Pyrosequencing of IDH1 and IDH2 Mutations in Brain Tumors and Non-neoplastic Conditions : Diagnostic Molecular Pathology

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00019606-201212000-00003ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2012 Lippincott Williams & Wilkins, Inc.21December 2012 p 214–220Pyrosequencing of IDH1 and IDH2 Mutations in Brain Tumors and Non-neoplastic ConditionsOriginal ArticlesCykowski, Matthew D. MD; Allen, Richard A. MS; Fung, Kar-Ming MD, PhD; Harmon, Michael A. BS; Dunn, Samuel T. PhDDepartment of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OklahomaThe authors declare no conflict of interest.Reprints: Samuel T. Dunn, PhD, Department of Pathology, The University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., BMSB 451, Oklahoma City, Oklahoma 73104 (e-mail: [email protected]).AbstractThe molecular profiling of brain tumors, including testing for MGMT promoter methylation and chromosome 1p/19q deletion, can provide both diagnostic and prognostic information that may guide treatment. Isocitrate dehydrogenase (IDH) mutation testing is a recent addition to this armamentarium of molecular pathology tools that similarly provides both diagnostic (eg, glioma vs. gliosis) and prognostic information. Herein, we describe a pyrosequencing-based approach to IDH1 and IDH2 mutation testing and its application to 139 neoplastic and non-neoplastic central nervous system specimens. Several technical issues encountered in the development of the assay, particularly with regard to the optimization of the sequencing reaction, are described. Mutations in IDH1 codon 132 or IDH2 codon 172 were identified in 31.2% of all screened cases and 46.2% of screened World Health Organization grade I to IV gliomas (n=93), with mutations arising exclusively in grade II to IV oligodendroglial, astrocytic, or mixed oligoastrocytic neoplasms. Examination of the relationship between the mutation status and other pertinent variables demonstrated a significant male predominance among IDH1-mutated gliomas, most notably in grade III to IV astrocytic neoplasms. A significant association between IDH1/IDH2 mutation and 1p/19q deletion was also seen (Kendall τ coefficient=0.26, P=0.018), although several cases with 1p/19q deletion were IDH1/IDH2 wild type.Isocitrate dehydrogenase 1 (IDH1) is an enzyme involved in the protection of cells against oxidizing stresses by indirectly facilitating the regeneration of the antioxidant protein glutathione.1 Recently, an unexpected role of the IDH1 gene in glioma tumorigenesis was identified in an analysis of mutations and copy number alterations in glioblastoma multiforme (GBM).2 Somatic IDH1 mutations were demonstrated in 11% of tumors with a guanine to adenine change at nucleotide position 395 of codon 132 in most cases, leading to the replacement of arginine by histidine (R132H). IDH2 codon 172 mutations were subsequently identified in IDH1 wild-type gliomas, albeit less frequently than IDH1 mutations and very rarely in GBM.3,4 Combined, however, IDH1 or IDH2 mutations are present in 90%, 84%, and 100% of World Health Organization grade II astrocytomas, oligodendrogliomas, and oligoastrocytomas, respectively.4 These mutations are not routinely seen in grade I gliomas, such as pilocytic astrocytoma, ependymoma, pituitary adenoma, and meningioma.5Several approaches to IDH1/IDH2 mutation testing are utilized in diagnostic surgical neuropathology to assist in difficult differential diagnoses such as infiltrating glioma versus reactive gliosis and to provide critical prognostic information to patients.4 Methods introduced to date include dideoxynucleotide sequencing, immunohistochemical (IHC) detection of mutant protein in formalin-fixed paraffin-embedded (FFPE) tissue, Western blot,6 melt curve analysis,7 and pyrosequencing (PS).8 Dideoxynucleotide sequencing has been the most widely used,2 offering specificity and the advantage of long-read length sequences capable of identifying less common sequence variants. However, this approach is restricted by the reduced sensitivity and lower throughput compared with other methods. The IHC method typically utilizes a mouse monoclonal antibody against IDH1R132H, producing a granular cytoplasmic staining pattern of mutant protein.9 The IHC method also allows testing of unstained slides from FFPE tissue. However, this method may suffer