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We previously reported a patient who had developed 2 glioblastomas at the age of 54 and 64 years, respectively. The first glioblastoma in the right frontal lobe was treated with surgery and radiotherapy. Ten years later, the patient developed a second, left frontal glioblastoma. Discordant patterns of TP53 and PTEN mutations suggested that the second tumor was not a recurrence but an independently developed glioblastoma. To determine the molecular mechanism underlying this enigmatic case with 10-year survival, we performed whole-exome sequencing. We found that both tumors were IDH-wildtype, excluding the possibility of secondary glioblastomas that developed from a less malignant astrocytic precursor lesion. We here report that the patient carried a heterozygous germline mutation [c.3305_3306insT; p.1102-fs-insT(Gly1105/TrpfsX3)] in the MSH6 mismatch repair gene. Further sequencing revealed that in addition to the germline MSH6 mutation, the first glioblastoma showed loss of the MSH6 wild-type allele, and the second glioblastoma carried a somatic MSH6 mutation [c.1403G>A; p.Arg468His]. Our results indicate that both glioblastomas had 2 hits in the MSH6 gene, and that loss of MSH6 function was the key event in the pathogenesis of these 2 independent primary glioblastomas.

Precise classification of nervous system tumors into coherent clinicopathologic entities is the necessary first step for the appropriate management of patients. While this has been traditionally performed by histopathology only, recent accelerations in our understanding of the molecular features of nervous system tumors have led to the integration of molecular characteristics into diagnostic criteria. The updated WHO 2016 classification of tumors of the central nervous system (CNS)1 thus includes both histologic and molecular features—primarily mutational status of individual genes and copy number alterations—facilitating the precise diagnosis of CNS tumors. While this has led to increased objectivity and reproducibility, classification progress is still required, especially for rare and for poorly understood entities.

The majority of glioblastomas develop rapidly with a short clinical history (primary glioblastoma IDH wild-type), whereas secondary glioblastomas progress from diffuse astrocytoma or anaplastic astrocytoma. IDH mutations are the genetic hallmark of secondary glioblastomas. Gliosarcomas and giant cell glioblastomas are rare histological glioblastoma variants, which usually develop rapidly. We determined the genetic patterns of 36 gliosarcomas and 19 giant cell glioblastomas. IDH1 and IDH2 mutations were absent in all 36 gliosarcomas and in 18 of 19 giant cell glioblastomas analyzed, indicating that they are histological variants of primary glioblastoma. Furthermore, LOH 10q (88%) and TERT promoter mutations (83%) were frequent in gliosarcomas. Copy number profiling using the 450k methylome array in 5 gliosarcomas revealed CDKN2A homozygous deletion (3 cases), trisomy chromosome 7 (2 cases), and monosomy chromosome 10 (2 cases). Giant cell glioblastomas had LOH 10q in 50% and LOH 19q in 42% of cases. ATRX loss was detected immunohistochemically in 19% of giant cell glioblastomas, but absent in 17 gliosarcomas. These and previous results suggest that gliosarcomas are a variant of, and genetically similar to, primary glioblastomas, except for a lack of EGFR amplification, while giant cell glioblastoma occupies a hybrid position between primary and secondary glioblastomas.

The revised fourth edition of the WHO classification of Tumours of the Central Nervous System, published in 2016, comprises several newly recognized tumour entities, and a significant restructuring of the classification, mainly based on genetic profiling. Glioblastomas are now classified into two major types. Isocitrate dehydrogenase (IDH)-wildtype glioblastoma (primary glioblastoma IDH-wildtype) develops rapidly de novo without a recognizable precursor lesion. IDH-mutant glioblastoma (secondary glioblastoma IDH-mutant) develops more slowly through malignant progression from diffuse or anaplastic astrocytoma. Medulloblastomas are now defined by combining histological patterns (classic, desmoplastic/nodular, extensive nodularity, anaplastic) and genetic hallmarks (WNT-activated; SHH-activated, TP53-mutant; SHH-activated, TP53-wildtype; non-WNT/non-SHH). Other newly recognized tumour entities include diffuse midline glioma, H3 K27M-mutant; ependymoma, RELA fusion-positive; and embryonal tumour with multilayered rosettes. The new classification is a significant step forward and will facilitate the development of novel targeted therapies of brain tumours.

Major discoveries in the biology of nervous system tumors have raised the question of how non-histological data such as molecular information can be incorporated into the next World Health Organization (WHO) classification of central nervous system tumors. To address this question, a meeting of neuropathologists with expertise in molecular diagnosis was held in Haarlem, the Netherlands, under the sponsorship of the International Society of Neuropathology (ISN). Prior to the meeting, participants solicited input from clinical colleagues in diverse neuro-oncological specialties. The present "white paper" catalogs the recommendations of the meeting, at which a consensus was reached that incorporation of molecular information into the next WHO classification should follow a set of provided "ISN-Haarlem" guidelines. Salient recommendations include that (i) diagnostic entities should be defined as narrowly as possible to optimize interobserver reproducibility, clinicopathological predictions and therapeutic planning; (ii) diagnoses should be "layered" with histologic classification, WHO grade and molecular information listed below an "integrated diagnosis"; (iii) determinations should be made for each tumor entity as to whether molecular information is required, suggested or not needed for its definition; (iv) some pediatric entities should be separated from their adult counterparts; (v) input for guiding decisions regarding tumor classification should be solicited from experts in complementary disciplines of neuro-oncology; and (iv) entity-specific molecular testing and reporting formats should be followed in diagnostic reports. It is hoped that these guidelines will facilitate the forthcoming update of the fourth edition of the WHO classification of central nervous system tumors.

Telomerase reverse transcriptase (TERT) is up-regulated in a variety of human neoplasms. Mutations in the core promoter region of the TERT gene, which increases promoter activity, have been reported in melanomas and a variety of human neoplasms, including gliomas. In the present study, we screened for TERT promoter mutations by direct DNA sequencing in a population-based collection of 358 glioblastomas. TERT promoter mutations (C228T, C250T) were detected in 55 % glioblastomas analysed. Of these, 73 % had a C228T mutation, and 27 % had a C250T mutation; only one glioblastoma had both C228T and C250T mutations. TERT promoter mutations were significantly more frequent in primary (IDH1 wild-type) glioblastomas (187/322; 58 %) than in secondary (IDH1 mutated) glioblastomas (10/36, 28 %; P = 0.0056). They showed significant inverse correlations with IDH1 mutations (P = 0.0056) and TP53 mutations (P = 0.043), and a significant positive correlation with EGFR amplification (P = 0.048). Glioblastoma patients with TERT mutations showed a shorter survival than those without TERT mutations in univariate analysis (median, 9.3 vs. 10.5 months; P = 0.015) and multivariate analysis after adjusting for age and gender (HR 1.38, 95 % CI 1.01-1.88, P = 0.041). However, TERT mutations had no significant impact on patients' survival in multivariate analysis after further adjusting for other genetic alterations, or when primary and secondary glioblastomas were separately analysed. These results suggest that the prognostic value of TERT mutations for poor survival is largely due to their inverse correlation with IDH1 mutations, which are a significant prognostic marker of better survival in patients with secondary glioblastomas.

