一种用于一步检测脑胶质瘤中 12 种 IDH1/2 突变的新敏感 PCR 检测方法。

A new sensitive PCR assay for one-step detection of 12 IDH1/2 mutations in glioma.

机构信息

QIAGEN Marseille, Av, de Luminy, Marseille, France.

出版信息

Acta Neuropathol Commun. 2014 Jun 2;2:58. doi: 10.1186/2051-5960-2-58.

DOI:10.1186/2051-5960-2-58

PMID:24889502

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4229941/

Abstract

INTRODUCTION

Mutations in isocitrate dehydrogenase genes IDH1 or IDH2 are frequent in glioma, and IDH mutation status is a strong diagnostic and prognostic marker. Current IDH mutation screening is performed with an immunohistochemistry (IHC) assay specific for IDH1 R132H, the most common mutation. Sequencing is recommended as a second-step test for IHC-negative or -equivocal cases. We developed and validated a new real-time quantitative polymerase chain reaction (PCR) assay for single-step detection of IDH1 R132H and 11 rare IDH1/2 mutations in formalin-fixed paraffin-embedded (FFPE) glioma samples. Performance of the IDH1/2 PCR assay was compared to IHC and Sanger sequencing.

RESULTS

The IDH1/2 PCR assay combines PCR clamping for detection of 7 IDH1 and 5 IDH2 mutations, and Amplification Refractory Mutation System technology for specific identification of the 3 most common mutations (IDH1 R132H, IDH1 R132C, IDH2 R172K). Analytical sensitivity of the PCR assay for mutation detection was <5% for 11/12 mutations (mean: 3.3%), and sensitivity for mutation identification was very high (0.8% for IDH1 R132H; 1.2% for IDH1 R132C; 0.6% for IDH2 R172K). Assay performance was further validated on 171 clinical glioma FFPE samples; of these, 147 samples met the selection criteria and 146 DNA samples were successfully extracted. IDH1/2 status was successfully obtained in 91% of cases. All but one positive IDH1 R132H-IHC cases were concordantly detected by PCR and 3 were not detected by sequencing. Among the IHC-negative cases (n = 72), PCR detected 12 additional rare mutations (10 IDH1, 2 IDH2). All mutations detected by sequencing (n = 67) were concordantly detected by PCR and 5/66 sequencing-negative cases were PCR-positive (overall concordance: 96%). Analysis of synthetic samples representative of the 11 rare IDH1/2 mutations detected by the assay produced 100% correct results.

CONCLUSIONS

The new IDH1/2 PCR assay has a high technical success rate and is more sensitive than Sanger sequencing. Positive concordance was 98% with IHC for IDH1 R132H detection and 100% with sequencing. The PCR assay can reliably be performed on FFPE samples and has a faster turnaround time than current IDH mutation detection algorithms. The assay should facilitate implementation of a comprehensive IDH1/2 testing protocol in routine clinical practice.

摘要

简介

异柠檬酸脱氢酶基因 IDH1 或 IDH2 的突变在神经胶质瘤中很常见，IDH 突变状态是一个强有力的诊断和预后标志物。目前的 IDH 突变筛选是使用针对 IDH1 R132H 的免疫组织化学（IHC）检测进行的，IDH1 R132H 是最常见的突变。对于 IHC 阴性或可疑的病例，建议进行测序作为第二步检测。我们开发并验证了一种新的实时定量聚合酶链反应（PCR）检测方法，用于在福尔马林固定石蜡包埋（FFPE）神经胶质瘤样本中一步检测 IDH1 R132H 和 11 种罕见的 IDH1/2 突变。IDH1/2 PCR 检测方法的性能与 IHC 和 Sanger 测序进行了比较。

结果

IDH1/2 PCR 检测方法结合了 PCR 夹心法检测 7 种 IDH1 和 5 种 IDH2 突变，以及扩增受阻突变系统技术特异性鉴定 3 种最常见的突变（IDH1 R132H、IDH1 R132C 和 IDH2 R172K）。PCR 检测方法对 12 种突变（平均：3.3%）的突变检测分析灵敏度<5%，对突变鉴定的灵敏度非常高（IDH1 R132H 为 0.8%；IDH1 R132C 为 1.2%；IDH2 R172K 为 0.6%）。该检测方法在 171 例临床神经胶质瘤 FFPE 样本中进一步得到验证；其中，147 例样本符合选择标准，146 例 DNA 样本成功提取。91%的病例成功获得了 IDH1/2 状态。除了一个 IDH1 R132H-IHC 阳性病例外，所有病例均通过 PCR 检测到，并通过测序检测到 3 例未检测到。在 IHC 阴性病例（n=72）中，PCR 检测到 12 种额外的罕见突变（10 种 IDH1，2 种 IDH2）。通过测序检测到的所有突变（n=67）均通过 PCR 检测到，66 例测序阴性病例中有 5 例 PCR 阳性（总一致性：96%）。对该检测方法检测到的 11 种罕见 IDH1/2 突变的代表性合成样本进行分析，结果均为 100%正确。

结论

新的 IDH1/2 PCR 检测方法具有较高的技术成功率，比 Sanger 测序更敏感。IDH1 R132H 的检测阳性一致性为 98%，与 IHC 检测为 100%，与测序检测为 100%。PCR 检测方法可可靠地在 FFPE 样本上进行，且比当前 IDH 突变检测算法具有更快的周转时间。该检测方法应有助于在常规临床实践中实施全面的 IDH1/2 检测方案。