Mutations of both the IDH1 and IDH2 (isocitratedehydrogenase enzyme 1 and 2) genes have recently been described in cases of human glioma. Since IDH1 mutations have been associated with better clinical outcome , they are suitable predictive markers for adult glioma patients. We have developed a pyrosequencing assay that allows both the sensitive and rapid detection of mutant IDH1 alleles in DNA extracted from formalin-fixed , paraffin-embedded tissues. PCR products that span exon 4 of IDH1 were used as a template for pyrosequencing. For validation , PCR products were additionally cloned and sequenced conventionally by Sanger sequencing. Sensitivity was measured by titration of wild-type and mutant sequences. PCR kinetic experiments were performed to investigate the influences of PCR cycle number on the accuracy of the assay. We found that a minimum of 5% of mutant IDH1 alleles can easily be detected with the pyrosequencing approach. So far, there are few data regarding IDH1 mutation status in high-grade gliomas of childhood. Therefore , we applied this assay to 47 pediatric high-grade glioma samples (age range 6 weeks to 23 years). Mutations were found in 5/14 astrocytoma III and in 6/33 glioblastomas. In conclusion , we have developed a pyrosequencing-based assay for the detection of mutations at the hotspot regions of IDH1 and provide proof for its applicability as a molecular diagnostic assay for clinical samples.

Advances in understanding the molecular basis of primitive neuroectodermal tumors of the central nervous system (CNS-PNET) biology are critical to improve patient outcome. Recently, new data on their molecular features have been reported, suggesting that supratentorial PNET (s-PNET) in adult patients may represent a specific tumor entity among CNS-PNETs. In this study, we analyzed the clinicopathologic and molecular features of 12 cases of s-PNET in adult patients. The follow-up analysis showed that these tumors have an aggressive clinical behavior. At the histopathologic level, they resembled their pediatric counterpart, showing a variable spectrum of neuronal differentiation. These cases did not show astrocytic differentiation; therefore, they did not qualify for the differential diagnosis of glioblastoma variants. The tumors were also screened for mutation of TP53, IDH1, IDH2, and β-catenin, using single strand conformation polymorphism-based and sequencing assays, and were analyzed for c-myc/N-myc gene copy numbers with a quantitative polymerase chain reaction-based method. The strand conformation polymorphism-based mutational analysis showed that 5 tumors harbored TP53 mutations. In 2 cases, a mutation at codon 132 of the IDH1 gene was also found. No mutations of the β-catenin or IDH2 genes were identified. No cases presented c-myc or N-myc amplifications. Only 1 case presented overexpression of epidermal growth factor receptor. In conclusion, our data show a high incidence of TP53 mutations in this group of tumors and show, in comparison with pediatric s-PNET, the absence of amplification of the c-myc/N-myc genes, indicating that s-PNET in adult patients may represent a specific subset of tumors among CNS-PNETs.