from technical issues,10 and more significantly, targets only the most common mutant protein (R132H).The PS approach provides a rapid turnaround time comparable to IHC, a sensitivity similar to conventional sequencing, and, in contrast to IHC, detects common and infrequent mutation types. A recent study using a PS-based assay for IDH1/IDH2 mutation detection in a series of gliomas was sensitive enough to detect IDH1 mutations even in the infiltrating margins of a tumor and demonstrated a detection limit of 5% mutant allele for the c.395G>A IDH1 mutation.8 A second study applied a PS method to pediatric gliomas, similarly identifying a lower limit of detection of 5% IDH1 R132H mutant to IDH1 wild-type alleles.11Here, we describe the validation of a PS assay for the detection of IDH1/IDH2 mutations and report the results of its application in the screening of 139 neoplastic and non-neoplastic lesions, highlighting several challenges that arose in optimizing the sequencing reaction. We also examine the relationship between IDH1/IDH2 mutations in gliomas, patient age, sex, and tumor 1p/19q chromosome deletion.MATERIALS AND METHODSCasesSources utilized to identify 162 patient samples (blocks, paraffin sections, and/or slides) included archived material (years, 1998 to 2011) and active clinical cases. Of these cases, 139 unique patient samples were identified, the large majority of which were generated by resections performed at the University of Oklahoma Health Sciences Center. The 139 samples included FFPE tissue blocks (majority), unstained hematoxylin and eosin-stained FFPE sections from cases signed-out up to 8 years before, and unstained paraffin sections previously generated for 1p/19q deletion testing by fluorescence in situ hybridization (FISH) and stored at −20°C for up to 5 years. Repeated samples were available for 20 patients, representing either multiple samples from within a neurosurgical case or repeat resections. Sample identification, block retrieval, and all subsequent testing were performed with the approval of the University of Oklahoma Health Sciences Center Institutional Review Board.Histologic CategoriesPatient tissue samples included glial (n=93), glioneuronal (n=3), and nonglial tumors (n=30), and non-neoplastic brain lesions (n=13). Tumors were graded according to the standard World Health Organization classification and included 10 grade II oligodendrogliomas, 20 grade III (anaplastic) oligodendrogliomas, 1 oligodendroglioma of uncertain grade, 10 pilocytic astrocytomas, 8 grade II diffuse astrocytomas, 2 grade II gemistocytic astrocytomas, 12 grade III astrocytomas, 14 GBMs (including 2 gliosarcomas), 1 grade II pleomorphic xanthoastrocytoma, 2 grade I subependymal giant cell astrocytomas, 3 grade II oligoastrocytomas, 1 grade III oligoastrocytoma, 1 grade I myxopapillary ependymoma, 6 grade II ependymomas, 1 grade I subependymoma, 1 grade II mixed ependymoma/subependymoma, 2 grade I ganglion cell tumors, 1 grade II/III ganglion cell tumor, 6 grade IV medulloblastomas, 1 primitive neuroectodermal tumor, 11 meningiomas (1 secretory type, 2 choroid variants, 2 atypical), 4 choroid plexus tumors (including 1 atypical choroid plexus papilloma), 1 hemangioblastoma, and 7 schwannomas. Non-neoplastic lesions in the series included 4 cases of vascular malformation, and 1 case each of corticobasal degeneration, diffuse Lewy body/Alzheimer disease, progressive supranuclear palsy, Alzheimer disease (Braak stage 5), gliotic scar, radiation necrosis, abscess, infarction, and epilepsy.Tissue Selection and DNA IsolationA neuropathologist (K.-M.F.) reviewed the histologic materials, selected representative tissue, and demarcated tumor when necessary. Five to 12 sections of 20 μm thickness were subjected in toto for DNA isolation. Sections requiring enrichment for tumor cells were cut at 5 to 10 μm, floated on a water bath, and mounted on noncharged slides for microdissection. Tissues were deparaffinized in 3 changes of xylenes (1 mL, 10 min each) followed by 3 changes of 100% ethanol (1 mL, 10 min each). Tissues were pelleted by centrifugation between incubations. DNA isolation from tissue pellets was performed utilizing the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA) as per the manufacturer’s protocol.Polymerase Chain Reaction AmplificationPortions of the IDH1 and IDH2 genes were amplified spanning