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Background: NF1 inactivation is associated with sensitivity to MEK inhibitor targeted therapy in low-grade and some high-grade gliomas. NF1 loss may also be a harbinger of exploitable vulnerabilities in IDH-wildtype glioblastoma (GBM). Accurate and consistent detection of NF1 loss, however, is fraught given the large gene size, challenges with complete coverage and variant calling upon sequencing, and mechanisms of mRNA and protein regulation that result in early degradation in the absence of genomic alterations. Here, we seek to perform a composite analysis for NF1 loss accounting for genomic alterations and protein expression via immunohistochemistry. We also characterize the landscape of NF1 alterations in GBM., Methods: We assembled a single-institution, retrospective cohort of 542 IDH-wildtype GBM with somatic next generation sequencing to investigate the frequency and nature of detected NF1 alterations. We selected 69 GBMs from which to build a tissue microarray (TMA) of 44 NF1-wildtype and 25 NF1-mutant cases. We performed NF1 immunohistochemistry using two different NF1 antibodies (NFC, Sigma-Aldrich; and iNF-07E, iNFixion Bioscience) and correlated results with clinical, genomic, and other immunohistochemical features., Results: In our retrospective cohort, we identified 88 IDH-wildtype GBM with NF1 alterations (16%). NF1 alterations were mutually exclusive with EGFR and MDM2 alterations (p-adj < 0.001, 0.05, respectively), but co-occurred with PIK3R1 alterations (Log 2 (OR) = - 1.6, p-adj = 0.03). Of the 63 scorable sporadic GBMs in the TMA, 14 harbored NF1 inactivating alterations and of those, 12 (86%) demonstrated minimal NF1 immunoreactivity by NFC antibody, compared to 8 (57%) by iNF-07E antibody. Among the 42 scorable NF1-wildtype GBM in the TMA, NF1 immunostaining was minimal in 18 (43%) by NFC antibody compared to 4 (10%) by iNF-07E antibody, potentially reflecting false positives or differential protein regulation. Minimal immunoreactivity by NFC antibody was associated with decreased median overall survival (8.5 vs. 16.4 months, p = 0.011). Cox proportional hazards model correcting for prognostic variables in this subset revealed HR 3.23 (95% CI 1.29-8.06, p = 0.01) associated with decreased NF1 expression by IHC., Conclusion: NF1 immunostaining may serve as a sensitive surrogate marker of NF1 genomic inactivation and a valuable extension to next-generation sequencing for defining NF1 status. Minimal NF1 immunoreactivity is a poor prognostic marker, even in IDH-wildtype glioblastoma without apparent NF1 genomic alterations, but the underlying molecular mechanism requires further investigation., (© 2024. The Author(s).)