Glioblastoma is the most frequent and malignant brain tumor. The vast majority of glioblastomas (∼90%) develop rapidly de novo in elderly patients, without clinical or histologic evidence of a less malignant precursor lesion (primary glioblastomas). Secondary glioblastomas progress from low-grade diffuse astrocytoma or anaplastic astrocytoma. They manifest in younger patients, have a lesser degree of necrosis, are preferentially located in the frontal lobe, and carry a significantly better prognosis. Histologically, primary and secondary glioblastomas are largely indistinguishable, but they differ in their genetic and epigenetic profiles. Decisive genetic signposts of secondary glioblastoma are IDH1 mutations, which are absent in primary glioblastomas and which are associated with a hypermethylation phenotype. IDH1 mutations are the earliest detectable genetic alteration in precursor low-grade diffuse astrocytomas and in oligodendrogliomas, indicating that these tumors are derived from neural precursor cells that differ from those of primary glioblastomas. In this review, we summarize epidemiologic, clinical, histopathologic, genetic, and expression features of primary and secondary glioblastomas and the biologic consequences of IDH1 mutations. We conclude that this genetic alteration is a definitive diagnostic molecular marker of secondary glioblastomas and more reliable and objective than clinical criteria. Despite a similar histologic appearance, primary and secondary glioblastomas are distinct tumor entities that originate from different precursor cells and may require different therapeutic approaches. Clin Cancer Res; 19(4); 764–72. ©2012 AACR.

A single dose of N-ethyl-N-nitrosourea (ENU) during late prenatal or early postnatal development induces a high incidence of malignant schwannomas and gliomas in rats. Although T->A mutations in the transmembrane domain of the Neu (c-ErbB-2) gene are the driver mutations in ENU-induced malignant schwannomas, the molecular basis of ENU-induced gliomas remains enigmatic. We performed whole-genome sequencing of gliomas that developed in three BDIV and two BDIX rats exposed to a single dose of 80 mg ENU/kg body weight on postnatal day one. T:A->A:T and T:A->C:G mutations, which are typical for ENU-induced mutagenesis, were predominant (41% to 55% of all somatic single nucleotide mutations). T->A mutations were identified in all five rat gliomas at Braf codon 545 (V545E), which corresponds to the human BRAF V600E. Additional screening revealed that 33 gliomas in BDIV rats and 12 gliomas in BDIX rats all carried a Braf V545E mutation, whereas peritumoral brain tissue of either strain had the wild-type sequence. The gliomas were immunoreactive to BRAF V600E antibody. These results indicate that Braf mutation is a frequent early event in the development of rat gliomas caused by a single dose of ENU.

International audience; The 2016 World Health Organization Classificationof Tumors of the Central Nervous System is both aconceptual and practical advance over its 2007 predecessor.For the first time, the WHO classification of CNS tumors uses molecular parameters in addition to histology to define many tumor entities, thus formulating a concept for how CNS tumor diagnoses should be structured in the molecular era. As such, the 2016 CNS WHO presents major restructuring of the diffuse gliomas, medulloblastomas and other embryonal tumors, and incorporates new entities that are defined by both histology and molecular features, including glioblastoma, IDH-wildtype and glioblastoma, IDH-mutant; diffuse midline glioma, H3 K27M–mutant; RELA fusion–positive ependymoma; medulloblastoma, WNT-activated and medulloblastoma, SHH-activated; and embryonal tumour with multilayered rosettes, C19MC-altered. The 2016 edition has added newly recognized neoplasms, and has deleted some entities, variants and patterns that no longer have diagnostic and/or biological relevance. Other notable changes include the addition of brain invasion as a criterion for atypical meningioma and the introduction of a soft tissue-type grading system for the now combined entity of solitary fibrous tumor / hemangiopericytoma— a departure from the manner by which other CNS tumors are graded. Overall, it is hoped that the 2016 CNS WHO will facilitate clinical, experimental and epidemiological studies that will lead to improvements in the lives of patients with brain tumors.

The current World Health Organization classification recognizes three histological types of grade II low-grade diffuse glioma (diffuse astrocytoma, oligoastrocytoma, and oligodendroglioma). However, the diagnostic criteria, in particular for oligoastrocytoma, are highly subjective. The aim of our study was to establish genetic profiles for diffuse gliomas and to estimate their predictive impact. In this study, we screened 360 World Health Organization grade II gliomas for mutations in the IDH1, IDH2, and TP53 genes and for 1p/19q loss and correlated these with clinical outcome. Most tumors (86%) were characterized genetically by TP53 mutation plus IDH1/2 mutation (32%), 1p/19q loss plus IDH1/2 mutation (37%), or IDH1/2 mutation only (17%). TP53 mutations only or 1p/19q loss only was rare (2 and 3%, respectively). The median survival of patients with TP53 mutation ± IDH1/2 mutation was significantly shorter than that of patients with 1p/19q loss ± IDH1/2 mutation (51.8 months vs. 58.7 months, respectively; P = 0.0037). Multivariate analysis with adjustment for age and treatment confirmed these results (P = 0.0087) and also revealed that TP53 mutation is a significant prognostic marker for shorter survival (P = 0.0005) and 1p/19q loss for longer survival (P = 0.0002), while IDH1/2 mutations are not prognostic (P = 0.8737). The molecular classification on the basis of IDH1/2 mutation, TP53 mutation, and 1p/19q loss has power similar to histological classification and avoids the ambiguity inherent to the diagnosis of oligoastrocytoma.

Gliomas are the most common primary brain tumors. They account for more than 70% of all neoplasms of the central nervous system and vary considerably in morphology, location, genetic alterations, and response to therapy. Most frequent and malignant are glioblastomas. The vast majority (>90%) develops rapidly after a short clinical history and without evidence of a less malignant precursor lesion (primary or de novo glioblastoma). Secondary glioblastomas develop more slowly through progression from low-grade or anaplastic astrocytoma. These glioblastoma subtypes constitute distinct disease entities that affect patients of different age, develop through distinct genetic pathways, show different RNA and protein expression profiles, and may differ in their response to radio- and chemotherapy. Recently, isocitrate dehydrogenase 1 (IDH1) mutations have been identified as a very early and frequent genetic alteration in the pathway to secondary glioblastomas as well as that in oligodendroglial tumors, providing the first evidence that low-grade astrocytomas and oligodendrogliomas may share common cells of origin. In contrast, primary glioblastomas very rarely contain IDH1 mutations, suggesting that primary and secondary glioblastomas may originate from different progenitor cells, despite the fact that they are histologically largely indistinguishable. In this review, we summarize the current status of genetic alterations and signaling pathways operative in the evolution of astrocytic and oligodendroglial tumors.

Medulloblastoma (WHO grade IV) is an embryonal tumour of the cerebellum and the most common malignant central nervous system tumour in children. Despite significant advances in treatment, 5-year survival rates are still less than 70%, suggesting the presence of subgroups with different response to radio/chemotherapy. In the present study, we re-evaluated a series of 347 medulloblastomas from the SIOP II clinical trial of the International Society of Paediatric Oncology to identify features predictive of clinical outcome. Relapse free survival for medulloblastomas with severe anaplasia [5-year rate: S(60)=49.5%], was significantly shorter than for tumours with moderate or mild anaplasia S(60)=65.4%; P=0.001). The difference between both groups was even larger when the presence or absence of extensive apoptosis was included (46.5 vs. 66.7%; P=0.0216). Other histological features including nodularity, necrosis, vascular proliferation and the presence of beta-catenin mutations (7% of cases) were not predictive for relapse free survival. These findings indicate that degree of anaplasia is the most significant histologic feature predictive of the survival of medulloblastoma patients.

