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Abstract：
BACKGROUND: The World Health Organization (WHO) classification of central nervous system (CNS) tumors necessitates testing of isocitrate dehydrogenase (IDH) 1/2 gene mutation in patients with adult-type diffuse glioma (ADG) for better disease management. In clinical practice, the testing of IDH1 is primarily achieved using immunohistochemistry (IHC) specific to IDH1-R132, which carries a sensitivity of 80% and specificity of 100%. However, in some cases, non-specific background staining or regional heterogeneity in the protein expression of IDH1 may necessitate confirmatory genetic analysis. Robust and reliable assays are needed for IDH1/2 mutation testing. The aim of the current study was to detect IDH1 mutation in cfDNA and tissue of adult-type diffuse glioma with allele-specific qPCR.
METHODS: In the current study, IDH1-R132H mutation was analyzed in tumor tissue with paired cell-free DNA (cfDNA) in patients with ADG (n = 45) using IHC and competitive allele-specific Taqman PCR (CAST-PCR). Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue and matched serum for cfDNA using commercially available kits. CAST-PCR with IHC for the detection of IDH1-R132H mutation was also compared.
RESULTS: The IDH1-R132H mutation was detected in 46.67% (21/45) cases and 57.78% (26/45) cases using IHC and allele-specific CAST-PCR. In cfDNA of matched IDH1-mutant FFPE tissue DNA, IDH1-R132H mutation was detected in 11.54% (3/26) using CAST-PCR. The concordance rate for IDH1-R132Hmutation between IHC and CAST-PCR was 80.77% (21/26).
CONCLUSIONS: The CAST-PCR assay is more precise and sensitive for IDH1-R132Hdetection than traditional IHC, and IDH1-R132H mutation detection using cfDNA may add to the current methods of glioma genomic characterization.
METHODS: In the current study, IDH1-R132H mutation was analyzed in tumor tissue with paired cell-free DNA (cfDNA) in patients with ADG (n = 45) using IHC and competitive allele-specific Taqman PCR (CAST-PCR). Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue and matched serum for cfDNA using commercially available kits. CAST-PCR with IHC for the detection of IDH1-R132H mutation was also compared.
RESULTS: The IDH1-R132H mutation was detected in 46.67% (21/45) cases and 57.78% (26/45) cases using IHC and allele-specific CAST-PCR. In cfDNA of matched IDH1-mutant FFPE tissue DNA, IDH1-R132H mutation was detected in 11.54% (3/26) using CAST-PCR. The concordance rate for IDH1-R132Hmutation between IHC and CAST-PCR was 80.77% (21/26).
CONCLUSIONS: The CAST-PCR assay is more precise and sensitive for IDH1-R132Hdetection than traditional IHC, and IDH1-R132H mutation detection using cfDNA may add to the current methods of glioma genomic characterization.
摘要：
背景：世界卫生组织（WHO）对中枢神经系统（CNS）肿瘤的分类要求对成人型弥漫性神经胶质瘤（ADG）患者进行异柠檬酸脱氢酶（IDH）1/2基因突变检测，以更好地管理疾病。在临床实践中,IDH1的检测主要是使用IDH1-R132特异性的免疫组织化学(IHC)进行的,其敏感性为80%,特异性为100%.然而,在某些情况下,IDH1蛋白表达的非特异性背景染色或区域异质性可能需要进行确证性遗传分析.IDH1/2突变测试需要稳健和可靠的测定。本研究的目的是通过等位基因特异性qPCR检测成年型弥漫性神经胶质瘤的cfDNA和组织中的IDH1突变。
方法：在目前的研究中，使用IHC和竞争性等位基因特异性TaqmanPCR（CAST-PCR）分析ADG患者（n=45）中具有配对无细胞DNA（cfDNA）的肿瘤组织中的IDH1-R132H突变。使用可商购的试剂盒从福尔马林固定的石蜡包埋(FFPE)组织中提取基因组DNA并匹配cfDNA的血清。还比较了用IHC检测IDH1-R132H突变的CAST-PCR。
结果：使用IHC和等位基因特异性CAST-PCR，在46.67％（21/45）例和57.78％（26/45）例中检测到IDH1-R132H突变。在匹配的IDH1突变体FFPE组织DNA的cfDNA中，使用CAST-PCR检测到IDH1-R132H突变11.54%(3/26)。IHC与CAST-PCR的IDH1-R132H突变符合率为80.77%(21/26)。
结论：与传统的IHC相比，CAST-PCR对IDH1-R132H检测更精确和灵敏，使用cfDNA检测IDH1-R132H突变可能会增加目前的神经胶质瘤基因组表征方法。
方法：在目前的研究中，使用IHC和竞争性等位基因特异性TaqmanPCR（CAST-PCR）分析ADG患者（n=45）中具有配对无细胞DNA（cfDNA）的肿瘤组织中的IDH1-R132H突变。使用可商购的试剂盒从福尔马林固定的石蜡包埋(FFPE)组织中提取基因组DNA并匹配cfDNA的血清。还比较了用IHC检测IDH1-R132H突变的CAST-PCR。
结果：使用IHC和等位基因特异性CAST-PCR，在46.67％（21/45）例和57.78％（26/45）例中检测到IDH1-R132H突变。在匹配的IDH1突变体FFPE组织DNA的cfDNA中，使用CAST-PCR检测到IDH1-R132H突变11.54%(3/26)。IHC与CAST-PCR的IDH1-R132H突变符合率为80.77%(21/26)。
结论：与传统的IHC相比，CAST-PCR对IDH1-R132H检测更精确和灵敏，使用cfDNA检测IDH1-R132H突变可能会增加目前的神经胶质瘤基因组表征方法。