codon 132 (IDH1, 75 bp amplicon) and codon 172 (IDH2, 102 bp amplicon) mutation sites, respectively. Forward primers were 5′-GCTTGTGAGTGGATGGGTAAA-3′ (IDH1) and 5′-TCCGGGAGCCCATCATCT-3′ (IDH2) and biotinylated reverse primers were 5′-TTGCCAACATGACTTACTTGATC-3′ (IDH1) and 5′-CCTGGCCTACCTGGTCGC-3′ (IDH2). Polymerase chain reaction (PCR) was performed in a 50 μL total volume containing a final concentration of 1× colorless GoTaq Flexi Buffer (Promega, Madison, WI), 2.5 mM MgCl2, 0.125 mM of each dNTP, 0.2 μM of the appropriate primers, 1.5 U of GoTaq Flexi DNA polymerase, and 50 ng of genomic DNA. Thermal cycling consisted of 45 cycles with denaturing (95°C, 10 s), annealing (53°C,10 s), and elongation (72°C, 10 s) steps, preceded by an initial denaturation step (95°C, 120 s) and succeeded by a final elongation step (72°C, 120 s).PSPS was performed utilizing a PyroMark Q24 pyrosequencer (Qiagen). Initially, 10 or 20 μL of the same PCR were added to the Sepharose bead mix [70 or 60 μL, respectively, comprised of 2 μL of Streptavidin Sepharose High Performance beads (GE Healthcare, Piscataway, NJ), 40 μL PyroMark Binding Buffer (Qiagen), and 28 or 18 μL of type I water]. From these initial experiments, it was determined that 20 µL of amplicons increased the signal strength substantially without increasing the background beyond acceptable levels.Two sequencing primers were initially used in the development of the IDH1 mutation assay. The first IDH1 primer (5′-GGGTAAAACCTATCATCATA-3′) was selected to provide sequence data that included codon 131 and the first nucleotide of codon 132. The second IDH1 primer (5′-AAAACCTATCATCATAGGTC-3′) began with the second nucleotide of codon 132 with the expectation of increased sensitivity to mutations at that position. As described below in the Results (see the Sequencing optimization section), the former primer was utilized for all sequencing reactions reported herein. The sequencing primer selected for IDH2 codon 172 analysis (5′-AGCCCATCACCATTG-3′) was utilized in all sequencing reactions after resolving an issue of aberrant height of peaks in the pyrogram (see the Sequencing optimization section).A master sepharose bead mix (60 μL/reaction, prepared as above) was added to a 24-well PCR plate per PS run. PCR products (20 µL) were added to the wells, and then shaken at room temperature for 10 minutes to bind the biotinylated amplicons to the sepharose beads. Biotinylated PCR amplicons captured on the beads were then processed through washes of 70% ethanol (30 to 40 s), 0.2 NaOH for denaturation (30 to 40 s), and PyroMark Wash Buffer to remove nonbiotinylated DNA strands (50 to 80 s) using the PyroMark Q24 Vacuum Prep Workstation. Biotinylated DNA strands were then released into the sequencing primer solution (25 µL at 1.0 µM of primer) in a PyroMark Q24 Plate. The solution was then heated at 80°C for 2 minutes, and then cooled at room temperature (5 min). PyroMark Gold reagents (enzymes, substrates, dNTPs) were used in the PS reactions.Sequence to analyze (S2A) and dispensation order (DO) using PyroMark Q24 software were determined for each sequencing primer and nucleotide position. Nucleotide positions 1 and 2 of IDH1 codon 132 were interrogated using the DO of CGTGCAGTCAT. For nucleotide position 1, the S2A was set as GGTNGTCATGCTTAT (N standing for any base and GGT being the sequence of codon 131). A second S2A of GGTCNTCATGCTTAT was used to assess nucleotide position 2. Base changes at position 3 were not specifically targeted for interrogation as these would not change the wild-type amino acid arginine. A single DO of CGTCACTGTACACG was used for IDH2 codon 172 with a S2A of GCNGGCACGCCCATGG for nucleotide position 1, GCANGCACGCCCATGG for nucleotide position 2, and GCAGNCACGCCCATGG for nucleotide position 3. This allowed mutations at any of the nucleotide positions of IDH2 codon 172 to be detected.Sensitivity and negative controls were run with all patient samples. Sensitivity controls comprised DNA from IDH1 and IDH2 mutant-positive cases diluted with DNA from wild-type cases. PCR-negative controls comprised water substituted for DNA.Sequence data were analyzed using PyroMark Q24 software. Pyrogram peak heights were reviewed with an expected average of 100 relative light units; specimens failing to achieve this standard were repeated. Sequence variations at nucleotide 2 of both IDH1 codon 