CD95 (Fas/APO‐1) and its ligand (CD95L) belong to a growing cytokine and cytokine receptor family that includes nerve growth factor (NGF) and tumor necrosis factor (TNF) and their corresponding receptors. CD95 expression increases during malignant progression from low‐grade to anaplastic astrocytoma and is most prominent in perinecrotic areas of glioblastoma. There is, however, no evidence that CD95 expression in malignant gliomas is triggered by hypoxia or ischemia. Agonistic antibodies to CD95, or the natural ligand, CD95L, induce apoptosis in human malignant glioma cells in vitro. Glioma cell sensitivity to CD95‐mediated apoptosis is regulated by CD95 expression at the cell surface and by the levels of intracellular apoptosis‐regulatory proteins, including bcl‐2 family members. Several cytotoxic drugs synergize with CD95L to kill glioma cells. For as yet unknown reasons, glioma cells may co‐express CD95 and CD95L in vitro without undergoing suicide or fratricide. Yet, they kill T cells via CD95/CD95L interactions and are sensitive to exogenously added CD95L. Since CD95L is expressed in gliomas in vivo, too, forced induction of CD95 expression might promote therapeutic apoptosis in these tumors. That glioma cells differ from non‐transformed T cells in their sensitivity to CD95 antibodies or recombinant ligand, may allow the development of selective CD95 agonists with high antitumor activity that spare normal brain tissue. A family of death ligand/receptor pairs related to CD95L/CD95, including APO2L (TRAIL) and its multiple receptors is beginning to emerge. Although several issues regarding glioma cell sensitivity to CD95L/CD95‐mediated apoptosis await elucidation, CD95 is a promising target for the treatment of malignant glioma.

Gliomas of astrocytic, oligodendroglial and ependymal origin account for more than 70% of all brain tumors. The most frequent (65%) and most malignant histological type is the glioblastoma. Since the introduction of computerized tomography and magnetic resonance imaging, the incidence rates of brain tumors have been rather stable, with a tendency of higher rates in highly developed, industrialized countries. Some reports indicate that Caucasians have higher incidence than black or Asian populations, but to some extent, this may reflect socio-economic differences and under-ascertainment in some regions, rather than a significant difference in genetic susceptibility. With the exception of pilocytic astrocytomas, the prognosis of glioma patients is still poor. Less than 3% of glioblastoma patients are still alive at 5 years after diagnosis, higher age being the most significant predictor of poor outcome. Brain tumors are a component of several inherited tumor syndromes, but the prevalence of these syndromes is very low. Several occupations, environmental carcinogens, and diet (N-nitroso compounds) have been reported to be associated with an elevated glioma risk, but the only environmental factor unequivocally associated with an increased risk of brain tumors, including gliomas, is therapeutic X-irradiation. In particular, children treated with X-irradiation for acute lymphoblastic leukemia show a significantly elevated risk of developing gliomas and primitive neuroectodermal tumor (PNET), often within 10 years after therapy. TP53 mutations are frequent in low-grade gliomas and secondary glioblastomas derived therefrom. Approximately 60% of mutations are located in the hot spot codons 248 and 273, and the majority of these are G:C→A:T transitions at CpG sites. TP53 mutations are significantly more frequent in low-grade astrocytomas with promoter methylation of the O 6 -methylguanine-DNA methyltransferase repair gene, suggesting that, in addition to deamination of 5-methylcytosine, exogenous or endogenous alkylation in the O 6 position of guanine may contribute to the formation of these mutations.

We conducted a population-based study on glioblastomas in the Canton of Zurich, Switzerland (population, 1.16 million) to determine the frequency of major genetic alterations and their effect on patient survival. Between 1980 and 1994, 715 glioblastomas were diagnosed. The incidence rate per 100,000 population/year, adjusted to the World Standard Population, was 3.32 in males and 2.24 in females. Observed survival rates were 42.4% at 6 months, 17.7% at 1 year, and 3.3% at 2 years. For all of the age groups, younger patients survived significantly longer, ranging from a median of 8.8 months (80 years). Loss of heterozygosity (LOH) 10q was the most frequent genetic alteration (69%), followed by EGFR amplification (34%), TP53 mutations (31%), p16INK4a deletion (31%), and PTEN mutations (24%). LOH 10q occurred in association with any of the other genetic alterations and was predictive of shorter survival. Primary (de novo) glioblastomas prevailed (95%), whereas secondary glioblastomas that progressed from low-grade or anaplastic gliomas were rare (5%). Secondary glioblastomas were characterized by frequent LOH 10q (63%) and TP53 mutations (65%). Of the TP53 mutations in secondary glioblastomas, 57% were in hotspot codons 248 and 273, whereas in primary glioblastomas, mutations were more equally distributed. G:C→A:T mutations at CpG sites were more frequent in secondary than primary glioblastomas (56% versus 30%; P = 0.0208). This suggests that the acquisition of TP53 mutations in these glioblastoma subtypes occurs through different mechanisms.

The histological diagnosis of low-grade astrocytomas and oligodendrogliomas (WHO grade II) is often challenging, particularly in cases that show both astrocytic and oligodendroglial differentiation. We carried out gene expression profiling on 17 oligodendrogliomas (93% with LOH 1p and/or 19q) and 15 low-grade astrocytomas (71% with a TP53 mutation), using a cDNA array containing 1176 cancer-related genes. In oligodendrogliomas, 40 genes showed on average higher expression (at least a two-fold increase) than in astrocytomas, including DES, TDGF1, TGF-beta, GABA-BR1A, Histone H4, CDKN1A, PCDH43, Rho7 and Jun-D, while 39 genes were expressed at lower levels (at least a two-fold decrease), including JNK2, ITGB4, JNK3A2, RhoC, IFI-56K, AAD14 and EGFR. Immunohistochemistry revealed nuclear staining of Jun-D in oligodendrogliomas, in contrast to the immunoreactivity of cytoplasm and cell processes in low-grade astrocytomas. Partial least-squares analysis of the 79 genes at least two-fold differentially expressed between oligodendrogliomas and low-grade astrocytomas demonstrated perfect separation of oligodendrogliomas from low-grade astrocytomas and normal cerebral white matter. Clustering analysis based on the entire gene set divided the 17 subjects with oligodendrogliomas into two subgroups with significantly different survival (log-rank test, P=0.0305; survival to 5-years, 80 vs 0%, P=0.048). These results demonstrate that oligodendrogliomas and low-grade astrocytomas differ in their gene expression profiles, and that there are subgroups of oligodendroglioma with distinct expression profiles related to clinical outcome.

Several national and international health agencies have established programs with the aim of identifying agents and exposures that cause cancer in humans. Carcinogen identification is an activity grounded in the scientific evaluation of the results of human epidemiologic studies, long-term bioassays in experimental animals, and other data relevant to an evaluation of carcinogenicity and its mechanisms. In this commentary, after a brief discussion of the science basis common to the evaluation of carcinogens across different programs, we discuss in more detail the principles and procedures currently used by the IARC Monographs program.

EGFR amplification is a frequent genetic alteration in primary (de novo) glioblastomas, and is often associated with structural alterations. Most common is variant III (EGFRvIII), which results from a non-random 801 bp in-frame deletion of exons 2 to 7 of the EGFR gene. We assessed amplification and overexpression of EGFRvIII and wild-type EGFR in 30 glioblastoma biopsies. Immunohistochemically, EGFR overexpression was observed in 20 (67%) of 30 glioblastomas. Eight (27%) cases also showed immunoreactivity to an EGFRvIII antibody. In 6 of these cases, the pattern of EGFR and EGFRvIII overexpression was compared in serial sections: In 4 cases, areas with immunoreactivity to EGFRvIII largely coincided with wild-type EGFR expression. In the other 2 cases, the areas immunoreactive to EGFRvIII were significantly less extensive than EGFR-positive areas. To assess whether EGFRvIII is predominantly amplified in tumors with concurrent wild-type EGFR amplification, we carried our real-time quantitative PCR using 2 sets of primers located in exon 2 and intron 15 of the EGFR gene. A > 5-fold ratio of relative copy numbers between intron 15 (present both in wild-type EGFR and EGFRvIII) and exon 2 (present only in wild-type EGFR, but missing in EGFRvIII) suggested predominant amplification of EGFRvIII in only 3 (10%) of 30 glioblastomas. The observation that intratumoral wild-type EGFR overexpression is often more extensive and that predominant amplification of EGFRvIII is a rare event would limit the effectiveness of therapeutic approaches based on selective targeting of EGFRvIII.

