Alternative lengthening of telomeres is enriched in,and impacts survival of TP53 mutant pediatric malignant brain tumors

Although telomeres are maintained in most cancers by telomerase activation, a subset of tumors utilize alternative lengthening of telomeres (ALT) to sustain self-renewal capacity. In order to study the prevalence and significance of ALT in childhood brain tumors we screened 517 pediatric brain tumors using the novel C-circle assay. We examined the association of ALT with alterations in genes found to segregate with specific histological phenotypes and with clinical outcome. ALT was detected almost exclusively in malignant tumors (p = 0.001). ALT was highly enriched in primitive neuroectodermal tumors (12 %), choroid plexus carcinomas (23 %) and high-grade gliomas (22 %). Furthermore, in contrast to adult gliomas, pediatric low grade gliomas which progressed to high-grade tumors did not exhibit the ALT phenotype. Somatic but not germline TP53 mutations were highly associated with ALT (p = 1.01 × 10^?8). Of the other alterations examined, only ATRX point mutations and reduced expression were associated with the ALT phenotype (p = 0.0005). Interestingly, ALT attenuated the poor outcome conferred by TP53 mutations in specific pediatric brain tumors. Due to very poor prognosis, one year overall survival was quantified in malignant gliomas, while in children with choroid plexus carcinoma, five year overall survival was investigated. For children with TP53 mutant malignant gliomas, one year overall survival was 63 ± 12 and 23 ± 10 % for ALT positive and negative tumors, respectively (p = 0.03), while for children with TP53 mutant choroid plexus carcinomas, 5 years overall survival was 67 ± 19 and 27 ± 13 % for ALT positive and negative tumors, respectively (p = 0.07). These observations suggest that the presence of ALT is limited to a specific group of childhood brain cancers which harbor somatic TP53 mutations and may influence the outcome of these patients. Analysis of ALT may contribute to risk stratification and targeted therapies to improve outcome for these children.     

The clinicopathological and prognostic significance of TP53 alteration in K27M mutated gliomas: an individual-participant data meta-analysis

This study aimed to investigate the impact of TP53 alteration on survival and clinicopathological features of glioma patients with H3K27M mutations. An individual-participant-data (IPD) meta-analysis was performed to investigate the impact of TP53 alteration on survival and clinicopathological features of patients with H3K27M mutations. Three hundred thirty-one individual records from 12 eligible glioma studies involving the H3K27M mutation were finally included in our meta-analysis, and a pooled hazard ratio (HR) of 1.53 (95%CI, 1.10–2.11; P?=?0.01) indicated that TP53 alterations were associated with a shorter overall survival. The pooled odds ratios (ORs) indicated that TP53 alterations were significantly associated with the age at diagnosis ≥?7 years (OR?=?1.97, 95%CI?=?1.15–3.38, P =?0.01), the status of histone H3.3 mutations (OR?=?9.15, 95%CI?=?4.18–20.06, P < 0.00001), and high WHO grade histology (III?+?IV) (OR?=?2.70, 95%CI?=?1.33–5.48, P?=?0.006). However, no association was found between TP53 alterations and gender or tumor location. This IPD meta-analysis suggests that TP53 alteration is a valuable predictor for the prognosis of patients with H3K27M mutated gliomas. TP53 alteration may be used for identifying a subset of patients who potentially benefit from targeted reactivation of TP53 activity.     

Frequent ATRX mutations and loss of expression in adult diffuse astrocytic tumors carrying IDH1/IDH2 and TP53 mutations