132 and IDH2 codon 172 were analyzed first (the most common mutation sites). Thus, pyrograms were first examined for mutations from guanine→N (deviation from normal CGT sequence at IDH1 codon 132) and from guanine→N (deviation from normal AGG sequence at IDH2 codon 172), respectively. S2As were then changed in the software such that the first nucleotide of IDH1 codon 132 was examined for a mutation from C→N, the first nucleotide of IDH2 codon 172 for an A→N, and the third nucleotide of IDH2 codon 172 for a G→N. Thresholds of 5% and 10% were set empirically as the lower limits of detection for true-positive IDH1 and IDH2 mutations, respectively (Figs. 1A, B).JOURNAL/dimp/04.03/00019606-201212000-00003/figure1-3/v/2021-02-17T200047Z/r/image-jpegPyrograms of wild-type and mutant IDH1 samples using GGTCNTCATGCTTAT as the sequence to analyze, including codon 131 (GGT) and codon 132 (CGT). The shaded segments indicate the 4 nucleotides dispensed to analyze codon 132. The mutant sample demonstrates a c.CGT>CAT substitution with 44% mutant allele.Concordance AnalysesSix samples were sent to a CLIA-approved laboratory for IDH1/IDH2 testing using real-time high-resolution PCR with melt curve analysis and result confirmation with dideoxynucleotide sequencing. There was perfect concordance of PS results between labs (4/6 cases with IDH1 mutation, 0/6 with IDH2 mutation). One sample harboring a known IDH2 mutation detected by testing at the outside laboratory was also sent to our lab; PS within our lab demonstrated the identical mutation. No concordance analyses were performed for any of the cases in this study using the IHC approach (mouse monoclonal antibody against IDH1R132H).Statistical AnalysesA 2-tailed Student t test (unequal variances) was utilized to test for a significant difference in patient age between IDH1 mutated and wild-type glial and glioneuronal tumors (n=95), and gliomas (n=71). In the latter subset of cases, the relationship between patient sex and the IDH1 mutation status was also tested using a Yates-corrected χ2 analysis. A Kendall τ rank correlation coefficient was utilized to assess the relationship between the 1p/19q deletion status and IDH1 mutation for those cases with both sets of data available (n=41).RESULTSSequencing OptimizationTwo sequencing primers were initially utilized in the IDH1 codon 132 assay (see PS section). A comparison of background peaks between the 2 primers was performed for 71 wild-type IDH1 and 43 IDH1 codon 132 CGT>CAT mutated samples. The highest background peak amplitude (as a percentage of reference peaks) for the first IDH1 primer was 3.14% for wild-type and 0.66% for mutant IDH1 samples. For the second primer, these background peak values were 5.5% and 5.25%, respectively; the first primer was thus utilized in this study (Fig. 2). A separate issue identified in the development of the IDH2 assay was the presence of aberrant height peaks (ie, reference peaks for bases in codons 173 and 174 were taller than expected relative to other peaks in codons 171 and 172). This issue was resolved using increased concentration of the IDH2 sequencing primer (Figs. 3A, B and Discussion).JOURNAL/dimp/04.03/00019606-201212000-00003/figure2-3/v/2021-02-17T200047Z/r/image-jpegPyrogram generated using a sequencing primer designed to analyze the most commonly altered second nucleotide of codon 132 (CGT) and NTCATGCTTAT as the sequence to analyze. Note the aberrant T peak in this wild-type sample. Signal-to-noise characteristics were improved by using an alternate sequencing primer (Fig. 1), which encompassed codon 131, and the entirety of codon 132 and was used in the final sequencing reactions.JOURNAL/dimp/04.03/00019606-201212000-00003/figure3-3/v/2021-02-17T200047Z/r/image-jpegPyrograms demonstrating the effect of sequencing primer concentrations on peak height in the IDH2 assay. Note the inconsistency in the amplitude of reference peaks on either side of the shaded segment when using 0.5 µM sequencing primer (A). Reference peak heights are proportionate when using 1.0 µM (B). When using a sequencing primer concentration of 0.3 µM (general recommendation of the manufacturer), there was further divergence in the amplitude of individual reference peaks that caused the pyrogram software analysis to fail.IDH1andIDH2Mutation AnalysesIDH1 and IDH2 mutations were identified in 42 of the 138 cases (43.8%) and 1 of the 138 cases (0.7%) that were successfully sequenced, respectively. One of the 139 cases failed to sequence for both IDH1 and IDH2. This was a grade II oligodendroglioma with DNA isolated from tissue prepared for 1p/19q deletion (FISH) and stored at −20°C for 2 years. Results of IDH1 and IDH2 testing by tumor type are listed in Table 1. The frequency of IDH1 codon 132 mutations observed in this study relative to that reported in the literature is summarized in Table 2. The only IDH2 mutation observed at codon 172 in this series was AGG>AAG.JOURNAL/dimp/04.03/00019606-201212000-00003/table1-3/v/2021-02-17T200047Z/r/image-tiffIDH1 and IDH2 Mutations by Histopathologic DiagnosisJOURNAL/dimp/04.03/00019606-201212000-00003/table2-3/v/2021-02-17T200047Z/r/image-tiffIDH1 R132 mutations in comparison with prior reportsFor IDH1 CGT>CAT and CGT>CTT mutations, the median percentage of mutant allele at position 2 of the codon was 39% (minimum value of 14.2%, maximum value of 63%). The percentage of mutant allele at position 2 in IDH1 CGT>GGT mutations was higher (77% and 74.9%), consistent with the effect of 2 successive guanine nucleotides being incorporated. The other IDH1 mutation identified in this study (CGT>AGT) was associated with mutant allele frequencies of 17.0% and 26.2% at position 1 of the codon. For the single case demonstrating an IDH2 mutation (AGG>AAG), the mutant allele frequency at position 2 was 50.5%.Several patients had multiple samples within a case (n=16) or repeat surgical specimens (n=4) (see the Sample retrieval in Methods section). For those cases with an IDH1 mutation (n=14, all of which were c.395G>A), the mutation was detectable on all tested samples. Wild-type cases also demonstrated consistent results across multiple and/or repeat samples.IDH1Mutation Status and Other Patient VariablesAmong glial and glioneuronal tumors (n=95), the age of patients with IDH1-mutated (mean=39.9 y, SD=10.1) versus IDH1 wild-type (mean=33.5 y, SD=19.3) gliomas differed significantly (t=2.08, P=0.04, v=82). In contrast, analysis on a restricted subset of gliomas (n=71, see the Methods section) identified no significant difference in age (t=0.44, P=0.66, v=43). In the same restricted subset, a significant difference was seen in patient sex between IDH1 mutated (69% male) and IDH1 wild-type (62% female) gliomas (χ2=5.5, P=0.019). For cases with both IDH1 mutation status and 1p/19q deletion data (n=41), a significant correlation was identified between the presence of the mutation and 1p/19q deletion (Kendall τ=0.26, Z=2.37, P=0.018). The single grade II oligodendroglioma with an IDH2 mutation also demonstrated a 1p/19 deletion.DISCUSSIONThe PS approach proved effective at identifying mutations in the IDH1 (codon 132) and IDH2 (codon 172) genes among the 139 neoplastic and non-neoplastic cases analyzed. A total of 42 cases demonstrated IDH1 mutations and a single case of grade II oligodendroglioma had an IDH2 mutation. Mutations were most commonly seen in grades II and III oligoastrocytoma (100% of cases), grade II astrocytoma (87.5%), grade III anaplastic oligodendroglioma (70%), grade II oligodendroglioma (66.7%), and grade III astrocytoma (58.3%). IDH1 mutations were detected infrequently in grade IV astrocytoma (GBM) (16.7%), consistent with the hypothesis that IDH1-mutated GBMs represent a less common “secondary” form of GBM that arises from lower-grade gliomas.5 Mutations were not identified in any grade I glial neoplasm, glioneuronal tumors, nonglial tumors, or non-neoplastic entities. These results are consistent with prior reports.4,5,8The majority of cases in this study had adequate FFPE tissue for DNA isolation and PS performed satisfactorily even with suboptimal tissue samples. For example, sequencing was successful using DNA isolated from 8-year-old unstained glass slides scraped for tumor, and in another case, an R132H IDH1 mutation was identified in a 2-year-old FFPE section frozen at −20°C. PS was also successful with limited samples such as tissue scraped from hematoxylin and eosin-stained slides. However, a single grade II oligodendroglioma failed to sequence successfully for both IDH1 and IDH2 despite repeat analysis. The tissue comprised an unused FFPE section previously obtained for 1p/19q deletion testing by FISH and frozen for 2 years at −20°C. The reason underlying this failure is not