The tumor suppressor gene PTEN on chromosome 10q23.3 regulates the Akt signaling pathway and modulates cell growth and apoptosis. The PTEN gene is mutated in 20-40% of glioblastomas. In this study, we assessed whether loss of PTEN expression is also caused epigenetically. Methylation-specific PCR revealed that CpG islands of the PTEN promoter were methylated in 27 of 77 (35%) glioblastomas and in 4 of 11 (36%) glioblastoma cell lines. Only two glioblastomas showed loss of PTEN immunoreactivity in the entire biopsy; both had a missense PTEN mutation and LOH at the PTEN locus, but lacked PTEN methylation. In biopsy specimens with focal loss of PTEN expression, DNA samples extracted from microdissected foci showed PTEN methylation only in areas with loss of PTEN expression. These results suggest that PTEN methylation occurs frequently in glioblastomas and may be associated with focal loss of PTEN expression. However, the correlation between PTEN methylation, PTEN mutations, LOH at the PTEN locus, and loss of PTEN protein expression was inconsistent. Possible reasons for discrepancies between gene status and protein expression include differences in the biological effect of specific PTEN mutations and the possibility that the processed PTEN pseudogene on 9p21 is expressed in glioblastomas and co-reacts with the PTEN antibody.

Object. The incidence of pilocytic astrocytomas and the rate of patient survival were analyzed in a population-based study in the canton of Zürich, Switzerland. Methods. Between 1980 and 1994, 987 astrocytic and oligodendroglial tumors were diagnosed, of which 55 (5.5%) were pilocytic astrocytomas. The incidence rate, adjusted to the World Standard Population, was 4.8 per 1 million per year. The mean age at clinical diagnosis was 19.6 ± 12.7 years, and the male/female ratio was 1.12. The most frequent tumor sites were the cerebellum (40%), followed by supratentorial locations (35%), the optic pathway and hypothalamus (11%), and the brainstem (9%). The mean follow-up period was 12 years. Observed survival rates were 100% at 5 years and 95.8% at 10 years after diagnosis (relative survival rate at 10 years: 96.8%). Seven patients (13%) received postoperative radiotherapy, but this did not significantly affect survival. In all patients the tumors were histologically classified as WHO Grade I, except in two patients who had anaplastic pilocytic astrocytoma (Grade III), one of whom died after 7 years, whereas the other was still alive after 10 years. Conclusions. This population-based study shows that, because of the benign biological behavior of pilocytic astrocytomas and advances in microneurosurgery, the survival rates for patients with these tumors are excellent, regardless of postoperative radiotherapy.

The most frequent and malignant brain tumor is the glioblastoma, which may develop de novo (primary glioblastoma) or through progression from low-grade or anaplastic astrocytoma (secondary glioblastoma). These glioblastoma subtypes constitute distinct disease entities that affect patients at different. age, and evolve through different genetic pathways. Primary glioblastornas develop in older patients (mean age, 55 years) and typically show EGFR amplification / overexpression, LOH on the entire chromosome 10, PTEN mutations and, occasionally, MDM2 amplification. Secondary glioblastomas develop in younger patients (mean age, 40 years) and typically contain TP53 mutations and/or p14 A R F promoter methylation as earliest detectable alterations. Additional changes in the pathway leading to secondary glioblastomas include LOH on 19q and 10q, and RB1 promoter methylation. Common to both primary and secondary glioblastoma is LOH on 10q, distal to the PTEN locus; a putative suppressor gene at 10q25-qter may be largely responsible for the glioblastoma phenotype. The etiology of human gliomas is largely unknown. Hereditary diseases predisposing to the development of gliomas e.g. Li-Fraumeni syndrome, Turcot syndrome, NF1, and NF2 syndromes are rare and cannot explain the development, of most of human gliomas. The presence of SV40 large T sequence has been observed in a variety of human brain tumors including gliomas, and they are likely be originated from the contamination of SV40 in poliovaccine between 1955-1962. However, there is no direct evidence that SV40 infection is associated with pathogenesis of human brain tumors. There is recent evidence that G:C→A:T transition mutations at CpG sites in the TP53 gene are significantly more frequent in astrocytic tumors with promoter methylation of the O 5 -methylguanine-DNA methyltrasferase (MGMT) than in those without methylation. This may suggest that endogenous alkylating agents that produce O 6 -methylguanine or related adducts recognized by MGMT may be involved in the development of astrocytic brain tumors.

Oligodendrogliomas typically show loss of heterozygosity (LOH) on chromosomes 1p and 19q, which correlates with their response to chemotherapy, whereas low-grade astrocytomas are characterized by frequent TP53 mutations and lack of sensitivity to alkylating therapeutic agents. Unequivocal histological distinction of low-grade diffuse astrocytomas from oligodendrogliomas and oligoastrocytomas is often difficult. To elucidate the relationships between morphological phenotype and genetic profile, we screened 19 oligodendrogliomas (WHO grade II) and 23 low-grade diffuse astrocytomas (WHO grade II) for TP53 mutations and LOH on 1p and 19q. In oligodendrogliomas, LOH on chromosomes 1p and/or 19q was found in 15 cases (79%) and TP53 mutation was detected in 4 cases (21%). The presence of a typical perinuclear halo in50% of tumour cells and a chicken-wire vascular pattern were significantly associated with LOH on 1p or 19q (93% of cases). This suggests that oligodendrogliomas with classical histologic features are likely to have a better prognosis. In low-grade diffuse astrocytomas, LOH on chromosomes 1p and/or 19q was found in three cases (13%) and TP53 mutation was detected in ten cases (43%). Histologically, five low-grade astrocytomas (22%) contained small areas with oligodendroglial differentiation, but this did not correlate with the presence of TP53 mutations or LOH on 1p and 19q. In both oligodendrogliomas and astrocytomas, LOH on chromosomes 1p or 19q and TP53 mutation were mutually exclusive. Methylation of the promoter of the gene for O (6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein, which confers resistance to chemotherapy with alkylating agents, was detected in 47% of oligodendrogliomas and 48% of low-grade diffuse astrocytomas. There was no correlation with LOH on chromosomes 1p/19q, suggesting that MGMT may not be a prognostic marker for oligodendrogliomas.

