutations may represent a prognostic signature in liver cancer.Telomerase reactivation during hepatocarcinogenesis is recurrently caused by two point mutations occurring most frequently at the nucleotide −124 (95%) and occasionally at the nucleotide −146 (<5%) upstream of the TERT translational start site in hepatocellular carcinoma (HCC). In this study, we designed a droplet digital PCR (ddPCR) assay to detect TERT promoter (TERTp) nucleotide change G>A at position −124 and to quantify the mutant allele frequency (MAF) in 121 primary liver cancers, including 114 HCC along with 23 autologous cirrhotic tissues, five cholangiocarcinoma (CC), and two hepato-cholangiocarcinoma (HCC-CC). All cases were evaluated for tumour markers such as α-fetoprotein (AFP), carbohydrate antigen 19-9 (CA19-9), and carcinoembryonic antigen (CEA). We compared the sensitivity of ddPCR and Sanger sequencing and investigated the prognostic relevance of TERTp mutations. The TERTp G>A transition was identified in 63.6% and 52.1% of HCC samples by ddPCR and Sanger sequencing, respectively. One out of 23 (4.3%) peri-tumour tissues tested positive only by ddPCR. One out of five CC (20%) and none of the HCC-CC were found concordantly mutated by the two methods. The TERTp MAF ranged from 2% to 66%, and the large majority (85.5%) of mutated samples showed a value above 20%. A statistically significant correlation was found between TERTp mutation and tumour size (p = 0.048), while an inverse correlation was observed with CA19-9 levels (p = 0.0105). Moreover, HCC patients with TERTp −124A had reduced survival. In conclusion, the single nucleotide variation G>A at position −124 in TERTp, detected either by ddPCR or by Sanger sequencing, showed a remarkable high frequency in HCC. Such mutation is associated with lower levels of CA19-9 and reduced survival in HCC patients suggesting that the TERTp status may represent a distinct signature of liver cancer subgroups.
Highlights
Primary liver cancer with a global burden of 905,677 new cases and 830,180 deaths in is the second and the sixth leading cause of cancer mortality in men and in women, respectively [1]
We developed an in-house-designed assay to identify and quantify the nucleotide variation G>A at position −124 upstream the TERT gene ATG start site by droplet digital PCR (ddPCR)
The sensitivity of the assay was measured by performing serial dilutions of TERT promoter (TERTp) mutated genomic DNA into wildtype genomic DNA, extracted from liver control tissue that allowed us to set the limit of detection (LOD) to 0.2% mutant allele frequency (MAF)
Summary
Primary liver cancer with a global burden of 905,677 new cases and 830,180 deaths in is the second and the sixth leading cause of cancer mortality in men and in women, respectively [1]. Inactivation of the TP53-RB pathway observed in approximately 30% of HBV-related HCC is a common event due to frequent mutations in TP53 gene as well as in ATM and RPS6KA3, encoding p53-activating kinases [17,18,19]. Cancer-specific nucleotide variations in the promoter region of TERT gene, first identified by Horn et al (2013) and Huang et al (2013), were shown to occur more frequently than any other observed somatic mutation in several cancer types including HCC [21,22,23]. End-point PCR and Sanger sequencing are defined as gold standard for the detection of mutations in target DNA regions, this method is not quantitative and has low sensitivity for the identification of rare mutants [31]. Pathological parameters of liver cancer patients included in this study
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