clear as other tissues procured and stored similarly performed well; a role for tissue/DNA degradation cannot be excluded. Overall, the PS approach was robust with limited samples, suggesting that it would perform well in small stereotactic biopsy specimens with limited reserve tissue (eg, a single FFPE block or unstained sections of frozen tissue).The threshold for a true positive in this study, using mutant:normal allele ratios, was 5% and 10% for the IDH1 and the IDH2 genes, respectively. As shown in Tables 1 and 2, this approach yielded results comparable to prior studies using non-PS methodologies. Further, Felsberg et al8 utilized a similar threshold (5%) for mutation detection in their PS assay. In practice, however, the minimum value of mutant allele detected in our study was 14.2% among the 42 cases with an IDH1 mutation and the median frequency was 39%. The study by Felsberg et al8 found a similar range of 23% to 61% with a median mutant allele frequency of 40%. A threshold for a PS-based IDH1 mutation assay could therefore be set at 5%, although as demonstrated in both of these PS-based studies, the minimum percentage of mutant allele in clinical specimens typically exceeds 10% in practice.Two unexpected technical challenges arose in the development of the assay relating to PS optimization. The first issue was related to primer choice in the IDH1 assay. In this case, the primer selected was one that included codons 131 and 132 in the PS output as this had improved signal-to-noise characteristics as determined by background peak amplitudes (as a percentage of reference peaks). A primer designed to begin at the second nucleotide of codon 132 was anticipated to demonstrate optimal sensitivity. However, background noise in the reference peak data was unacceptably high. Therefore, the former sequencing primer including all nucleotides of codon 131 and the first nucleotide of codon 132 (ie, 5′-GGGTAAAACCTATCATCATA-3′) was utilized for the studies reported here. A second issue was the presence of aberrant height peaks identified in the development of the IDH2 assay. Specifically, the reference peaks for bases in codons 173 and 174 were taller than expected relative to other peaks in codons 171 and 172 and these exaggerated reference peaks caused the analysis software to fail. Different sets of PCR primers and sequencing primers did not alleviate the aberrant peak heights, and gel electrophoresis of amplicons demonstrated that amplified IDH2 gene fragments were specific and of the expected size. Additional attempts to rectify the exaggerated peak heights included: (1) raising the denaturing temperature from 80°C to 85°C before annealing of the sequencing primer to the template DNA; (2) more than doubling the annealing time of the sequencing primers from 5 to 12 minutes; and (3) increasing the concentration of the sequencing primer above the 0.3 µM indicated by Qiagen. Only the last of these experiments impacted the peak height; a primer concentration of 1.0 µM brought the peaks heights into alignment. All subsequent testing for IDH1/IDH2 was performed with this higher concentration of sequencing primer.The relationship between IDH1 mutation status and pertinent patient variables such as age, sex, and 1p/19q deletion status were also tested with this data set and largely conformed to prior reports. Of the 14 grade IV gliomas (GBM and gliosarcoma) in this study, CGT>CAT mutations were identified in a 37- and a 45-year-old patient. This is consistent with prior results, whereby IDH1 mutations are seen in younger adult patients with GBMs.5,8 Similarly, 3 adolescent patients diagnosed with GBM (all ≤18 y old) were IDH1 wild-type consistent with a similar finding in 22/22 pediatric GBMs.12 For grade II to IV gliomas overall, there was no significant relationship between patient age and the IDH1 mutation status. This finding is consistent with the observation that age differences in IDH1 mutated/IDH1 wild-type tumors emerge only in high-grade entities (GBM, grade III astrocytoma, and oligoastrocytoma),5,13 although this phenomenon has no satisfactory explanation at present. An unexpected finding was the significantly higher rate of male patients with an IDH1 mutation among grade II to IV gliomas in this study (29/40 male patients with mutation vs. 13/29 female patients in the grade II to IV glioma category). The most marked