Purpose A single dose of N-ethyl-N-nitrosourea (ENU) during late prenatal or early postnatal development induces a high incidence of malignant schwannomas and gliomas in rats. Although T -> A mutations in the transmembrane domain of the neu (c-ErbB-2) gene are the driver mutations in ENU-induced malignant schwannomas, the molecular basis of ENU-induced gliomas was unknown. The objectives of this study were to identify driver mutations in ENU-induced rat gliomas. Methods We performed whole-genome sequencing of gliomas that developed in three BDIV and two BDIX rats exposed to a single dose of 80 mg ENU/kg body weight on postnatal day one. Results T:A->A:T and T:A->C:G mutations, which are typical for ENU-induced mutagenesis, were predominant (41-55% of all somatic single nucleotide mutations). T->A mutations were detected in all 5 rat gliomas at Braf codon 545 (V545E), which corresponds to the human BRAF V600E. Additional screening revealed that 33 gliomas in BDIV rats and 12 gliomas in BDIX rats all carried a Braf V545E mutation, while peritumoral brain tissue of either strain (n=16) had the wild-type sequence. The gliomas were immunoreactive to BRAF V600E antibody. Conclusions Braf mutation is a frequent early event in the development of rat gliomas caused by a single dose of ENU. Citation Format: Kaishi Satomi, Qi Wang, Ji Eun Oh, Barbara Hutter, Benedikt Brors, Nicolle Diessl, Hai-Kun Liu, Stephan Wolf, Otmar Wiestler, Paul Kleihues, Bernd Koelsch, Andrea Kindler-Rohrborn, Hiroko Ohgaki. Braf mutations initiate the development of rat gliomas induced by postnatal exposure to N-ethyl-N-nitrosourea (ENU) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4807. doi:10.1158/1538-7445.AM2017-4807

Gemistocytic astrocytoma (World Health Organization grade II) is a rare variant of diffuse astrocytoma that is characterized by the presence of neoplastic gemistocytes and has a significantly less favorable prognosis. Other than frequent TP53 mutations (>80%), little is known about its molecular profile. Here, we show that gemistocytic astrocytomas carry a lower frequency of IDH mutations than fibrillary astrocytomas (74% vs 92%; p = 0.0255) but have profiles similar to those of fibrillary astrocytomas with respect to TERT promoter mutations (5% vs 0%), 1p/19q loss (10% vs 8%), and loss of heterozygosity 10q (10% vs 12%). Exome sequencing in 5 gemistocytic astrocytomas revealed homozygous deletion of genes at 19q13 (i.e. RRAS [related RAS viral oncogene homolog; 2 cases] and ERCC1 [excision repair cross-complementing rodent repair deficiency, complementation group 1; 1 case]). Further screening showed RRAS homozygous deletion in 7 of 42 (17%) gemistocytic astrocytomas and in 3 of 24 (13%) IDH1 mutated secondary glioblastomas. Patients with gemistocytic astrocytoma and secondary glioblastoma with an RRAS deletion tended to have shorter survival rates than those without deletion. Differential polymerase chain reaction and methylation-specific polymerase chain reaction revealed an ERCC1 homozygous deletion or promoter methylation in 10 of 42 (24%) gemistocytic astrocytomas and in 8 of 24 (33%) secondary glioblastomas. Alterations in RRAS and ERCC1 appear to be typical in gemistocytic astrocytomas and secondary glioblastomas, since they were not present in 49 fibrillary astrocytomas or 30 primary glioblastomas.

Oligodendrogliomas are characterized by frequent loss of heterozygosity (LOH) on chromosomes 1p and 19q, but additional genetic alterations are likely to be involved. In this study, we screened 28 oligodendrogliomas (WHO grade II) and 20 anaplastic oligodendrogliomas (WHO grade III) for alterations in the RB1/CDK4/p16INK4a/p15INK4b and TP53/p14ARF/MDM2 pathways. In oligodendrogliomas, hypermethylation of RB1 (1 case) and p14ARF (6 cases) were the only detectable genetic changes (7/28, 25%). In anaplastic oligodendrogliomas, the RB1/CDK4/p16INK4a/p15INK4b signaling pathway regulating the G1 -->3 S transition of the cell cycle was altered in 13/20 (65%) cases, by either RBI alteration, CDK4 amplification, or p16IN4a/p15INK4b homozygous deletion or promoter hypermethylation. Further, 50% (10/20) of anaplastic oligodendrogliomas showed alterations in the TP53 pathway through promoter hypermethylation or homozygous deletion of the p14ARF gene and, less frequently, through TP53 mutation or MDM2 amplification. Of 13 anaplastic astrocytomas with an altered RB1 pathway, 9 (69%) also showed a dysregulated TP53 pathway. Thus, simultaneous disruption of the RB1/CDK4/p16INK4a/p15INK4b and the TP53/p14ARF/MDM2 pathways occurs in 45% (9/20) of anaplastic oligodendrogliomas, suggesting that these phenomena contribute to their malignant phenotype.

O(6)-Methylguanine-DNA methyltransferase (MGMT) is a repair protein that specifically removes promutagenic alkyl groups from the O(6) position of guanine in DNA. Repair of O(6)-alkylguanine adducts by tumour cells has been implicated in drug resistance since it reduces the cytotoxicity of alkylating chemotherapeutic agents. We assessed promoter methylation of the MGMT gene in astrocytic brain tumours by methylation-specific PCR. MGMT promoter methylation was detected in 26 of 54 (48%) low-grade diffuse astrocytomas (WHO grade II) and in 12 of 16 (75%) of secondary glioblastomas (WHO grade IV) that had progressed from low-grade astrocytomas. The frequency of MGMT methylation was significantly lower in primary (de novo) glioblastomas (13 of 36, 36%, P = 0.0155). The majority of low-grade astrocytomas with MGMT methylation (24/26, 92%) contained a TP53 mutation, whereas only 11 out of 28 (39%) cases without MGMT methylation carried a TP53 mutation (P0.0001). Furthermore, G:C --A:T transition mutations at CpG sites were significantly more frequent in low-grade astrocytomas with MGMT methylation (15/26, 58%) than in those without (3/28, 11%, P = 0.0004). These results suggest that loss of MGMT expression as a result of promoter methylation, which frequently occurs at an early stage in the pathway leading to secondary glioblastomas, appears to be associated with increased frequency of TP53 mutations, in particular G:C --A:T transitions.

International audience; Glioblastomas, the most malignant human brain tumors, are characterized by marked aneuploidy, suggesting chromosomal instability which may be caused by a defective mitotic spindle checkpoint. We screened 22 glioblastomas for mutations in the mitotic spindle check-point genes hBUB1, hBUBR1 and hBUB3. DNA sequencing revealed a silent mutation at codon 144 of hBUB1 (CAG-->CAA, Gln-->Gln) in one glioblastoma, a silent mutation at codon 952 of hBUBR1 (GAC-->GAT, Asp-->Asp) in another glioblastoma, and a silent mutation at codon 388 of the hBUBR1 gene (GCG-->GCA, Ala-->Ala) in 8 glioblastomas. We also observed a known polymorphism at hBUBR1 codon 349 (CAA/CGA, Gln/Arg), with an allelic frequency of 0.75 for Gln and 0.25 for Arg, which is similar to that among healthy Caucasian individuals (0.73 vs 0.27). The coding sequence of the hBUB3 gene did not contain any mutation, but in 4 glioblastomas (18%), a C-->T point mutation was detected at position -6 (6 nucleotides upstream of the ATG initiator codon). Analysis of blood DNA of these patients showed identical sequence alterations, indicating that this is a polymorphism. Again, the frequency in glioblastomas was similar to that in healthy Caucasians (15%). We further screened hBUB1 in 18 cases of giant cell glioblastoma, a variant characterized by a predominance of bizarre, multinucleated giant cells. There were no changes, except for a silent mutation at codon 144 in two cases. These results suggest that mutations in these mitotic spindle checkpoint genes do not play a significant role in the causation of chromosomal instability in glioblastomas.

