Department of Hepatology, Toranomon Hospital and Okinaka Memorial Institute for Medical Research, Tokyo, Japan.
Liver Research Laboratory, Toranomon Hospital, Tokyo, Japan.
J Med Virol. 2020 Dec;92(12):3604-3608. doi: 10.1002/jmv.25724. Epub 2020 Mar 1.
Telomerase reverse transcriptase (TERT) promoter mutation is the most frequent genetic alteration in hepatocellular carcinoma (HCC). However, there is currently no suitable highly sensitive method that can detect such mutation using serum cell-free DNA (cfDNA). We analyzed somatic point mutations that substitute cytosine for thymidine at position 228 (C228T), as one of the hotspots of TERT promoter mutations, in serum cfDNA using a highly sensitive detection method of wild-type blocking polymerase chain reaction (WTB-PCR) combined with Sanger sequencing. In TERT promoter mutation sensitivity study, synthetic oligonucleotides were prepared to determine the lowest detection limit of the WTB-PCR, using serial dilutions of mutant-type (MT) DNA in the background of wild-type (WT) DNA. Using this technique, we conducted a longitudinal study in one patient who developed HCC during the follow-up and determined the relationship between HCC and TERT C228T in serum cfDNA. In the sensitivity study, the mutant peak at position 228 was detected at 0.7% or higher but was not detected at 0.6%. Thus, sequencing analysis of WTB-PCR product demonstrated the limit of detection in excess of 0.7% MT DNA in the background of WT DNA. One male patient with HCV-related cirrhosis developed HCC during the follow-up. TERT C228T was negative before the diagnosis of HCC, positive at the diagnosis of HCC and did not increase with advancement of malignancy. We developed a highly sensitive method for detection of TERT promoter mutation using WTB-PCR combined with Sanger sequencing and demonstrated its clinical usefulness in the measurement of TERT C228T in serum cfDNA. Larger studies are needed to confirm these results and establish the clinical utility of this new method.
端粒酶逆转录酶 (TERT) 启动子突变是肝细胞癌 (HCC) 中最常见的遗传改变。然而，目前尚无合适的高度敏感方法可以使用血清无细胞 DNA (cfDNA) 检测这种突变。我们使用高度敏感的野生型阻断聚合酶链反应 (WTB-PCR) 联合 Sanger 测序检测血清 cfDNA 中 TERT 启动子突变热点之一的胞嘧啶替换胸腺嘧啶 228 位 (C228T) 的体细胞点突变。在 TERT 启动子突变敏感性研究中，我们使用 MT DNA 在 WT DNA 背景下的系列稀释液制备合成寡核苷酸，以确定 WTB-PCR 的最低检测限。使用该技术，我们对一名在随访期间发生 HCC 的患者进行了纵向研究，并确定了 HCC 和血清 cfDNA 中 TERT C228T 之间的关系。在敏感性研究中，在 0.7%或更高的位置可以检测到 228 位的突变峰，但在 0.6%时无法检测到。因此，WTB-PCR 产物的测序分析表明 WT DNA 背景下的 MT DNA 检测限超过 0.7%。一名患有 HCV 相关肝硬化的男性患者在随访期间发生 HCC。在 HCC 诊断之前，TERT C228T 为阴性，在 HCC 诊断时为阳性，并且随着恶性程度的增加而没有增加。我们开发了一种使用 WTB-PCR 联合 Sanger 测序检测 TERT 启动子突变的高度敏感方法，并在测量血清 cfDNA 中的 TERT C228T 方面证明了其临床实用性。需要更大的研究来证实这些结果并确立这种新方法的临床实用性。