difference was identified among anaplastic (grade III) astrocytomas (3/3 male and 3/9 female patients tested). This finding has not previously been reported to our knowledge and needs to be replicated independently in a larger number of samples per glioma subtype.The well-known association between 1p/19q deletion and IDH1 mutation1,4 also held true in this study. Deletions of chromosomes 1p and 19q (including cases with definitive deletion at 1 site and equivocal deletion at the other site) were identified in 21 cases, including grades II to III oligodendroglioma (18 cases), a single grade II diffuse astrocytoma, and 2 GBMs with oligodendroglial components. Of these 21 cases, 17 harbored IDH1 mutations, all but one of which had the common c.395G>A IDH1 mutation. Yan et al4 reported 1p/19q deletion in 85% of their IDH1/IDH2 mutated oligodendroglial neoplasms. Unlike this prior study, however, we observed several cases of 1p/19q-deleted tumors that were IDH1/IDH2 wild-type (all 1p/19q-deleted tumors in their study demonstrated the IDH1 mutation).In summary, IDH1/IDH2 mutation analyses can provide diagnostic support for difficult differential diagnoses, particularly in small biopsy specimens, and prognostic information that may be useful in treatment planning and patient counseling.1 Relative to the latter point, even among grade III anaplastic astrocytomas, IDH1 mutation confers significant survival benefit relative to wild-type tumors (65 and 20 mo median survivals, respectively).4 Therefore, IDH1/IDH2 testing may join MGMT promoter methylation and 1p/19q deletion testing in the molecular phenotyping of gliomas to assist in treatment planning.3 The utilization of high-throughput, sensitive, and robust assays for IDH1/IDH2 mutations is therefore likely to increase in the practice of diagnostic surgical neuropathology. In our experience, as outlined here, the PS-based approach is well suited to this task.REFERENCES1. Sanson M, Marie Y, Paris S, et al. Isocitrate dehydrogenase 1 codon 132 mutation is an important prognostic biomarker in gliomas. J Clin Oncol. 2009;27:4150–4154[Context Link][Full Text][CrossRef][Medline Link]2. Parsons DW, Jones S, Zhang X, et al. An integrated genomic analysis of human glioblastoma multiforme. Science. 2008;321:1807–1812[Context Link][Full Text][CrossRef][Medline Link]3. Bourne TD, Schiff D. Update on molecular findings, management and outcome in low-grade gliomas. Nat Rev Neurol. 2010;6:695–701[Context Link][Full Text][Medline Link]4. Yan H, Parsons DW, Jin G, et al. IDH1 and IDH2 mutations in gliomas. N Engl J Med. 2009;360:765–773[Context Link][Full Text][CrossRef][Medline Link]5. Balss J, Meyer J, Mueller W, et al. Analysis of the IDH1 codon 132 mutation in brain tumors. Acta Neuropathol. 2008;116:597–602[Context Link][Full Text][CrossRef][Medline Link]6. 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The shaded segments indicate the 4 nucleotides dispensed to analyze codon 132. The mutant sample demonstrates a c.CGT>CAT substitution with 44% mutant allele.Pyrogram generated using a sequencing primer designed to analyze the most commonly altered second nucleotide of codon 132 (CGT) and NTCATGCTTAT as the sequence to analyze. Note the aberrant T peak in this wild-type sample. Signal-to-noise characteristics were improved by using an alternate sequencing primer (Fig. 1), which encompassed codon 131, and the entirety of codon 132 and was used in the final sequencing reactions.Pyrograms demonstrating the effect of sequencing primer concentrations on peak height in the IDH2 assay. Note the inconsistency in the amplitude of reference peaks on either side of the shaded segment when using 0.5 µM sequencing primer (A). Reference peak heights are proportionate when using 1.0 µM (B). When using a sequencing primer concentration of 0.3 µM (general recommendation of the manufacturer), there was further divergence in the amplitude of individual reference peaks that caused the pyrogram software analysis to fail.IDH1 and IDH2 Mutations by Histopathologic DiagnosisIDH1 R132 mutations in comparison with prior reportsPyrosequencing of <em xmlns:mrws="">IDH1</em> and <em xmlns:mrws="">IDH2</em> Mutations in Brain Tumors and Non-neoplastic ConditionsCykowski Matthew D. MD; Allen, Richard A. MS; Fung, Kar-Ming MD, PhD; Harmon, Michael A. BS; Dunn, Samuel T. PhDOriginal ArticlesOriginal Articles421p 214-220