Loss of expression of the retinoblastoma gene (RB1) has been shown to occur in up to 25% of glioblastomas (WHO Grade IV). To elucidate the underlying mechanism, we assessed RB1 promoter hypermethylation using methylation-specific polymerase chain reaction and RB1 expression by immunohistochemistry in 35 primary (de novo) glioblastomas and in 21 secondary glioblastomas that had progressed from low-grade diffuse astrocytoma (WHO Grade II) or anaplastic astrocytoma (WHO Grade III). Promoter hypermethylation was significantly more frequent in secondary (9 of 21, 43%) than in primary glioblastomas (5 of 35, 14%; p = 0.0258). There was a clear correlation between loss of RB1 expression and promoter hypermethylation. In the majority of glioblastomas with loss of RB1 expression, there was promoter hypermethylation (11 of 13, 85%), whereas 93% of tumors with RB1 expression had a normal RB1 gene status (p < 0.0001). In three glioblastomas, areas with and without RB1 expression were microdissected; promoter hypermethylation was detected only in areas lacking RB1 expression. In patients with multiple biopsies, methylation of the RB1 promoter was not detectable in the less malignant precursor lesions, ie, low-grade diffuse and anaplastic astrocytoma. These results indicate that promoter hypermethylation is a late event during astrocytoma progression and is the major mechanism underlying loss of RB1 expression in glioblastomas.

Invasion of spheroids from 20 human primary glioblastomas into precultured fetal rat brain tissue in culture has been studied and quantified. Between 30 and 98 percent of the normal brain tissue was destroyed by invading glioma cells within 4 days. The degree of invasion did not correlate with patient survival. A slightly higher invasiveness and shorter survival was seen in tumors with EGF receptor overexpression, and the opposite pattern was found for tumors with a TP53 mutation. The degree of invasiveness in vitro was far higher than would be expected from the dynamics of clinically observed tumor spread. This suggests that mechanisms suppressing invasion may be operative in the normal brain; alternatively the differences may be due to a higher permissiveness of the fetal brain tissue for invasion in vitro.

Glioblastomas develop rapidly de novo (primary glioblastomas) or slowly through progression from low-grade or anaplastic astrocytoma (secondary glioblastomas). Recent studies have shown that these glioblastoma subtypes develop through different genetic pathways. Primary glioblastomas are characterized by EGFR amplification/overexpression, PTEN mutation, homozygous p16 deletion, and loss of heterozygosity (LOH) on entire chromosome 10, whereas secondary glioblastomas frequently contain p53 mutations and show LOH on chromosome 10q. In this study, we analyzed LOH on chromosomes 19q, 1p, and 13q, using polymorphic microsatellite markers in 17 primary glioblastomas and in 13 secondary glioblastomas that progressed from low-grade astrocytomas. LOH on chromosome 19q was frequently found in secondary glioblastomas (7 of 13, 54%) but rarely detected in primary glioblastomas (1 of 17, 6%, p = 0.0094). The common deletion was 19q13.3 (between D19S219 and D19S902). These results suggest that tumor suppressor gene(s) located on chromosome 19q are frequently involved in the progression from low-grade astrocytoma to secondary glioblastoma, but do not play a major role in the evolution of primary glioblastomas. LOH on chromosome 1p was detected in 12% of primary and 15% of secondary glioblastomas. LOH on 13q was detected in 12% of primary and in 38% of secondary glioblastomas and typically included the RB locus. Except for 1 case, LOH 13q and 19q were mutually exclusive.

APO2 ligand (APO2L)/TRAIL is a novel member of the tumor necrosis factor cytokine family and a potent inducer of apoptosis in tumor cell lines. We recently reported that APO2L is consistently expressed in low-grade astrocytomas, anaplastic astrocytomas, glioblastomas, and cell lines derived thereof, and that malignant glioma cell lines are susceptible to APO2L-induced apoptosis. In this study, we investigated whether APO2L is expressed in medulloblastoma or neuroblastoma cell lines and whether these cells are sensitive to APO2L-induced apoptosis. Immunoblot analyses revealed full-length APO2L protein expression in one (DAOY) of three medulloblastoma cell lines but not in two neuroblastoma cell lines (SKN-BE and SKN-LE). Viability assay performed after exposure to soluble APO2L for 16 h showed that DAOY medulloblastoma cells were the most sensitive and that apoptosis induced by APO2L was greatly enhanced when protein synthesis was inhibited by cycloheximide. Neuroblastoma cell lines were almost completely resistant to APO2L-induced apoptosis. We also carried out APO2L immunohistochemistry in a total of 115 tumors of the nervous system with different histogenesis and biological behavior. In all 9 pilocytic astrocytomas, the areas of dense fibrillary network showed diffuse and strong APO2L expression. In oligodendrogliomas, APO2L expression was observed in areas with a significant admixture of astrocytic cells, but was absent in neoplastic oligodendrocytes. In 13 of 14 ependymomas, APO2L was expressed in perivascular pseudorosettes. In all 12 medulloblastomas, strong APO2L expression was observed in intra-tumoral-reactive astrocytes, but neoplastic cells did not show APO2L immunoreactivity. Thus, the pattern of APO2L expression was largely similar to that of glial fibrillary acidic protein (GFAP), except for choroid plexus tumors and 3 of 8 anaplastic meningiomas, in which APO2L was focally expressed without concomitant GFAP expression. APO2L expression was absent in meningiomas, neurocytomas, and schwannomas. Thus, there is considerable heterogeneity of APO2L expression and susceptibility to APO2L-induced apoptosis among human brain tumors.

Astrocytic brain tumors are the most frequent human gliomas and they include a wide range of neoplasms with distinct clinical, histopathologic, and genetic features. Diffuse astrocytomas are predominantly located in the cerebral hemispheres of adults and have an inherent tendency to progress to anaplastic astrocytoma and (secondary) glioblastoma. The majority of glioblastomas develop de novo (primary glioblastomas), without an identifiable less-malignant precursor lesion. These subtypes of glioblastoma evolve through different genetic pathways, affect patients at different ages, and are likely to differ in their responses to therapy. Primary glioblastomas occur in older patients and typically show epidermal growth factor receptor ( EGFR) overexpression, PTEN mutations, p16 deletions, and, less frequently, MDM2 amplification. Secondary glioblastomas develop in younger patients and often contain TP53 mutations as their earliest detectable alteration. Morphologic variants of glioblastoma were shown to have intermediate clinical and genetic profiles. The giant cell glioblastoma clinically and genetically occupies a hybrid position between primary (de novo) and secondary glioblastomas. Gliosarcomas show identical gene mutations in the gliomatous and sarcomatous tumor components, which strongly supports the concept that there is a monoclonal origin for gliosarcomas and an evolution of the sarcomatous component due to aberrant mesenchymal differentiation in a highly malignant astrocytic neoplasm.

Loss of heterozygosity on chromosome 10 (LOH#10) is the most frequent genetic alteration in glioblastomas and occurs in more than 80% of cases. We recently reported that PTEN (MMAC1) on 10q23.3 is mutated in approximately 30% of primary (de novo) glioblastomas but rarely in secondary glioblastomas that progressed from low-grade or anaplastic astrocytomas. Because secondary glioblastomas also show LOH#10, tumor suppressor genes other than PTEN are likely to be involved. We analyzed LOH on chromosomes 10 and 19, using polymorphic microsatellite markers in microdissected foci showing histologically an abrupt transition from low-grade or anaplastic astrocytoma to glioblastoma, suggestive of the emergence of a new tumor clone. When compared to the respective low-grade or anaplastic astrocytoma of the same biopsy, deletions were detected in 7 of 8 glioblastoma foci on 10q25-qter distal to D10S597, covering the DMBT1 and FGFR2 loci. Six of 8 foci showed LOH at one or two flanking markers of PTEN but did not contain PTEN mutations. LOH on 10p and 19q was found in only one case each. These data indicate that acquisition of a highly anaplastic glioblastoma phenotype with marked proliferative activity and lack of glial fibrillary acidic protein expression is associated with loss of a putative tumor suppressor gene on 10q25-qter.