Gliomas are the most common primary brain tumors in children and adults. We recently identified frequent alterations in chromatin remodelling pathways including recurrent mutations in H3F3A and mutations in ATRX (α-thalassemia/mental-retardation-syndrome-X-linked) in pediatric and young adult glioblastoma (GBM, WHO grade IV astrocytoma). H3F3A mutations were specific to pediatric high-grade gliomas and identified in only 3.4?% of adult GBM. Using sequencing and/or immunohistochemical analyses, we investigated ATRX alterations (mutation/loss of expression) and their association with TP53 and IDH1 or IDH2 mutations in 140 adult WHO grade II, III and IV gliomas, 17 pediatric WHO grade II and III astrocytomas and 34 pilocytic astrocytomas. In adults, ATRX aberrations were detected in 33?% of grade II and 46?% of grade III gliomas, as well as in 80?% of secondary and 7?% of primary GBMs. They were absent in the 17 grade II and III astrocytomas in children, and the 34 pilocytic astrocytomas. ATRX alterations closely overlapped with mutations in IDH1/2 (p?<?0.0001) and TP53 (p?<?0.0001) in samples across all WHO grades. They were prevalent in astrocytomas and oligoastrocytomas, but were absent in oligodendrogliomas (p?<?0.0001). No significant association of ATRX mutation/loss of expression and alternative lengthening of telomeres was identified in our cohort. In summary, our data show that ATRX alterations are frequent in adult diffuse gliomas and are specific to astrocytic tumors carrying IDH1/2 and TP53 mutations. Combined alteration of these genes may contribute to drive the neoplastic growth in a major subset of diffuse astrocytomas in adults.     

Clinicopathological Features and Molecular Analysis of Primary Glioblastomas in Moroccan Patients

Glioblastoma is the most frequent and most aggressive primary brain tumor. Primary and secondary glioblastomas develop through different genetic pathways. The aim of this study was to determinate the genetic and clinical features of primary glioblastoma in Moroccan patients. The blood and tumor samples were obtained from a group of 34 Moroccan patients affected with primary glioblastoma. The tumors were investigated for TP53, IDH1, and IDH2 mutations using PCR sequencing analysis. Clinicopathological data showed that the mean age at diagnosis of patients was 50.06 years, the sex ratio was 11 F/23 M, and the median of Karnofsky performance score was 60. About 18 % of patients were initially treated by total tumor resection, 41 % by subtotal, and 38 % by partial resection, but biopsy was performed for a single patient (3 %). Twenty-five patients (74 %) received radiotherapy. In addition, the median survival of the all patients was 13 months following diagnosis. There was a significant impact of higher Karnofsky performance score (KPS) (≥80) on overall survival, p-log-rank test?=?0.0002, whereas other parameters did not show any significant differences. The molecular analysis revealed TP53 mutations in 3/34 (8.82 %) cases; R273H, R306X, and Q136X. However, none of the analyzed samples contained the R132-IDH1 or R172-IDH2 mutations. These results showed the absence of IDH1 mutation in primary glioblastoma, confirming that this mutation is a hallmark of secondary glioblastoma. It can be used to distinguish primary from secondary glioblastomas. We found also that higher KPS was a significantly favorable factor in patients with primary glioblastoma.     

Analysis of the TP53 gene in laser-microdissected glioblastoma vasculature

Malignant transformation of human gliomas is accompanied by extensive proliferation of stromal blood vessels. Recent data suggest mesenchymal transdifferentiation of neoplastic cells in various human cancers, including colon and breast cancer as well as gliosarcoma. In this study, we have analyzed proliferating stromal blood vessels in glioblastoma multiforme for the presence of mutations in the tumor suppressor gene TP53. Using tissue arrays derived from glioblastoma specimens, cases with significant immunohistochemical p53 accumulation were selected for molecular genetic detection of TP53 mutations in exons 5 to 8. None of the tumors included in this series displayed properties of gliosarcoma. Proliferating glomeruloid stromal vessels were isolated by laser microdissection from paraffin sections. In six cases, single-strand conformation polymorphism analysis for mutations of the TP53 gene in stromal blood vessels compared with adjacent tumor cells and subsequent DNA sequencing of the resulting DNA fragments were carried out. Glioblastoma cells of these cases exhibited TP53 mutations in exons 5, 7 and 8. None of these tumors showed TP53 mutations in microdissected samples from glomeruloid vessels. The absence of TP53 mutations in vascular stromal components of glioblastoma multiforme supports the hypothesis that microvascular proliferations originate from the tumor stroma and are not derived from transdifferentiated glioblastoma cells.     