Background: Diffusion MRI estimates of the apparent diffusion coefficient (ADC) have been shown to be useful in predicting treatment response in patients with glioblastoma (GBM), with ADC elevations indicating tumor cell death. We aimed to investigate whether the ADC values measured before and after treatment with immune checkpoint inhibitors (ICIs) and the changes in these ADC values could predict overall survival (OS) in patients with recurrent IDH wild-type GBM., Methods: Forty-four patients who met the following inclusion criteria were included in this retrospective study: (i) diagnosed with recurrent IDH wild-type GBM and treated with either pembrolizumab or nivolumab and (ii) availability of diffusion data on pre- and post-ICI MRI. Tumor volume and the median relative ADC (rADC) with respect to the normal-appearing white matter within the enhancing tumor were calculated., Results: Median OS among all patients was 8.1 months (range, 1.0-22.5 months). Log-rank test revealed that higher post-treatment rADC was associated with a significantly longer OS (median, 10.3 months for rADC ≥ 1.63 versus 6.1 months for rADC < 1.63; P = .02), whereas tumor volume, pretreatment rADC, and changes in rADC after treatment were not significantly associated with OS. Cox regression analysis revealed that post-treatment rADC significantly influenced OS (P = .02, univariate analysis), even after controlling for age and sex (P =.01, multivariate analysis), and additionally controlling for surgery after ICI treatment (P = .045, multivariate analysis)., Conclusions: Elevated post-treatment rADC may be an early imaging biomarker for OS benefits in GBM patients receiving ICI treatment., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Subsets of high-grade gliomas, including glioblastoma (GBM), are known to utilize the alternative lengthening of telomeres (ALT) pathway for telomere length maintenance. However, the telomere maintenance profile of one subtype of GBM-giant cell GBM-has not been extensively studied. Here, we investigated the prevalence of ALT, as well as ATRX and SMARCAL1 protein loss, in a cohort of classic giant cell GBM and GBM with giant cell features. To determine the presence of ALT, a telomere-specific fluorescence in situ hybridization assay was performed on 15 cases of classic giant cell GBM, 28 additional GBMs found to have giant cell features, and 1 anaplastic astrocytoma with giant cell features. ATRX, SMARCAL1, and IDH1 protein status were assessed in a proportion of cases by immunohistochemistry and were compared to clinical-pathologic and molecular characteristics. In the overall cohort of 44 cases, 19 (43%) showed evidence of ALT. Intriguingly, of the ALT-positive cases, only 9 (47.4%) displayed loss of the ALT suppressor ATRX by immunohistochemistry. Since inactivating mutations in SMARCAL1 have been identified in ATRX wild-type ALT-positive gliomas, we developed an immunohistochemistry assay for SMARCAL1 protein expression using genetically validated controls. Of the 19 ALT-positive cases, 6 (31.5%) showed loss or mis-localization of SMARCAL1 by immunohistochemistry. Of these cases, four retained ATRX protein expression, while two cases also displayed ATRX loss. Additionally, we assessed five cases from which multiple temporal samples were available and ALT status was concordant between both tumor biopsies. In summary, we have identified a subset of giant cell GBM that utilize the ALT telomere maintenance mechanism. Importantly, in addition to ATRX loss, ALT-positive tumors harboring SMARCAL1 alterations are prevalent in giant cell GBM., (© 2021. The Author(s), under exclusive licence to United States & Canadian Academy of Pathology.)

Detection of BRAF V600E mutation in glioblastomas (GBMs) is important because of potential therapeutic implications. Still, the relative paucity of these mutations makes molecular detection in all GBMs controversial. In the present study, we analyzed clinical, radiographic and pathologic features of 12 BRAF V600E-mutant GBMs and 12 matched controls from 2 institutions. We found that a majority of BRAF V600E-mutant GBMs displayed a combination of well-circumscribed lesions, large cystic components with thin walls and solid cortical component on MRI, but with some overlap with matched BRAF wildtype controls (p = 0.069). BRAF V600E-mutant GBMs were also apt to gross total resection (83% vs 17%, p = 0.016) and morphologically displayed epithelioid features (83% vs 0%, p < 0.0001). Identification of these clinical, radiographic, and pathologic characteristics should prompt testing for BRAF V600E in IDH-wildtype GBM., (© 2021. The Japan Society of Brain Tumor Pathology.)

Background: Gliomas with 1p/19q-codeletion as well as mutation of isocitrate dehydrogenase (IDH) 1 are typically characterized as oligodendrogliomas with comparatively good response to treatment with radiation and chemotherapy., Case Presentation: We present the case of a 28-year-old man with an IDH1 and TP53 mutant high grade glioma with abnormalities in chromosomes 1 and 19 suggestive of anaplastic oligodendroglioma that rapidly progressed to widespread metastatic disease. Biopsy of a liver lesion confirmed metastasis of the patient's known brain primary and chemotherapy with temozolomide was initiated. The patient's rapidly growing tumor burden with fulminant liver failure and tumor lysis led to multisystem failure of which the patient died. Further molecular testing illustrated features more consistent with glioblastoma: multiple large chromosomal aberrations including loss of whole chromosome 1 and 2q; gain/amplification of MYCN, MET, and CDK4; loss of CDKN2A/B; and an ATRX mutation., Conclusion: This case illustrates the importance of higher level molecular diagnostic testing for patients with particularly aggressive disease progression that is not concordant with standard prognoses. Additional data on cases with atypical alterations of 1p and 19q are needed to better understand the distinct biology of these cancers so that appropriate therapies can be developed.

