**7.1 Tumor protein 53 mutations**

Since Tp53 mutations are broadly distributed, exons 5-8 need to be screened by singlestrand conformational polymorphism analysis followed by direct sanger sequencing of samples exhibiting mobility shifts. In unremarkable cases, sequencing analysis is usually extended to exons 4, 9 and 10. Because of these efforts, Tp53 molecular analysis is not part of routine diagnosis unlike p53 immunostaining. A Tp53 mutation in diffuse astrocytomas grade II WHO is observed in 52-60% of the tumors, while Tp53 mutations are present in 35- 44% of oligoastrocytomas and in 10% oligodendrogliomas (Okamoto et al., 2004, Kim et al., 2010). Especially astrocytomas with gemistocytic tumor cell morphology may contain Tp53 mutations in up to 82% of tumors (Watanabe et al., 1998). Since Tp53 mutations are acquired early, their frequency does not increase much further during tumor progression (Watanabe 1997). In pediatric glioblastomas, Tp53 mutations were found in 60% of tumors examined (Srivastava et al., 2010). Grade II WHO diffuse astrocytomas may show in 49% a combined TP53 mutation and IDH1 or IDH2 mutation (Kim et al., 2010). A Tp53 mutation without associated IDH1/2 mutation is rare (3%), thus indicating that IDH mutations occur at an earlier stage of tumorgenesis.

#### **7.2 Isocitrate dehydrogenase mutations**

The NADP-dependent enzymes IDH1 and IDH2 catalyze the conversion from isocitrate in alpha-ketoglutarate. Mutations of the catalytic center in gliomas result in accumulation of the oncogenic metabolite D-2-hydroxyglutarate (Dang et al., 2009). It is thought that the reduced NADPH levels in IDH mutated gliomas could sensitize tumors to radiation and chemotherapy (Bleeker et al., 2010). The frequency of IDH mutations is high in diffuse astrocytomas, anaplastic astrocytomas and secondary glioblastomas evolving from these precursor lesions, while presence of IDH mutations is seen in only 3-7% primary glioblastomas (Hartmann et al., 2009). The vast majority of IDH1 mutations are point mutations leading to a distinct amino acid substitution on codon 132 (Arg132His) for which an specific antibody has been developed (Capper et al., 2010). The other amino exchange mutations can be detected either by direct sanger sequencing or restriction-endonuclease based PCR (Meyer et al., 2010).

IDH1 mutations are found in 59-88% diffuse astrocytomas, 50-78% anaplastic astrocytomas and 50-88% secondary glioblastomas, IDH2 mutations are present in 1-7% diffuse astrocytomas, 1-4% anaplastic astrocytomas and seem to be absent in secondary glioblastomas (Bourne et al., 2010). The rate of IDH2 mutations in oligodendrogliomas is higher as in astrocytomas (4-8% in oligodendrogliomas grade II and grade III, 1-6% of oligoastrocytomas grade II and grade III) but still lower than number of IDH1 mutations (68-82% oligoadendrogliomas grade II, 49-75% anaplastic oligodendrogliomas grade III, 50- 100% oligoastrocytomas, 63-100% anaplastic oligoastrocytomas grade III) (Bourne et al., 2010). In addition the IDH1 R132C mutation is strongly associated with an astrocytoma phenotype (Hartmann et al., 2009).

#### **7.3 MGMT methylation status**

The DNA repair enzyme O-methylguanine-DNA methyltransferase (MGMT) removes alkyl groups from the O6 position resulting in an increased tumor resistance to alkylating agents therapy. Methylation of the MGMT promotor region results in decreased MGMT activity which in turn increases glioblastoma tumor cell sensivity to therapy with temozolomide and

Since Tp53 mutations are broadly distributed, exons 5-8 need to be screened by singlestrand conformational polymorphism analysis followed by direct sanger sequencing of samples exhibiting mobility shifts. In unremarkable cases, sequencing analysis is usually extended to exons 4, 9 and 10. Because of these efforts, Tp53 molecular analysis is not part of routine diagnosis unlike p53 immunostaining. A Tp53 mutation in diffuse astrocytomas grade II WHO is observed in 52-60% of the tumors, while Tp53 mutations are present in 35- 44% of oligoastrocytomas and in 10% oligodendrogliomas (Okamoto et al., 2004, Kim et al., 2010). Especially astrocytomas with gemistocytic tumor cell morphology may contain Tp53 mutations in up to 82% of tumors (Watanabe et al., 1998). Since Tp53 mutations are acquired early, their frequency does not increase much further during tumor progression (Watanabe 1997). In pediatric glioblastomas, Tp53 mutations were found in 60% of tumors examined (Srivastava et al., 2010). Grade II WHO diffuse astrocytomas may show in 49% a combined TP53 mutation and IDH1 or IDH2 mutation (Kim et al., 2010). A Tp53 mutation without associated IDH1/2 mutation is rare (3%), thus indicating that IDH mutations occur at an

