**7. Molecular biology**

Because of their favourable prognostic value 1p19q codeletion, MGMT promoter methylation and isocitrate dehydrogenase mutations are considered important clinical biomarkers for diffuse gliomas. In addition p53 is useful for diagnostic purposes. These markers are requested with increasing frequency and are discussed in detail below. Even when the diagnosis of a specific glioma type is readily apparent in histological stains, pathologist need to take care, that sufficient tissue is available for future molecular analysis.

Diagnostic Evaluation of Diffuse Gliomas 213

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

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

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

Generally, tumor grade increases with age and younger age of onset is one of the strongest predictive factor of prolonged survival (Kita et al., 2009) and thus heavily influences all other markers found. Despite this fact, many publications do not take patients age into account when analyzing biomarkers on patient survival. In astrocytomas, MIB-1 proliferation values above 5% are considered to be associated with a shorter survival (Jaros et al., 1992). Because of study population heterogeneity, predictive data on Tp53 mutations are limited. Some authors see p53 immunoreactivity to be associated with a shorter survival or shorter time to malignant progression (Jaros et al., 1992, Ständer et al., 2004). It is noteworthy, that not all p53 immunoreactive tumors contain mutations in the TP53 gene (Kösel et al., 2001). Further contrasting to immunohistochemistry data, a molecular study on 159 grade II astrocytomas and oligoastrocytomas did not found an influence on overall survival, but reported a significant shorter progression-free survival (Peraud et al., 2002). In glioblastomas p53 mutation status does not correlate with patients outome (Weller et al.,

2009). Thus, p53 is only useful as a diagnostic marker but not prognostic.

contains several loci along the chromosomal arms (Riemenschneider et al. 2010).

**7.4 Loss of 1p/19q** 

homogeneity.

**8. Prognostic implications 8.1 Immunohistochemistry** 