Purpose: Clinical factors and neuro-imaging in patients with glioblastoma who appear to progress following standard chemoradiation are unable to reliably distinguish tumor progression from pseudo-progression. As a result, surgery is commonly recommended to establish a final diagnosis. However, studies evaluating the pathologists' agreement on pathologic diagnoses in this setting have not been previously evaluated., Methods: A hypothetical clinical history coupled with images of histological sections from 13 patients with glioblastoma who underwent diagnostic surgery for suspected early recurrence were sent to 101 pathologists from 50 NCI-designated Cancer Centers. Pathologists were asked to provide a final diagnosis (active tumor, treatment effect, or unable to classify) and to report on percent active tumor, treatment effect, and degree of cellularity and degree of mitotic activity., Results: Forty-eight pathologists (48%) from 30 centers responded. In three cases > 75% of pathologists diagnosed active tumor. In two cases > 75% diagnosed treatment effect. However, in the remaining eight cases the disparity in diagnoses was striking (maximum agreement on final diagnosis ranged from 36 to 68%). Overall, only marginal agreement was observed in the overall assessment of disease status [kappa score 0.228 (95% CI 0.22-0.24)]., Conclusions: Confidence in any clinical diagnostic assay requires that very similar results are obtained from identical specimens evaluated by sophisticated clinicians and institutions. The findings of this study illustrate that the diagnostic agreement between different cases of repeat resection for suspected recurrent glioblastoma can be variable. This raises concerns as pathological diagnoses are critical in directing standard and experimental care in this setting.

There has been controversy about the presence and potential role of aquaporin-4 (AQP4) in glioblastoma (GBM). We analyzed tissue from 22 patients with newly-diagnosed GBM as well as matching tissue from 17 of these cases who underwent repeat resection for suspected recurrence and performed immunohistochemical analysis for AQP-4 expression. While some degree of AQP4 expression was detected in all 22 cases (39 samples), there was no clear relationship between staining pattern and disease status (active versus inactive GBM) between baseline and time of repeat biopsy. In addition, there was no clear relationship between AQP4 expression and degree of edema.

The majority of glioblastomas can be classified into molecular subgroups based on mutations in the TERT promoter (TERTp) and isocitrate dehydrogenase 1 or 2 (IDH). These molecular subgroups utilize distinct genetic mechanisms of telomere maintenance, either TERTp mutation leading to telomerase activation or ATRX-mutation leading to an alternative lengthening of telomeres phenotype (ALT). However, about 20% of glioblastomas lack alterations in TERTp and IDH. These tumors, designated TERTp WT -IDH WT glioblastomas, do not have well-established genetic biomarkers or defined mechanisms of telomere maintenance. Here we report the genetic landscape of TERTp WT -IDH WT glioblastoma and identify SMARCAL1 inactivating mutations as a novel genetic mechanism of ALT. Furthermore, we identify a novel mechanism of telomerase activation in glioblastomas that occurs via chromosomal rearrangements upstream of TERT. Collectively, our findings define novel molecular subgroups of glioblastoma, including a telomerase-positive subgroup driven by TERT-structural rearrangements (IDH WT -TERT SV ), and an ALT-positive subgroup (IDH WT -ALT) with mutations in ATRX or SMARCAL1.

Purpose: Reports of cytomegalovirus (CMV) detection in high-grade gliomas (HGG)/glioblastoma have been conflicting. We undertook a comprehensive approach to determine the presence or absence of CMV in tissue, plasma, and serum of HGG patients. Experimental Design: In a retrospective arm, 25 fresh frozen tissues from glioblastoma patients were tested for CMV by real-time PCR. Tissue microarrays from 70 HGG patients were tested by IHC and 20 formalin-fixed paraffin-embedded (FFPE) glioblastoma tissues by IHC and chromogenic in situ hybridization (CISH), targeting CMV-encoded IE1/2 and pp65. In a prospective arm, 18 patients with newly diagnosed HGG provided tissue and blood samples. Results: All retrospectively collected tissues were negative for CMV by all methods. In the prospective cohort, 18 patients with newly diagnosed HGG provided blood samples at the time of diagnosis and during follow-up. Of 38 plasma specimens, CMV DNA was detected in 3 of 18 samples at baseline and 1 of 20 follow-up samples. Serum CMV IgG was positive in 8 of 15 (53%) of patients. Among the FFPE samples tested in the prospective arm, all were negative for CMV by IHC, CISH, and PCR. Conclusions: Utilizing 6 highly sensitive assays with three orthogonal technologies on multiple specimens and specimen types, no evidence for CMV in glioblastoma tissues was found. Our findings call for multicenter blinded analyses of samples collected from different geographical areas with agreed upon study designs and determination of causality or lack thereof of CMV in HGG/glioblastoma for future guidance on the necessary antiviral and/or CMV-based therapies. Clin Cancer Res; 23(12); 3150-7. ©2016 AACR ., (©2016 American Association for Cancer Research.)

