Two hot spot mutations (C228T, C250T) in the telomerase reverse transcriptase (TERT) gene are frequently identified in glioblastoma and oligodendroglioma. TERT mutations predicts an aggressive clinical course in isocitrate dehydrogenase (IDH) wild-type astrocytic tumors. Therefore, it is important to accurately detect TERT promoter mutations in glioma. Sanger DNA sequencing is the currently standard method for analyzing TERT mutations. However, PCR amplification in the first step of the sequencing has proven technically difficult because of the high GC content around the TERT mutation. In this report, we described a novel droplet digital PCR (ddPCR) assay to evaluate TERT hot spot mutations in fresh frozen and formalin-fixed paraffin-embedded (FFPE) specimens of glioma and verified the difference in results from the Sanger DNA sequencing results. We obtained the mutant allele fraction for TERT mutations of in a single ddPCR run in all cases, including the micro-dissected FFPE sections. On the contrary, up to twice the DNA sequences were required from fresh frozen tissue to obtain the results, consistent with ddPCR assay. When FFPE specimens were used, more time was required to evaluate TERT mutations through DNA sequencing. DdPCR is an effective and sensitive assay compared to the conventional standard Sanger DNA sequencing.

端粒酶逆转录酶 ( TERT ) 基因中的两个热点突变（C228T、C250T）在胶质母细胞瘤和少突胶质细胞瘤中经常被发现。TERT突变预示着异柠檬酸脱氢酶 ( IDH ) 野生型星形细胞肿瘤的侵袭性临床过程。因此，准确检测胶质瘤中的TERT启动子突变非常重要。Sanger DNA 测序是目前分析TERT突变的标准方法。然而，由于TERT突变周围的高 GC 含量，测序的第一步中的 PCR 扩增在技术上被证明是困难的。在本报告中，我们描述了一种新的液滴数字 PCR (ddPCR) 分析来评估神经胶质瘤新鲜冷冻和福尔马林固定石蜡包埋 (FFPE) 标本中的TERT热点突变，并验证了与 Sanger DNA 测序结果的差异。我们在所有情况下（包括微解剖的 FFPE 切片）都获得了单次 ddPCR 运行中TERT突变的突变等位基因分数。相反，需要从新鲜冷冻组织中提取多达两倍的 DNA 序列才能获得结果，这与 ddPCR 分析一致。当使用 FFPE 标本时，需要更多时间通过 DNA 测序评估TERT突变。与传统的标准 Sanger DNA 测序相比，DdPCR 是一种有效且灵敏的检测方法。