Possible explanations for this phenomenon include in vivo localization of the mutant protein to nucleus or by antigen diffusion (i.e., the penetration of the soluble protein into the nucleus during tissue processing).32,40 Heterogeneity of staining with only a portion of tumor cells taking up the stain was noted in 46.6% (14/30) cases. 88% (44/50) cases. All 6 cases with discrepancy were immunopositive with DIA-H09 antibody. While in 3 of these 6 cases, DNA sequencing failed to reveal any mutations, R132L (arginine replaced by leucine) mutation was found in the rest 3 cases. Interestingly, of the Egf immunopositive cases, 46.6% (14/30) showed immunostaining in only a fraction of tumor cells. Conclusions IHC is an easy and quick method of detecting IDH1-R132H mutations, but there may be some discrepancies between IHC and DNA sequencing. Although there were no false-negative cases, cross-reactivity with IDH1-R132L was seen in 3, a obtaining not reported thus far. Because of more universal availability of IHC over genetic testing, cross-reactivity and staining heterogeneity may have bearing over its use in detecting IDH1-R132H mutation in gliomas. strong class=”kwd-title” Keywords: diffuse gliomas, DNA sequencing, IDH1-R132H, IDH1-R132L, immunohistochemistry The IDH1 gene on chromosome 2q33.3 encodes for isocitrate dehydrogenase 1 (IDH1), Benzyl isothiocyanate located in the cytoplasm and the peroxisomes. This enzyme catalyzes NADPH production via oxidative decarboxylation of isocitrate to alpha-ketoglutarate in the Krebs citric acid cycle.1 In 2008, for the first time, Parsons et al introduced to the medicine world the role of IDH1 in the pathogenesis of glioblastoma multiforme (GBM). In their genome-wide sequencing analysis, recurrent somatic mutations specifically involving the amino acid arginine at position 132 were detected in 12% of Benzyl isothiocyanate the GBM specimens.2 Subsequent studies have shown that IDH1 mutation is an early step in gliomagenesis and has been reported to occur in grades II and III astrocytomas, oligodendrogliomas (OG), oligoastrocytomas (OA), and secondary GBM.3C12 Hartmann et al, in their analysis of 1010 diffuse glioma tumors, demonstrated that most cases of diffuse astrocytomas (DA; 72.7%, 165/227), anaplastic astrocytomas (AA; 64.0%, 146/228), OG (82.0%, 105/128), anaplastic oligodendrogliomas (AOG; 69.5%, 121/174), OA (81.6%, 62/76), and anaplastic oligoastrocytomas (AOA; 66.1%, 117/177) experienced IDH1 mutations.13 Of importance, these mutations appear to be specific for these tumors as main GBM, pilocytic astrocytoma World Health Business (WHO) grade I and other Benzyl isothiocyanate central nervous system (CNS) and non-CNS neoplasms, with the exception of acute myeloid leukemia and cartilaginous neoplasms, harbor this genetic alteration much less frequently.3C11,14C19 Different types of mutations have been described, and the most frequent is G to A transitions at position 395 of the IDH1 transcript. This results in substitution of the amino acid arginine with histidine (R132H). Rarer ones include R132C, R132S, R132G, R132L, R132V and R132P.2,4,5,8C11,13,17,20,21 Mutations involving IDH2, a homologous gene, have also been detected in gliomas but at a much lower frequency ranging from 2% to 5%.8,10,13,17,21,22 Although these mutations are rare in the pediatric age group, in patients aged 18 years, they seem to be associated with younger age at presentation and have a favorable impact on the overall and progression-free survival associated with grade II-IV gliomas.2,6,7,10,12,13,22C28 IDH1 testing is being used as a standard diagnostic tool in many neuropathology laboratories. It is useful in differentiating gliomas from nonneoplastic CNS lesions,21,29C31 diffuse astrocytoma WHO grade II from pilocytic astrocytoma grade I,15,32 anaplastic astrocytomas WHO grade III from GBM,32 main from secondary GBM,6,32 and astrocytomas from ependymomas.32 Most studies of IDH mutations are based on DNA sequencing, which is labor intensive, requiring trained personnel and sophisticated gear, not available at every center. Moreover, false-negative results may be obtained in cases of inadequate tumor DNA Benzyl isothiocyanate availability because of small biopsy samples, considerable necrosis, or admixture with normal tissue elements. Alternate rapid methods, some based on routinely processed tissue specimens, have been recently suggested.17,21,26,29C36 Of these, a significant development was the introduction of mAb H0933 and IMab-134 antibodies, which are specific for the most common IDH1 mutation: R132H. In the present study, which is a continuation of our previous study on IDH1 mutation as assessed by direct DNA sequencing,11 we compared the results of immunohistochemistry (IHC) using mAb H09 for IDH1-R132 mutations with those of DNA sequencing in different types and grades of gliomas. Materials and Methods Tumor Specimens Tumor samples were acquired fresh during surgery through the operation theatre from the Neurosurgery Division in the All India Institute of.