Glioblastoma (GBM) contains a population of stem-like cells that promote tumor invasion and resistance to therapy. Identifying and targeting stem cell factors in GBM may lead to the development of more effective therapies. High Mobility Group AT-hook 2 (HMGA2) is a transcriptional modulator that mediates motility and self-renewal in normal and cancer stem cells. We identified increased expression of HMGA2 in the majority of primary human GBM tumors and cell lines compared to normal brain. Additionally, HMGA2 expression was increased in CD133+ GBM neurosphere cells compared to CD133- cells. Targeting HMGA2 with lentiviral short hairpin RNA (shRNA) led to decreased GBM stemness, invasion, and tumorigenicity. Ectopic expression of HMGA2 in GBM cell lines promoted stemness, invasion, and tumorigenicity. Our data suggests that targeting HMGA2 in GBM may be therapeutically beneficial., Competing Interests: None., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Src-family kinase (SFK) signaling impacts multiple tumor-related properties, particularly in the context of the brain tumor glioblastoma. Consequently, the pan-SFK inhibitor dasatinib has emerged as a therapeutic strategy, despite physiologic limitations to its effectiveness in the brain. We investigated the importance of individual SFKs (Src, Fyn, Yes, and Lyn) to glioma tumor biology by knocking down individual SFK expression both in culture (LN229, SF767, GBM8) and orthotopic xenograft (GBM8) contexts. We evaluated the effects of these knockdowns on tumor cell proliferation, migration, and motility-related signaling in culture, as well as overall survival in the orthotopic xenograft model. The four SFKs differed significantly in their importance to these properties. In culture, Src, Fyn, and Yes knockdown generally reduced growth and migration and altered motility-related phosphorylation patterns while Lyn knockdown did so to a lesser extent. However the details of these effects varied significantly depending on the cell line: in no case were conclusions about the role of a particular SFK applicable to all of the measures or all of the cell types examined. In the orthotopic xenograft model, mice implanted with non-target or Src or Fyn knockdown cells showed no differences in survival. In contrast, mice implanted with Yes knockdown cells had longer survival, associated with reduced tumor cell proliferation. Those implanted with Lyn knockdown cells had shorter survival, associated with higher overall tumor burden. Together, our results suggest that Yes signaling directly affects tumor cell biology in a pro-tumorigenic manner, while Lyn signaling affects interactions between tumor cells and the microenvironment in an anti-tumor manner. In the context of therapeutic targeting of SFKs, these results suggest that pan-SFK inhibitors may not produce the intended therapeutic benefit when Lyn is present., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Aims: Pleomorphic xanthoastrocytoma (PXA) is a rare astrocytic neoplasm with a relative circumscribed architecture that typically arises superficially in the cerebral hemispheres of teenagers and young adults. Our aim is to highlight unconventional clinical presentations of this distinct neoplasm., Materials and Methods: We report two cases of PXA with unconventional clinical features, including clinical, pathologic, and immunohistochemical features., Results: The first case developed in the left frontal lobe of a 20-year-old female with neurofibromatosis type 1 (NF1). Focal anaplastic features were present. The neoplastic cells were immunoreactive for GFAP, S-100 protein and focally for synaptophysin, with a MIB-1/Ki-67 proliferative labelling index of 16%. The second case developed in a 39-year-old female as a suprasellar neoplasm. The neoplastic cells expressed GFAP, S-100 protein and focally CD34. The adenohypophysis was positive for synaptophysin and pituicytes for TTF1. Molecular studies were negative for BRAF (V600E) mutation in both cases., Conclusion: PXA is a distinct circumscribed neoplasm that may present in unexpected locations or clinical backgrounds. Neuropathologists must be aware of these unconventional presentations in order to provide a precise diagnosis leading to appropriate treatment.