APO2 ligand (APO2L) is a CD95 ligand (CD95L)-related cytokine of the tumor necrosis factor family that interacts with agonistic (DR4, DR5) and antagonistic (DcR1, DcR2) receptors. Cultured malignant glioma cells preferentially express agonistic receptors and are susceptible to APO2L-induced apoptosis. Here, we report that 8 of 8 human glioma cell lines expressed APO2L mRNA and protein in vitro. Immunohistochemistry using a monoclonal antibody to APO2L revealed that all 23 primary astrocytic brain tumors analyzed, including low-grade astrocytomas and glioblastomas, express APO2L in vivo. With the exception of reactive astrocytes, non-neoplastic glia and neurons in the cerebrum lacked immunoreactivity of APO2L. Thus, in addition to the CD95/CD95L system, a second death ligand/death receptor pair may regulate susceptibility to apoptosis in human glial neoplasms.

Glioblastomas may develop de novo (primary glioblastomas) or through progression from low-grade or anaplastic astrocytomas, (secondary glioblastomas). These subtypes of glioblastoma constitute distinct disease entities that evolve through different genetic pathways, affect patients at different ages, and are likely to differ in prognosis and response to therapy. Primary glioblastomas develop in older patients and typically show EGFR overexpression, PTEN (MMAC1) mutations, CDKN2A (p16) deletions, and less frequently, MDM2 amplification. Secondary glioblastomas develop in younger patients and often contain TP53 mutations as the earliest detectable alteration. These characteristics are derived largely from patients selected on the basis of clinical history and sequential biopsies. Currently available data are insufficient for a substitution of histologic classification and grading of astrocytic tumors by genetic typing alone. More subtypes of glioblastomas may exist with intermediate clinical and genetic profiles, a factor exemplified by the giant-cell glioblastoma that clinically and genetically occupies a hybrid position between primary (de novo) and secondary glioblastomas. Future research should aim at the identification of criteria for a combined clinical, histologic, and genetic classification of astrocytic tumors.

Phenacetin abuse and smoking are established risk factors for transitional cell carcinomas of the urinary tract. In the present study, we analysed exposure and the clinical course of patients who underwent nephrectomy for transitional cell carcinoma of the renal pelvis. PCR-SSCP of archival, paraffin-embedded histological sections followed by direct DNA sequencing revealed that 29 of 89 (33%) renal pelvic carcinomas contained a p53 mutation. Double mutations were found in 4 tumours and triple mutations in 1 tumour. The incidence of p53 mutations was significantly higher in tumours with grades 3 and 4 than in those with grades 1 and 2 and higher in invasive than in non-invasive tumours. Furthermore, patients with carcinomas carrying a p53 mutation showed poorer survival than those without mutation. The type of p53 mutation in renal pelvic carcinomas was similar to that reported for bladder cancer, G:C-->A:T transition mutations being most frequent (45%, 33% of these at CpG sites), followed by G:C-->T:A and G:C-->C:G transversions. The incidence and type of p53 mutation did not differ significantly in patients with a history of phenacetin abuse, smoking or neither of these habits. This was also true for G:C-->T:A transversions (17.5% of mutations), which are considered typical of smoking-induced carcinomas at other sites, e.g., lung, oral cavity and oesophagus. Our results indicate that the frequency and pattern of p53 mutations are similar in transitional cell carcinomas of the bladder and the renal pelvis and do not reflect exposure to phenacetin and/or smoking. The frequency of genetic polymorphism in genes coding for carcinogen-metabolising enzymes (CYP1A1, NAT1, GSTT1 and GSTM1) was also independent of exposure. Although the sample size of our study does not allow definite conclusions, these data are compatible with chronic tissue damage as a causative factor in the evolution of urothelial carcinomas rather than pointing to a direct mutagenic effect of phenacetin and tobacco-specific carcinogens.

Loss of heterozygosity (LOH) on chromosome 10 is the most frequent genetic alteration associated with the evolution of malignant astrocytic tumors and it may involve several loci. The tumor suppressor gene PTEN (MMAC1) on chromosome 10q23 is mutated in approximately 30% of glioblastomas (WHO Grade IV). In this study, we assessed the frequency of PTEN mutations in primary glioblastomas, which developed clinically de novo, and in secondary glioblastomas, which evolved from low-grade (WHO Grade II) or anaplastic astrocytomas (WHO Grade III). Nine of 28 (32%) primary glioblastomas contained a PTEN mutation and an additional case showed a homozygous PTEN deletion. This indicates that after overexpression/amplification of the EGF receptor, loss of PTEN function is the most common alteration in primary glioblastomas. In this series, 5 of 28 (18%) primary glioblastomas showed both a PTEN mutation and EGFR amplification. In contrast, only 1 of 25 (4%) secondary glioblastomas contained a PTEN mutation, and none of them showed a homozygous PTEN deletion. The secondary glioblastoma with a PTEN mutation developed from an anaplastic astrocytoma that already carried the mutation. The observation that secondary glioblastomas have a p53 mutation as a genetic hallmark but rarely contain a PTEN mutation supports the concept that primary and secondary glioblastomas develop differently on a genetic level.

Glioblastomas may develop rapidly without clinical and histopathological evidence of a less malignant precursor lesion (de novo or primary glioblastoma) or through progression from low-grade or anaplastic astrocytoma (secondary glioblastoma). Primary glioblastomas typically show overexpression of EGFR, but rarely p53 mutations, while secondary glioblastomas frequently carry a p53 mutation, but usually lack overexpression of EGFR, suggesting that these glioblastoma subtypes develop through distinct genetic pathways. In the present study, we assessed the expression of Fas/APO-1 (CD95), an apoptosis-mediating cell membrane protein, and its relation to necrosis phenotype in primary and secondary glioblastomas. Large areas of ischemic necroses were observed in all 18 primary glioblastomas, but were significantly less frequent in secondary glioblastomas (10 of 19, 53%; p = 0.0004). Fas expression was predominantly observed in glioma cells surrounding large areas of necrosis and was thus significantly more frequent in primary glioblastomas (18 of 18, 100%) than in secondary glioblastomas (4 of 19, 21%; p < 0.0001), suggesting that these clinically and genetically defined subtypes of glioblastoma differ in the extent and mechanism of necrogenesis. Necrosis and microvascular proliferation are histologic hallmarks of the glioblastoma. Following incubation of glioblastoma cell lines under hypoxic/anoxic conditions for 24-48 hours, Fas mRNA levels remained unchanged, whereas VEGF expression was markedly upregulated. This suggests that in contrast to VEGF Fas expression is not induced by ischemia/hypoxia. Analysis of Fas mRNA levels in a glioblastoma cell line containing a p53 mutation and an inducible wild-type p53 gene showed little difference under induced and noninduced conditions, suggesting that in glioblastomas, Fas expression is not directly linked to the p53 status.

