Clone 3c6

The paraffin specimens were made into 3–5 μm sections. For immunohistochemical staining, paraffin sections were deparaffinized in xylene and rehydrated in a graded series of ethanol. After blocking peroxidase activity with hydrogen peroxide and antigen retrieval by heat induced using citrate buffer (pH 9.0), the sections were incubated at 4°C with primary antibodies against EGFR (1:500, clone 3C6, Ventana Medical Systems Inc., Tucson, AZ, USA), C-erb B2 (1:500, A0485, Dako Cytomation, Glostrup, Denmark), Ki-67 (1:100, MIB-1, Dako Cytomation, Glostrup, Denmark), and p53 (1:100, clone DO-7, Dako Cytomation, Glostrup, Denmark). Immunostaining was conducted using the ImmPRESS (MP7410-50, Vector, Burlingame, CA, USA) system and the diaminobenzidine (DAB) kit (Sk4100, Vector). After the reactions, the sections were counterstained with Meyer’s hematoxylin, dehydrated, cleared, and mounted.

Neuropathological diagnosis including molecular markers of the tumor was routinely assessed using standard histopathology, immunohistochemistry, in situ hybridization, and sequencing technologies. For molecular classification, following markers, among others, were determined in routine analysis: IDH1 status, MGMT methylation status and EGFR amplification status. The MGMT methylation status was assessed by pyrosequencing using the Therascreen MGMT Pyro Kit (QIAGEN, Hilden, Germany). Cases with a mean methylation percentage of more than 8% were considered to be MGMT methylated [20 (link)]. Immunohistochemistry was performed to assess EGFR amplification status (clone 3C6; Ventana, Oro Valley, AZ, USA) and IDH1 mutation status (clone DIA-H09; Dianova, Hamburg, Germany) [21 (link),22 (link)]. Presence of oligodendroglial component was excluded by 1p19q codeletion status.
Because of clinical and prognostic impact, in this study we focused only on MGMT and EGFR alterations in IDH1 wild type GBM.