Growing evidence supports a role for the unfolded protein response (UPR) in carcinogenesis; however, the precise molecular mechanisms underlying this phenomenon remain elusive. Herein, we identified the circadian clock PER1 mRNA as a novel substrate of the endoribonuclease activity of the UPR sensor IRE1α. Analysis of the mechanism shows that IRE1α endoribonuclease activity decreased PER1 mRNA in tumor cells without affecting PER1 gene transcription. Inhibition of IRE1α signaling using either siRNA-mediated silencing or a dominant-negative strategy prevented PER1 mRNA decay, reduced tumorigenesis, and increased survival, features that were reversed upon PER1 silencing. Clinically, patients showing reduced survival have lower levels of PER1 mRNA expression and increased splicing of XBP1, a known IRE-α substrate, thereby pointing toward an increased IRE1α activity in these patients. Hence, we describe a novel mechanism connecting the UPR and circadian clock components in tumor cells, thereby highlighting the importance of this interplay in tumor development., (©2013 AACR.)

A heterogeneous population of uncommon neoplasms of the central nervous system (CNS) cause significant morbidity and mortality. To explore their genetic origins, we sequenced the exomes of 12 pleomorphic xanthoastrocytomas (PXA), 17 non-brainstem pediatric glioblastomas (PGBM), 8 intracranial ependymomas (IEP) and 8 spinal cord ependymomas (SCEP). Analysis of the mutational spectra revealed that the predominant single base pair substitution was a C:G>T:A transition in each of the four tumor types. Our data confirm the critical roles of several known driver genes within CNS neoplasms, including TP53 and ATRX in PGBM, and NF2 in SCEPs. Additionally, we show that activating BRAF mutations play a central role in both low and high grade glial tumors. Furthermore, alterations in genes coding for members of the mammalian target of rapamycin (mTOR) pathway were observed in 33% of PXA. Our study supports the hypothesis that pathologically similar tumors arising in different age groups and from different compartments may represent distinct disease processes with varied genetic composition.

Microvascular proliferation is a key biological and diagnostic hallmark of human glioblastoma, one of the most aggressive forms of human cancer. It has recently been suggested that stem-like glioblastoma cells have the capacity to differentiate into functional endothelial cells, and that a significant proportion of the vascular lining in tumors has a neoplastic origin. In principle, this finding could significantly impact the efficacy and development of antiangiogenic therapies targeting the vasculature. While the potential of stem-like cancer cells to form endothelium in culture seems clear, in our clinical experience using a variety of molecular markers, neoplastic cells do not contribute significantly to the endothelial-lined vasculature of primary human glioblastoma. We sought to confirm this impression by analyzing vessels in glioblastoma previously examined using chromogenic in situ hybridization (CISH) for EGFR and immunohistochemistry for mutant IDH1. Vessels containing cells expressing these definitive neoplastic markers were identified in a small fraction of tumors, but only 10% of vessel profiles examined contained such cells and when identified these cells comprised less than 10% of the vascular cellularity in the cross section. Interestingly, these rare intravascular cells showing EGFR amplification by CISH or mutant IDH1 protein by immunohistochemistry were located in the middle or outer portions of vessel walls, but not amongst the morphologic boundaries of the endothelial lining. To more directly address the capacity of glioblastoma cells to contribute to the vascular endothelium, we performed double labeling (Immunofluorescence/FISH) for the endothelial marker CD34 and EGFR gene locus. This analysis did not identify EGFR amplified CD34+ endothelial cells within vascular linings, and further supports our observation that incorporation of glioblastoma cells into the tumor vessels is, at best, extremely rare of questionable clinical or therapeutic significance.