The NADP-dependent enzymes IDH1 and IDH2 catalyze the conversion from isocitrate in alpha-ketoglutarate. Mutations of the catalytic center in gliomas result in accumulation of the oncogenic metabolite D-2-hydroxyglutarate (Dang et al., 2009). It is thought that the reduced NADPH levels in IDH mutated gliomas could sensitize tumors to radiation and chemotherapy (Bleeker et al., 2010). The frequency of IDH mutations is high in diffuse astrocytomas, anaplastic astrocytomas and secondary glioblastomas evolving from these precursor lesions, while presence of IDH mutations is seen in only 3-7% primary glioblastomas (Hartmann et al., 2009). The vast majority of IDH1 mutations are point mutations leading to a distinct amino acid substitution on codon 132 (Arg132His) for which an specific antibody has been developed (Capper et al., 2010). The other amino exchange mutations can be detected either by direct sanger sequencing or restriction-endonuclease

IDH1 mutations are found in 59-88% diffuse astrocytomas, 50-78% anaplastic astrocytomas and 50-88% secondary glioblastomas, IDH2 mutations are present in 1-7% diffuse astrocytomas, 1-4% anaplastic astrocytomas and seem to be absent in secondary glioblastomas (Bourne et al., 2010). The rate of IDH2 mutations in oligodendrogliomas is higher as in astrocytomas (4-8% in oligodendrogliomas grade II and grade III, 1-6% of oligoastrocytomas grade II and grade III) but still lower than number of IDH1 mutations (68-82% oligoadendrogliomas grade II, 49-75% anaplastic oligodendrogliomas grade III, 50- 100% oligoastrocytomas, 63-100% anaplastic oligoastrocytomas grade III) (Bourne et al., 2010). In addition the IDH1 R132C mutation is strongly associated with an astrocytoma

The DNA repair enzyme O-methylguanine-DNA methyltransferase (MGMT) removes alkyl groups from the O6 position resulting in an increased tumor resistance to alkylating agents therapy. Methylation of the MGMT promotor region results in decreased MGMT activity which in turn increases glioblastoma tumor cell sensivity to therapy with temozolomide and

**7.1 Tumor protein 53 mutations** 

earlier stage of tumorgenesis.

based PCR (Meyer et al., 2010).

phenotype (Hartmann et al., 2009).

**7.3 MGMT methylation status** 

**7.2 Isocitrate dehydrogenase mutations** 

is therefore a predictive molecular marker (Hegi et al., 2005). MGMT expression in tumor cells of astrocytomas and glioblastomas can be determined by nuclear immunoreactivity of tumor cells (Capper et al., 2008). Together with other sophisticated methods such as realtime RT-PCR or methylation-specific pyrosequencing, they lack a valid definition for clinically relevant cut-off values (von Deimling et al., 2010). Usually MGMT is determined in formalin-fixated paraffin-embedded specimens through methylation-specific PCR, yet reliability and reproducibility are still limited in the current standard method (Preusser, et al., 2008b, Elezi et al., 2008). Not only is MGMT protein expression within tumors heterogenous, but also highly dependent on the method used and changes during therapy (Jung et al., 2010, Preusser et al., 2008a, Janzer et al., 2008). Thus reports on MGMT methylation range from 93% in frozen tissue sections in diffuse astrocytoma grade II (Everhard et al., 2006) to 30-35% in glioblastoma paraffin blocks (Tabatabai et al., 2010). In pediatric glioblastomas approximately half of the tumors are methylated (Srivastava et al., 2010). Despite these shortcomings MGMT analysis is essential for almost all clinical studies

**7.4 Loss of 1p/19q**  A loss of heterozygosity is usually assessed though use of microsatellite marker PCR. This method requires corresponding blood samples to determine allele status. Therefore use of fluorescent in situ hybridisation is preferred by some laboratories but carries the risk of misdiagnosing cases with only partial loss. This risk can be covered by additional PCR that contains several loci along the chromosomal arms (Riemenschneider et al. 2010).

and one of the most requested molecular analysis in neuropathology routine practice.

Loss of heterozygosity in 1p and 19q are found in 78% of oligodendrogliomas grade II, 44% of oligoastrocytomas and 17% of diffuse astrocytomas grade II WHO. Therefore 1p19q codeletion is strongly associated with a oligodendroglial tumor morphology and often used as a diagnostic marker. In addition in oligodendrogliomas up to 73% of codeleted tumors also show either additional IDH1 or IDH2 mutations (Kim et al., 2010). Not surprisingly journal reviewers often require 1p19q deletions in oligodendrogliomas for sample homogeneity.
