Department of Pathology, Nantes University Hospital, Nantes, France.
Int J Oncol. 2012 Jun;40(6):2058-62. doi: 10.3892/ijo.2012.1404. Epub 2012 Mar 16.
Previous studies have identified mutations of the isocitrate dehydrogenase 1 (IDH1) gene in more than 70% of World Health Organization (WHO) grade II and III gliomas. The most frequent mutation leads to a specific amino acid change from arginine to histidine at codon 132 (c.395G>A, p.R132H). IDH1 mutated tumors have a better prognosis than IDH1 non-mutated tumors. The aim of our study was to evaluate and compare the methods of mIDH1 R132H immunohistochemistry, allele-specific PCR and DNA sequencing for determination of IDH1 status. We performed a retrospective study of 91 patients with WHO grade II (n=43) and III (n=48) oligodendrogliomas. A fragment of exon 4 spanning the sequence encoding the catalytic domain of IDH1, including codon 132, was amplified and sequenced using standard conditions. Allele-specific amplification was performed using two forward primers with variations in their 3' nucleotides such that each was specific for the wild-type or the mutated variant, and one reverse primer. Immunohistochemistry was performed with mouse monoclonal mIDH1 R132H. DNA was extracted from FFPE sections following macrodissection. IDH1 mutations were found in 55/90 patients (61.1%) by direct sequencing. R132H mutations were found in 47/55 patients (85.4%). The results of the allele-specific PCR positively correlated with those from DNA sequencing. Other mutations (p.R132C, p.R132S and pR132G) were found by DNA sequencing in 3, 3 and 2 tumors, respectively (8/55 patients, 14.6%). mIDH1 R132H immunostaining was found in the 47 patients presenting the R132H mutation (sensitivity 47/47, 100% for this mutation). None of the tumors presenting a wild-type IDH1 gene were stained (specificity 35/35, 100%). Our results demonstrate that immunohistochemistry using the mIDH1 R132H antibody and allele-specific amplification are highly sensitive techniques to detect the most frequent mutation of the IDH1 gene.
先前的研究已经确定，在超过 70%的世界卫生组织（WHO）二级和三级胶质瘤中存在异柠檬酸脱氢酶 1（IDH1）基因突变。最常见的突变导致密码子 132（c.395G>A，p.R132H）处的精氨酸到组氨酸的特定氨基酸变化。IDH1 突变型肿瘤比 IDH1 非突变型肿瘤预后更好。我们的研究旨在评估和比较 mIDH1 R132H 免疫组化、等位基因特异性 PCR 和 DNA 测序用于确定 IDH1 状态的方法。我们对 91 例 WHO 二级（n=43）和三级（n=48）少突胶质细胞瘤患者进行了回顾性研究。使用标准条件扩增并测序包含 IDH1 催化结构域编码序列的外显子 4 的片段，包括密码子 132。使用两个正向引物进行等位基因特异性扩增，这两个引物的 3' 核苷酸存在差异，使其分别特异性针对野生型或突变型变体和一个反向引物。使用鼠单克隆 mIDH1 R132H 进行免疫组化。通过宏观切割从 FFPE 切片中提取 DNA。直接测序发现 90 例患者中的 55 例（61.1%）存在 IDH1 突变。在 55 例患者中的 47 例（85.4%）发现 R132H 突变。等位基因特异性 PCR 的结果与 DNA 测序的结果呈正相关。在另外 3、3 和 2 例肿瘤中分别通过 DNA 测序发现其他突变（p.R132C、p.R132S 和 pR132G）（8/55 例患者，14.6%）。在存在 R132H 突变的 47 例患者中发现 mIDH1 R132H 免疫染色（这种突变的敏感性为 47/47，100%）。未发现存在野生型 IDH1 基因的肿瘤染色（特异性 35/35，100%）。我们的结果表明，使用 mIDH1 R132H 抗体和等位基因特异性扩增的免疫组化是检测 IDH1 基因最常见突变的高度敏感技术。
