Germline MSH6 Mutation in a Patient With Two Independent Primary Glioblastomas.

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.

Braf Mutations Initiate the Development of Rat Gliomas Induced by Postnatal Exposure to N-Ethyl-N-Nitrosourea.

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.

The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary.

The 2016 World Health Organization Classification of Tumors of the Central Nervous System is both a conceptual 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.

Genetic Alterations in Gliosarcoma and Giant Cell Glioblastoma.

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.

Alterations of the RRAS and ERCC1 genes at 19q13 in gemistocytic astrocytomas.

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.

International Society Of Neuropathology--Haarlem consensus guidelines for nervous system tumor classification and grading.

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.

TP53, MSH4, and LATS1 germline mutations in a family with clustering of nervous system tumors.

Exome DNA sequencing of blood samples from a Li-Fraumeni family with a TP53 germline mutation (codon 236 deletion) and multiple nervous system tumors revealed additional germline mutations. Missense mutations in the MSH4 DNA repair gene (c.2480T>A; p.I827N) were detected in three patients with gliomas (two anaplastic astrocytomas, two glioblastomas). Two family members without a TP53 germline mutation who developed peripheral schwannomas also carried the MSH4 germline mutation, and in addition, a germline mutation of the LATS1 gene (c.286C>T; p.R96W). LATS1 is a downstream mediator of the NF2, but has not previously been found to be related to schwannomas. We therefore screened the entire coding sequence of the LATS1 gene in 65 sporadic schwannomas, 12 neurofibroma/schwannoma hybrid tumors, and 4 cases of schwannomatosis. We only found a single base deletion at codon 827 (exon 5) in a spinal schwannoma, leading to a stop at codon 835 (c.2480delG; p.*R827Kfs*8). Mutational loss of LATS1 function may thus play a role in some inherited schwannomas, but only exceptionally in sporadic schwannomas. This is the first study reporting a germline MSH4 mutation. Since it was present in all patients, it may have contributed to the subsequent acquisition of TP53 and LATS1 germline mutations.

TERT promoter mutations in primary and secondary glioblastomas.

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.

The definition of primary and secondary glioblastoma.

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.

Role of Biomarkers in the Clinical Management of Glioblastomas: What are the Barriers and How Can We Overcome Them?

Thousands of articles describing biomarkers predictive of treatment and prognostic of survival in cancer have been published, yet only a handful of biomarkers are currently used routinely in the clinic. Biomarkers need to be analytically standardized, validated, and clinically useful. This review will address the challenges and ways in which we can improve our discovery and translation of prospective biomarkers from the lab into validated diagnostic tests with a specific focus on patients diagnosed with glioblastoma and MGMT promoter methylation status. There has been long-held enthusiasm to use MGMT promoter methylation as a predictive biomarker for patients treated with the alkylating agent, temozolomide; however in the majority of centers around the world, this has not yet transpired.

Genetic profile of astrocytic and oligodendroglial gliomas.

Low-grade diffuse gliomas WHO grade II (diffuse astrocytoma, oligoastrocytoma, oligodendroglioma) are characterized by frequent IDH1/2 mutations (>80%) that occur at a very early stage. In addition, the majority of diffuse astrocytomas (about 60%) carry TP53 mutations, which constitute a prognostic marker for shorter survival. Oligodendrogliomas show frequent loss at 1p/19q (about 70% of cases), which is associated with longer survival. With respect to clinical outcome, molecular classification on the basis of IDH1/2 mutations, TP53 mutations, and 1p/19q loss showed a predictive power similar to histological classification. IDH1/2 mutations are frequent (>80%) in secondary glioblastomas that have progressed from low-grade or anaplastic astrocytomas. Primary (de novo) glioblastomas with IDH1/2 mutations are very rare (<5%); they show an age distribution and genetic profile similar to secondary glioblastomas and are probably misclassified. Using the presence of IDH1/2 mutations as a diagnostic criterion, secondary glioblastomas account for approximately 10% of all glioblastomas. IDH1/2 mutations are the most significant predictor of favorable outcome of glioblastoma patients. The high frequency of IDH1/2 mutations in oligodendrogliomas, astrocytomas, and secondary glioblastomas derived thereof suggests these tumors share a common progenitor cell population. The absence of this molecular marker in primary glioblastomas suggests a different cell of origin; both glioblastoma subtypes acquire a similar histological phenotype as a result of common genetic alterations, including the loss of tumor suppressor genes on chromosome 10q.

