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

Catteau et al. Acta Neuropathologica Communications 2014, 2:58 http://www.actaneurocomms.org/content/2/1/58 METHODOLOGY ARTICLE Open Access A new sensitive PCR assay for one-step detection of 12 IDH1/2 mutations in glioma Aurélie Catteau1, Hélène Girardi1, Florence Monville1, Cécile Poggionovo1, Sabrina Carpentier1, Véronique Frayssinet1, Jesse Voss3, Robert Jenkins3, Blandine Boisselier2, Karima Mokhtari2, Marc Sanson2, Hélène Peyro-Saint-Paul1* and Caterina Giannini3 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. Keywords: Glioma, IDH1/2, Quantitative real-time PCR Introduction Histopathological evaluation remains the gold standard for glioma classification [1] but the incorporation of emergent molecular biomarkers has been shown to improve diagnosis and prognosis of this heterogeneous disease. In addition to the established 1p/19q co-deletion and MGMT methylation, new biomarkers including IDH1/2, EGFR or BRAF mutations and FGFR gene fusions, are increasingly * Correspondence: 1 QIAGEN Marseille, Av. de Luminy, Marseille, France Full list of author information is available at the end of the article documented to play a role as diagnostic, prognostic or predictive markers, and should progressively be introduced in the diagnostic and treatment decision algorithm for glioma [2,3]. IDH1/2 mutations are highly frequent (up to 80%) in diffuse glioma [4-6]. Their identification in surgical neuropathology samples increases diagnostic accuracy of World Health Organization (WHO) grade II or III astrocytoma, oligodendroglioma, oligoastrocytoma, and WHO grade IV secondary glioblastoma (GBM) [5,7]. In addition, IDH mutations have been repeatedly shown to be associated with © 2014 Catteau et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Catteau et al. Acta Neuropathologica Communications 2014, 2:58 http://www.actaneurocomms.org/content/2/1/58 significantly better patient survival, thereby providing valuable prognostic information [8-12]. Based on these findings, IDH1 mutation status is becoming part of the standard diagnostic assessment of these tumors and will likely be included in the next WHO classification of diffuse gliomas [2]. In addition, IDH mutation status may predict benefit from alkylating agent when combined with MGMT promoter methylation assessment [13]. Very recently, updated data from the RTOG 9402 trial showed that the IDH mutation predicts the benefit of adjuvant chemotherapy in grade III glioma, even in absence of 1p19q co-deletion [14]. Recent data suggested that IDH1 mutation may also serve as a predictive marker to guide aggressive surgical resection of malignant astrocytomas [15]. Given the high IDH clinical relevance, stratification according to IDH mutation should be taken into account for more effective future clinical trials [2,16]. In addition, the discovery of IDH mutations has led to the development of novel therapies targeted against IDH alterations, using selective IDH inhibitors or by reversion of mutated-IDH induced hypermethylation with the use of DNA methyltransferase inhibitors [17-19]. IDH1 R132H mutation represents the most common IDH mutation (approximately 90%). Less common are the IDH mutations within the same IDH1 codon 132 (around 7%), and in the homologous IDH2 codon 172 (approximately 3%) [20]. An algorithm for IDH mutation screening has recently been proposed [21]. It is a two-step process implying initial search for the most common IDH1 R132H mutation using immunohistochemistry (IHC)-based assay, followed by DNA-based analysis on IHC-negative or -equivocal cases. The IDH1 R132H mutation-specific antibody shows high sensitivity and specificity [7,22], but the IHC technique can be problematic in some cases, as a result of background staining or regional heterogeneity of IDH1 R132H protein expression [23]. Regarding DNA-based analyses, Sanger sequencing still represents the gold standard for the identification of somatic mutations. However, sequencing sensitivity is low (around 15-20% mutation load) and this may lead to false negative results when analyzing tumor specimens with insufficient neoplastic cells in a background of normal cells. Moreover, this technology is also not readily available in all neuro (...truncated)