“Borderline Case” demonstrating that MGMT and BRAF are helpful additional molecular markers to differentiate PXA from gcGBM.

<p><b>A</b> Tumor specimen of a 51-years old female patient, located in the right parietal region: T1-weighted, contrast-enhanced MRI scan shows a well-circumscribed, vascularized and in-homogeneously contrasted tumor, suspicious for focal necrosis. <b>B</b> HE staining displays a pleomorphic astroglial tumor with giant cells, elevated mitotic activity and palisading necrosis. <b>C</b> Reticulin fiber dispositions are present around the vessel walls, while <b>D</b> CD34 expression is limited to the vessels. <b>E</b> As the proliferative index additionally accounts for 20% the tumor was initially classified as giant cell glioblastoma (WHO grade IV). <b>F</b> Pyrogram indicating a significant <i>MGMT</i> hypermethylation and G <i>BRAF</i> V600E mutation. For that reason the tumor was finally classified as PXA with anaplastic features (analogues to grade III).</p

PXA and PXA-A: Patients, histology, immunohistochemical and molecular markers.

<p>PXA and PXA-A: Patients, histology, immunohistochemical and molecular markers.</p

gcGBM: Patients, histology, immunohistochemical and molecular markers.

<p>gcGBM: Patients, histology, immunohistochemical and molecular markers.</p

Correlation of histological and molecular markers in temporal and non-temporal located PXA.

<p><i>BRAF</i> V600E mutations were found in 70.0% of all PXA with temporal localization (n = 7). 6 of these patients showed simultaneous reticulin and CD34 expression. Both PXA that harbored a methylation of the <i>MGMT</i> promoter were located in the temporal lobe. A slightly higher incidence of reticulin fiber disposition and CD34 expression was found in temporal tumors.</p

MGMT and BRAF V600 analysis in gcGBM and PXA.

<p><b>A</b> Hypermethylation of <i>MGMT</i> was detected in 2 PXA (10%) and 14 gcGBM (41,2%). <b>B</b> 5 PXA and 6 PXA-A (55%) reveal V600E mutations in the <i>BRAF</i> gene, while being absent in gcGBM.</p

<i>MGMT</i> Promoter Methylation and <i>BRAF</i> V600E Mutations Are Helpful Markers to Discriminate Pleomorphic Xanthoastrocytoma from Giant Cell Glioblastoma

<div><p>Giant Cell Glioblastoma (gcGBM) and Pleomorphic Xanthoastrocytoma (PXA) are rare astroglial tumors of the central nervous system. Although they share certain histomorphological and immunohistochemical features, they are characterized by different clinical behavior and prognosis. Nevertheless, few cases remain uncertain, as their histomorphological hallmarks and immunophenotypes do correspond to the typical pattern neither of gcGBM nor PXA. Therefore, in addition to the routinely used diagnostic histochemical and immunohistochemical markers like Gömöri, p53 and CD34, we analyzed if genetic variations like <i>MGMT</i> promoter methylation, mutations in the <i>IDH1/2</i> genes, or <i>BRAF</i> mutations, which are actually used as diagnostic, prognostic and predictive molecular markers in anaplastic glial tumors, could be helpful in the differential diagnostic of both tumor entities. We analyzed 34 gcGBM and 20 PXA for genetic variations in the above-named genes and found distinct distributions between both groups. <i>MGMT</i> promoter hypermethylation was observed in 3 out of 20 PXA compared to 14 out of 34 gcGBM (15% vs. 41.2%, p-value 0.09). <i>BRAF</i> V600E mutations were detected in 50% of the PXA but not in any of the gcGBM (50% vs. 0%, p-value < 0.001). <i>IDH1</i> R132 and <i>IDH</i> R172 mutations were not present in any of the PXA and gcGBM cases. Our data indicate, that in addition to the histological and immunohistochemical evaluation, investigation of <i>MGMT</i> promoter methylation and in particular <i>BRAF</i> V600E mutations represent reliable additional tools to sustain differentiation of gcGBM from PXA on a molecular basis. Based on these data specific BRAF kinase inhibitors could represent a promising agent in the therapy of PXA and their use should be emphasized.</p></div

Comparative marker distribution in gcGBM and PXA.

<p>Comparative marker distribution in gcGBM and PXA.</p

Expression of ZEB1 in human glial tumors and normal brain.

<p>(A) Biopsy samples of human glial tumors show abundant nuclear expression of ZEB1. Sections from non-neoplastic normal brain show weak cytoplasmic staining of neurons and nuclear expression in astrocytes. (B) Quantification of ZEB1 in full histological sections of gross total resections of human glial tumors. ZEB1 positive cells were quantified using automated image analysis. Dots represent the mean of multiple regions of interest (ROI) per tumor. Box plots show the distribution of the ZEB1 labelling in tumor with indicated integrated diagnosis according the 2016 WHO classification of CNS tumors [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185376#pone.0185376.ref029" target="_blank">29</a>]. (C) <i>ZEB1</i> mRNA expression in public GBM cDNA microarray datasets. Boxplots show distribution in normal brain vs. GBM. Data were retrieved via the GlioVis portal [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185376#pone.0185376.ref030" target="_blank">30</a>].</p

ZEB1 expression in the tumor microenvironment.

<p>(A) Cell type specific expression of ZEB1 in reactive brain tissue. Human brain biopsy samples from cases of seizure-induced reactive gliosis or subacute infarction were co-stained for ZEB1 and Iba1 for microglia, CD45 for leucocytes, CD68 for macrophages and GFAP for astrocytes, respectively. Scale bars 50 μm. (B) Co-labeling of ZEB1 and CD68 or HLA-DR, respectively in human GBM. (C,D) Correlation of microarray-based gene expression levels of <i>ZEB1</i> and myeloid cell markers <i>CD68</i> (C) and <i>AIF1</i> (D), respectively. Processed log₂-transformed intensities from 157 GBM cases studied by Gravendeel et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185376#pone.0185376.ref034" target="_blank">34</a>] were obtained via the GlioVis portal [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185376#pone.0185376.ref030" target="_blank">30</a>].</p

Intratumoral heterogeneity of ZEB1 expression.

<p>(A) Co-labeling of IDH1 R132H and ZEB1 in a case of IDH1 mutant glioblastoma. The image shown has been taken from one out of ten fields of view that were subjected to separate and blinded manual scoring of mutant IDH1 and ZEB1 expression. (B) ZEB1 gradient along the tumor edge of a mesenchymal GBM (BLN-7 parental tumor). ZEB1 IHC, overall cellularity (<i>blue</i>), the relative frequency of ZEB1+ cells (<i>red</i>) and the mean nuclear intensity of ZEB1+ cells (<i>green</i>) at the tumor edge are shown. (C,D) Expression of ZEB1 (<i>pink</i>) and CD68 (<i>brown</i>) in perinecrotic regions with (C) or without (D) pseudopalisades.</p