Molecular classification of low-grade diffuse gliomas.

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.

Intratumoral patterns of genomic imbalance in glioblastomas.

Glioblastomas are morphologically and genetically heterogeneous, but little is known about the regional patterns of genomic imbalance within glioblastomas. We recently established a reliable whole genome amplification (WGA) method to randomly amplify DNA from paraffin-embedded histological sections with minimum amplification bias [Huang et al (J Mol Diagn 11: 109-116, 2009)]. In this study, chromosomal imbalance was assessed by array comparative genomic hybridization (CGH; Agilent 105K, Agilent Technologies, Santa Clara, CA, USA), using WGA-DNA from two to five separate tumor areas of 14 primary glioblastomas (total, 41 tumor areas). Chromosomal imbalances significantly differed among glioblastomas; the only alterations that were observed in > or =6 cases were loss of chromosome 10q, gain at 7p and loss of 10p. Genetic alterations common to all areas analyzed within a single tumor included gains at 1q32.1 (PIK3C2B, MDM4), 4q11-q12 (KIT, PDGFRA), 7p12.1-11.2 (EGFR), 12q13.3-12q14.1 (GLI1, CDK4) and 12q15 (MDM2), and loss at 9p21.1-24.3 (p16(INK4a)/p14(ARF)), 10p15.3-q26.3 (PTEN, etc.) and 13q12.11-q34 (SPRY2, RB1). These are likely to be causative in the pathogenesis of glioblastomas (driver mutations). In addition, there were numerous tumor area-specific genomic imbalances, which may be either nonfunctional (passenger mutations) or functional, but constitute secondary events reflecting progressive genomic instability, a hallmark of glioblastomas.

IDH1 mutations as molecular signature and predictive factor of secondary glioblastomas.

PURPOSE: To establish the frequency of IDH1 mutations in glioblastomas at a population level, and to assess whether they allow reliable discrimination between primary (de novo) glioblastomas and secondary glioblastomas that progressed from low-grade or anaplastic astrocytoma. EXPERIMENTAL DESIGN: We screened glioblastomas from a population-based study for IDH1 mutations and correlated them with clinical data and other genetic alterations. RESULTS: IDH1 mutations were detected in 36 of 407 glioblastomas (8.8%). Glioblastoma patients with IDH1 mutations were younger (mean, 47.9 years) than those with EGFR amplification (60.9 years) and were associated with significantly longer survival (mean, 27.1 versus 11.3 months; P < 0.0001). IDH1 mutations were frequent in glioblastomas diagnosed as secondary (22 of 30; 73%), but rare in primary glioblastomas (14 of 377; 3.7%: P < 0.0001). IDH1 mutations as genetic marker of secondary glioblastoma corresponded to the respective clinical diagnosis in 95% of cases. Glioblastomas with IDH1 mutation diagnosed as primary had clinical and genetic profiles similar to those of secondary glioblastomas, suggesting that they may have rapidly progressed from a less malignant precursor lesion that escaped clinical diagnosis and were thus misclassified as primary. Conversely, glioblastomas without IDH1 mutations clinically diagnosed as secondary typically developed from anaplastic rather than low-grade gliomas, suggesting that at least some were actually primary glioblastomas, that may have been misclassified, possibly due to histologic sampling error. CONCLUSION: IDH1 mutations are a strong predictor of a more favorable prognosis and a highly selective molecular marker of secondary glioblastomas that complements clinical criteria for distinguishing them from primary glioblastomas.

Genetic alterations and signaling pathways in the evolution of gliomas.

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.

Selective acquisition of IDH1 R132C mutations in astrocytomas associated with Li-Fraumeni syndrome.

Mutations of the IDH1 gene are frequent in gliomas, with R132H (CGT-->CAT) being the most common (>85%). In astrocytomas, IDH1 mutations are typically co-present with, or precede, TP53 mutations. We assessed IDH1 mutations in brain tumors diagnosed in patients from three families with Li-Fraumeni syndrome. We identified IDH1 mutations in five astrocytomas that developed in carriers of a TP53 germline mutation. Without exception, all were R132C (CGT-->TGT), which in sporadic astrocytomas accounts for <5% of IDH1 mutations. This remarkably selective occurrence of R132C mutations may reflect differences in the sequence of genetic events, with a preference for R132C mutations in astrocytes or precursor cells that already carry a germline TP53 mutation.