Recent studies have shown that there are distinct genetic pathways leading to the most malignant astrocytic neoplasm, the glioblastoma. Primary (de novo) glioblastomas are characterized by amplification/overexpression of the EGF receptor (EGFR) and, less frequently, of the MDM2 gene. Another pathway, operative in the progression of low-grade or anaplastic astrocytomas to secondary glioblastomas, is characterized by the frequent occurrence of p53 mutations. In this study, we assessed p53 mutations and EGFR expression in the giant cell glioblastoma. This rare variant is characterized by unusually large, multinucleated giant cells, but tends to be more confined and has been reported to carry a somewhat more favorable prognosis. We analyzed biopsies from 16 patients (mean age at clinical manifestation, 40 years). DNA sequencing revealed that 12 of 16 (75%) giant cell glioblastomas contained a p53 mutation. In 7 patients with two or more surgical interventions, the p53 mutation was already detected in the first biopsy. Focal EGFR overexpression, including multinucleated giant cells, was observed immunohistochemically in 9 of 16 (56%) tumors. However, most tumor areas lacked immunoreactivity, indicating that EGFR overexpression does not play a significant role in the evolution of this glioblastoma variant. These results suggest that giant cell glioblastomas develop de novo with a short preoperative history (mean, 47 +/- 40 days), but contain genetic alterations similar to those observed in secondary glioblastomas.

Summary Glioblastoma multiforme, the most malignant human brain tumor, may develop de novo (primary glioblastoma) or through progression from low-grade or anaplastic astrocytoma (secondary glioblastoma). We present evidence that these subtypes of glioblastoma constitute distinct disease entities which evolve through different genetic pathways, affect patients at different age and are likely to differ in prognosis and response to therapy. Primary glioblastomas develop in older patients (mean, 55 years) and typically show EGFR overexpres-sion or, less frequently, MDM2 overexpression and pi6 deletion. Secondary glioblastomas develop in younger patients (mean, 40 years) and frequently contain TP53 mutations and, less consistently, loss of DCC expression. Although primary and secondary glioblastomas are considered to be histologically indistinguishable, we found that the pattern and pathogenesis of necrosis are different, large areas of ischaemic necrosis surrounded by Fas expressing tumor cells being a hallmark of primary glioblastomas. The giant cell glioblastoma occupies an intermediate position. Like the primary glioblastoma, it rapidly develops de novo but manifests in younger patients (including children) and has genetic alterations typical for secondary glioblastomas, i.e. frequent TP53 mutations and lack of EGFR overexpression.

The p16INK4a gene product acts as a negative regulator of the cell cycle by binding to cyclin-dependent kinases (CDKs) 4 and 6, thereby inhibiting the formation of an active CDK/cyclin D complex. Deletion of the p16 locus has been observed in tumor cell lines and, less frequently, in primary human neoplasms. We analyzed 31 glioblastomas and identified 6 cases with hemizygous and 6 with homozygous deletions of the p16 locus. Eight of these cases showed a concurrent amplification of the EGFR gene (epidermal growth factor receptor) while the overall frequency was 35%. This close correlation suggests that deletion of the p16 chromosomal region constitutes another genetic hallmark of the primary glioblastoma, which rapidly develops de novo, without a less malignant precursor lesion and for which EGFR amplification is a characteristic genetic change. The p16 protein was not detectable in 15 of 22 glioblastomas but only 4 of these showed homozygous deletion of the gene. The alternative transcript p16 beta, for which a growth-suppressing function has been suggested, was co-expressed with p16 alpha mRNA in most cases. Hypermethylation of CpG islands in the 5' region of the p16 gene was identified in only 1 case, suggesting that this alternative mechanism of gene silencing is rarely responsible for loss of p16 expression in glioblastomas. Likewise, only 1 glioblastoma carried a p16 mutation and in addition, unexpectedly, a homozygous deletion of p16 in approximately 80% of tumor cells. This mutation, Arg24Pro, has previously been identified in a melanoma kindred.

Glioblastoma multiforme is a rare neoplasm in children and is often located infratentorially, particularly in the brainstem: Pediatric glioblastomas arise frequently (here 60%) outside the cerebral hemispheres. We investigated 20 pediatric glioblastomas for mutational inactivation of the p53 tumor suppressor gene, loss of p16 protein expression and overexpression of the epidermal growth factor receptor (EGFR). Mutations in the p53 gene were identified in 5/20 (25%) glioblastomas, 4 of which occurred in primary glioblastomas with a clinical history of less than 4 months and neither clinical nor histologic evidence of a less malignant precursor lesion. Loss of p16 expression was detected in 11/18 (61%) glioblastomas. Overexpression of the EGFR was infrequent (2/19, 11%) and included 1 tumor with a p53 mutation. Of 4 secondary glioblastomas that progressed from histologically diagnosed lower grade tumors, one contained a p53 mutation. Our results are at variance with similar studies in adult patients in which primary and secondary glioblastomas are characterized by EGFR overexpression and p53 mutations, respectively, suggesting that the evolution of pediatric glioblastomas follows different genetic pathways.

Neoplastic transformation is often associated with aberrant gap junctional intercellular communication. We assessed mutations and expression of the connexin43 (Cx43) gene in 49 intracranial meningiomas. SSCP analyses followed by direct DNA sequencing showed GCG-->GTG (Ala-->Val) transition mutation in codon 253 of the cytoplasmic carboxyl terminal of the Cx43 gene in 1 of 31 (3%) benign meningiomas and 1 of 14 (7%) anaplastic meningiomas. The same base change was present in normal tissue from these patients and also in 4 of 80 (5%) DNA samples extracted from lymphocytes of healthy Europeans, suggesting that this constitutes a newly identified Cx43 polymorphism. Western blot analyses showed expression of phosphorylated P1 (45 kD) and P2 (47 kD) Cx43 as well as the unphosphorylated form (42 kD) in 11 of 14 (79%) benign meningiomas. In contrast, the P2 form was not detectable in the majority (7 of 9; 78%) of atypical and anaplastic meningiomas. Since the presence of the P2 form is often associated with optimal function of the Cx43, these results suggest that loss or impaired gap junctional cell to cell communication may be associated with meningiomas displaying more rapid growth and a less favorable prognosis.

The proliferative potential of central neurocytomas was determined in a biopsy series of 36 cases and compared with clinical outcome. The mean size of the growth fraction, as determined by MIB-1 labeling index (MIB-1 LI) at first biopsy, was 2.8 +/- 2.5 with a range of 0.1 to 8.6%. Neurocytomas with an MIB-1 LI > 2% comprised 39% of cases and showed a close correlation with the presence of vascular proliferation (p = 0.0006). The Kaplan-Meier analysis showed a highly significant difference in disease-free survival between the 2 groups (p = 0.0068). Over an observation time of 150 months, there was a 22% relapse among patients with an MIB-1 LI less than 2% and a 63% chance of relapse among those with an MIB-1 LI greater than 2%. We propose the term "atypical central neurocytoma" for the latter subset, corresponding to WHO grade II.

Two cases of Wernicke's encephalopathy with the rare phenomenon of ballooned neurons in the mamillary bodies are reported. Both patients suffered from acute Wernicke's symptoms starting approximately two weeks before death. The mamillary bodies contained grossly enlarged, ballooned neurons, in one case associated with focal necrosis. The affected neurons were immunoreactive for phosphorylated neurofilament (160 and 200 kDa), and synaptophysin. Ubiquitin and alpha beta-crystallin expression were not detected. The mamillo-thalamic tract appeared normal in both cases. There was a marked associated microglial reaction, as shown by the antibody Ki-MIP. It is concluded that the ballooning of mamillary neurons reflects an acute retrograde reaction to primarily axonal damage. Rather than being a rare manifestation of the disease, these cases may constitute a typical intermediate early stage (10-15 days) in the development of Wernicke's encephalopathy).