Background: Glioblastomas exhibit a remarkable tendency toward morphologic diversity. Although rare, pseudoepithelial components (adenoid or epithelioid) or true epithelial differentiation may occur, posing a significant diagnostic challenge., Methods: Hematoxylin and eosin-stained slides were reviewed, and immunohistochemistry and fluorescence in situ hybridization were performed., Results: The patients included 38 men and 20 women. The median age at diagnosis was 57 years (interquartile range [IQR], 50 years-67 years), and the median overall survival was 7 months (IQR, 4 months-11 months). "Adenoid" glioblastomas (A-GBM) predominated (48%). True epithelial glioblastomas (TE-GBM) were next most frequent based on morphology and immunohistochemistry (35%), followed by epithelioid glioblastomas (E-GBM) (17%). Overall, 25 (43%) tumors featured a sarcomatous component. Molecular cytogenetic abnormalities identified by fluorescent in situ hybridization in A-GBM, E-GBM, and TE-GBM, respectively, included p16 deletion/-9 (60%, 71%, 64%); chromosome 10 loss (40%, 63%, 57%), chromosome 7 gain without EGFR amplification (70%, 38%, 40%), EGFR amplification (10%, 50%, 27%), PTEN deletion (10%, 25%, 29%), PDGFRA amplification (10%, 25%, 0%), and RB1 deletion/-13q (50%, 0%, 14%). Abnormalities identified by immunohistochemistry included p21 immunonegativity (60%, 25%, 93%), which was most frequent in TE-GBM (P = .008), strong nuclear p53 staining (29%, 29%, 41%), strong membranous staining for epidermal growth factor receptor (EGFR) (21%, 63%, 19%), which was most frequent in E-GBM (P = .03), and an increased frequency of p27 immunonegativity in gliosarcomas (15% negative, 85% focal) compared with tumors without sarcoma (38% strongly positive) (P = .009)., Conclusions: Pseudoepithelial and true epithelial morphology are rare phenomena in GBM and may be associated with a similar poor prognosis. These tumors demonstrate proportions of molecular genetic abnormalities varying somewhat from conventional GBM.

O6-methylguanine-DNA methyltransferase (MGMT) expression has been recently proposed as a useful prognostic and/or predictive marker in glioblastoma patients receiving adjuvant therapy after the surgery. We studied samples from 50 patients with histologically confirmed GBM to evaluate MGMT expression by immunohistochemistry and its relation to promoter methylation status. Genomic DNA was extracted from scrapings of formalin-fixed, paraffin-embedded tissue corresponding to hematoxylin and eosin sections. Using the mouse monoclonal antibody MT3.1, MGMT expression was assessed and scored in tumor cells: (1=negative or limited to <10% positive tumor cells, 2=10% to 50%, 3=>50%). Methylation-specific polymerase chain reaction was performed after bisulfite treatment. Assessment of MGMT expression in neoplastic tissue required careful scrutiny because of its expression in a variety of non-neoplastic cells. MGMT expression was present in tumor cells with a score of 1, 2, and 3, respectively in 36 (72%), 13 (26%), and 1 (2%) cases. Methylation-specific polymerase chain reaction yielded interpretable results in 39 cases (78%). MGMT promoter methylation was detected in 15 cases (38.5%), whereas 24 (61.5%) were unmethylated. Among the methylated samples, 14 (of 15) had a score of 1, and 1 had a score of 3 by immunohistochemistry. Of the 24 unmethylated samples, 18 had a score of 1, and 6 of 2. There was no significant correlation between MGMT expression and methylation, and no significant survival difference was observed between patients whose tumors were negative versus positive for MGMT protein by immunohistochemistry. This study underscores some of the difficulties in applying immunohistochemistry to assess MGMT expression.

