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Figure 1: Exemplary images of immunohistochemical evaluation of isocitrate dehydrogenase (IDH‑1 [R132H]) and karyopherin a2 (KPNA2) expression as well as
nuclear accumulation of p53 protein. Immunohistochemical staining with antibodies against mutated IDH‑1 (R132H) shows a strong cytoplasmic immunoreactivity
in the tumor cells. Nuclear expression of KPNA2 is detected in a subpopulation of tumor cells. Nuclear p53 accumulation can be observed in > 50% of the tumor

a b
Figure 2: Pyrosequencing was used for quantitative analysis of O ‑methylguanine DNA methyltransferase promoter methylation. Pyrogram demonstrating (a) an
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unmethylated and (b) methylated glioblastoma tissue. Each colored box includes one of the four studied CpG positions (CpGs 9‑12). The incorporation of the bases
guanine and adenine represent the methylated and unmethlylated fractions, respectively. The percentages given in both pyrograms reflect the methylated fractions
(fractions over 10% define a methylated sample)
expression of CD11c (a-integrin) and CD123 (IL-3 assessed and visually scored independently by two
receptor a), respectively. Figures 1 and 2 of our previous experienced neuropathologists (PN, GHG) as previously
publication illustrate the classification/gating steps. described [Figure 1]. [14] Immunohistochemical staining
[4]
HLA-DR, CD11c, CD45, CD123 are co-stimulatory surface with a monoclonal antibody against p53 (clone DO-7,
molecules, which have been identified in the relevant Dako, Glostrup, Denmark), in a dilution of 1:150, were
literature as markers of maturation. [24-27] CD34 is a marker performed on the Ventana Immunostainer (Roche,
for all stem cells and the proportion of precursor DCs. [28] Mannheim, Germany), with a closed avidin-biotin
Therefore, HLA DR+/Lin-/CD34-/CD45+ were defined as complex Ventana Detection System (Ventana). Positive
total mature DCs. HLA DR+/Lin-/CD34-/CD45+/CD123-/ and negative controls were also performed using
CD11c- represent less mature DCs, whereas HLA DR+/ glioblastoma tissue with p53 overexpression [Figure 1].
Lin-/CD34-/CD45+/CD11c+/CD123- mDCs and HLA Tumors were assigned to immunoreactivity classes of
DR+/Lin-/CD34-/CD45+/CD123+/CD11c- pDCs mature KPNA2 and p53 based on the percentage of moderately
DCs in advanced stages of maturation. or strongly immunopositive cell nuclei (< 1%, 1%-< 5%,
5%‑< 10%, 10%‑< 20%, 20%‑< 50% and ≥ 50%).
Immunohistochemistry
Neuropathological analysis of glioblastomas Pyrosequencing
comprised hematoxylin/eosin staining as well as The quantitative analysis of MGMT promoter
immunohistochemistry with monoclonal antibodies methylation by pyrosequencing was performed as
directed against the microtubule-associated protein previously described. [29] Briefly, the first four CpG sites
2 (MAP2, Sigma, Steinheim, Germany), polyclonal are assayed for a primer extension reaction. Methylated
antibodies directed against glial fibrillary acid fractions > 10% at all positions define a methylated
protein (GFAP, Sigma, Steinheim, Germany) and sample [Figure 2].
monoclonal antibodies directed against Ki67 (MIB1,
Dako, Glastrop, Denmark). Statistical analysis
Statistical analyses of the data were performed using
IDH-1-R132H mutation status (monoclonal mouse commercially available software (SSPS 21.0, IBM
antibody H09 directed against mutated IDH-1 R132H Deutschland, Ehningen, Germany). Comparisons
mutation, Dianova, Hamburg, Germany) and KPNA2 of samples were performed using standard
immunoreactivity (goat polyclonal SC6917; Santa Cruz methods (Pearson’s Chi-square, Fisher’s exact test).
Biotechnology, Santa Cruz, USA; dilution 1:100) were P < 0.05 (two-tailed) were considered to be statistically

10 Neuroimmunol Neuroinflammation | Volume 2 | Issue 1 | January 15, 2015