Granular cell astrocytoma (GCA) is a rare adult infiltrating glioma subtype. We studied a series of 39 GCAs. Median age of presentation was 57.8 years and most cases developed in the frontal or temporal lobes. Tumors included grade II (n = 14), grade III (n = 11), and grade IV (n = 14) by WHO criteria. Granular cell morphology was diffuse in 31 (79%) cases and partial in eight (21%). Immunohistochemistry showed frequent positivity for GFAP (28 of 31), OLIG2 (16 of 16), and CD68 (27 of 30), but HAM56, CD163, and IBA‐1 histiocytic markers were all negative (22 of 22). IDH1(R132H) was negative in all the cases tested (16 of 16), while ATRX expression was retained (12 of 12). Cytogenetics demonstrated monosomy 10 (6 of 6) cases, +7 in 4 (of 6), −13q in 4 of 6, and −14 in 4 of 6. Next‐generation sequencing demonstrated mutations in PTEN/PIK3 genes in 6/13 (46%), NF1 in 3 of 10 (30%), TP53 in 3 of 13 (23%), PALB2 in 3 of 10 (30%), STAG2 in 3 of 10 (30%), EGFR mutation/amplification in 3 of 13 (23%), and AR in 2 of 10 (20%). CDKN2A/B deletion was identified in 5 of 13 (30%) cases (homozygous deletion in 4). The TERT C228T mutation was identified in 9 of 13 (69%). No mutations were encountered in IDH1,IDH2,CIC,FUBP1, H3F3A,BRAF or ATRX genes. The mean overall survival was 11.3 months. Patients >60 years old at diagnosis had a worse survival than patients <60 years (P = 0.001). There were no statistically significant differences in survival by WHO grade, extent of granular cell change, sex or MIB‐1 (P > 0.05). GCA is a variant of IDH‐wildtype diffuse glioma with aggressive behavior irrespective of grade and extent of granular cell morphology, and with molecular genetic features corresponding to primary glioblastoma. [ABSTRACT FROM AUTHOR]

Recent studies suggest that the telomere maintenance mechanism known as alternative lengthening of telomeres ( ALT) is relatively more common in specific glioma subsets and strongly associated with ATRX mutations. We retrospectively examined 116 high-grade astrocytomas (32 pediatric glioblastomas, 65 adult glioblastomas, 19 anaplastic astrocytomas) with known ALT status using tissue microarrays to identify associations with molecular and phenotypic features. Immunohistochemistry was performed using antibodies against ATRX, DAXX, p53 and IDH1R132H mutant protein. EGFR amplification was evaluated by fluorescence in situ hybridization ( FISH). Almost half of fibrillary and gemistocytic astrocytomas (44%) demonstrated ALT. Conversely all gliosarcomas (n = 4), epithelioid (n = 2), giant cell (n = 2) and adult small cell astrocytomas (n = 7) were ALT negative. The ALT phenotype was positively correlated with the presence of round cells ( P = 0.002), microcysts ( P < 0.0002), IDH1 mutant protein ( P < 0.0001), ATRX protein loss ( P < 0.0001), strong P53 immunostaining ( P < 0.0001) and absence of EGFR amplification ( P = 0.004). There was no significant correlation with DAXX expression. We conclude that ALT represents a specific phenotype in high-grade astrocytomas with distinctive pathologic and molecular features. Future studies are required to clarify the clinical and biological significance of ALT in high-grade astrocytomas. [ABSTRACT FROM AUTHOR]

Microvascular proliferation is a key biological and diagnostic hallmark of human glioblastoma, one of the most aggressive forms of human cancer. It has recently been suggested that stem-like glioblastoma cells have the capacity to differentiate into functional endothelial cells, and that a significant proportion of the vascular lining in tumors has a neoplastic origin. In principle, this finding could significantly impact the efficacy and development of antiangiogenic therapies targeting the vasculature. While the potential of stem-like cancer cells to form endothelium in culture seems clear, in our clinical experience using a variety of molecular markers, neoplastic cells do not contribute significantly to the endothelial-lined vasculature of primary human glioblastoma. We sought to confirm this impression by analyzing vessels in glioblastoma previously examined using chromogenic in situ hybridization (CISH) for EGFR and immunohistochemistry for mutant IDH1. Vessels containing cells expressing these definitive neoplastic markers were identified in a small fraction of tumors, but only 10% of vessel profiles examined contained such cells and when identified these cells comprised less than 10% of the vascular cellularity in the cross section. Interestingly, these rare intravascular cells showing EGFR amplification by CISH or mutant IDH1 protein by immunohistochemistry were located in the middle or outer portions of vessel walls, but not amongst the morphologic boundaries of the endothelial lining. To more directly address the capacity of glioblastoma cells to contribute to the vascular endothelium, we performed double labeling (Immunofluorescence/FISH) for the endothelial marker CD34 and EGFR gene locus. Although rare CD34 positive neoplastic cells unassociated with vessels were identified (<1%), this analysis did not identify EGFR amplified cells within vascular linings, and further supports our observations that incorporation of glioblastoma cells into the tumor vessels is at best extremely rare, and therefore of questionable clinical or therapeutic significance.