GFAP promoter driven transgenic expression of PDGFB in the mouse brain leads to glioblastoma in a Trp53 null background

Glioblastomas are the most common and malignant astrocytic brain tumors in human adults. The tumor suppressor gene TP53 is commonly mutated and/or lost in astrocytic brain tumors and the TP53 alterations are often found in combination with excessive growth factor signaling via PDGF/PDGFRα. Here, we have generated transgenic mice over‐expressing human PDGFB in brain, under control of the human GFAP promoter. These mice showed no phenotype, but on a Trp53 null background a majority of them developed brain tumors. This occurred at 2–6 months of age and tumors displayed human glioblastoma‐like features with integrated development of Pdgfrα⁺ tumor cells and Pdgfrβ⁺/Nestin⁺ vasculature. The transgene was expressed in subependymal astrocytic cells, in glia limitans, and in astrocytes throughout the brain substance, and subsequently, microscopic tumor lesions were initiated equally in all these areas. With tumor size, there was an increase in Nestin positivity and variability in lineage markers. These results indicate an unexpected plasticity of all astrocytic cells in the adult brain, not only of SVZ cells. The results also indicate a contribution of widely distributed Pdgfrα⁺ precursor cells in the tumorigenic process. © 2008 Wiley‐Liss, Inc.     

Analysis of p53 mutation and expression in pleomorphic xanthoastrocytoma

Pleomorphic xanthoastrocytoma (PXA) is a rare, superficially situated tumor that most frequently occurs in the temporal lobe of young adults and is often associated with seizures. It generally has a relatively favorable prognosis. Prior studies have shown that TP53 mutations may occur in up to 25% of PXAs, suggesting that PXA may have an etiology similar to diffuse astrocytoma rather than pilocytic astrocytoma. In the present study, we performed immunostaining for p53 protein and examined the mutation status of exons 5–8 of the p53 gene in 55 PXAs, 8 of which had undergone one or multiple recurrences. Of 55 primary PXAs, 35 (64%) showed staining in <1% of tumor cells, 15 (27%) in 1–10%, 4 (7%) in 11–50%, and only 1 (2%) in >50%. No significant increase in p53 protein expression was noted in recurrences, even when associated with increased histological anaplasia. We found a TP53 heterozygous mutation in exon 7 in 1 of 47 primary tumors that yielded useable DNA, and in its recurrence 3 years later. This tumor, a grade II PXA, did not show signs of anaplastic transformation at recurrence. Eleven additional recurrences from 7 patients, 5 of which showed signs of histological anaplasia, did not show TP53 mutations in exons 5–8. Based on our data, the p53 mutation appears to be an uncommon (2%) genetic event in PXA formation and does not appear to be involved in tumor progression. Consequently, our findings suggest that the genetic events that underlie PXA formation differ from those involved in diffuse astrocytoma. Electronic Publication     

The TP53-ARF tumor suppressor pathway is frequently disrupted in large/cell anaplastic medulloblastoma

We analyzed the TP53 and INK4A/ARF loci in 29 pediatric medulloblastomas. Mutually exclusive mutation in TP53, methylation of P14(ARF) or deletion of INK4A/ARF were identified in 21% (6/29) of tumors. Five of these alterations were detected in large cell/anaplastic medulloblastomas or tumors with significant anaplasia. Our data provide the first evidence that alterations within the TP53-ARF tumor suppressor pathway contribute to development of aggressive forms of medulloblastoma.     

Selective acquisition of <Emphasis Type="Italic">IDH1</Emphasis> 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.     

Analysis of TP53 and PTEN in gliomatosis cerebri

Gliomatosis cerebri (GC) is a rare glial neoplasm with extensive diffuse brain infiltration but relative preservation of the underlying architecture. Previous molecular studies, mostly analyzing biopsy samples, have suggested an astrocytic origin of GC, but a larger collective of autopsy tissue has not been investigated so far. Furthermore, whether the widespread neoplastic infiltration is based on a monoclonal process is still a matter of debate. In the present study, we screened paraffin-embedded brain tissue from different areas of 18 cases (8 autopsy cases and 10 biopsies) for alterations in the TP53 and PTEN genes. Nuclear accumulation of p53 protein was detected in 9 cases (50%). Somatic TP53 mutations occurred in two autopsy cases (11% of all cases). In the first case, a C-->T transition in codon 273 (Arg-->Cys) was detected in all tumor samples. In the second case, in tumor samples from one hemisphere, nuclear accumulation of p53 was caused by a G-->A transition in codon 244 (Gly-->Asp). In the present series, no mutations within the coding region of PTEN were found. Pten expression was observed in two autopsy cases (25%) and seven biopsy samples (70%). These data suggest that TP53 is affected in some cases, but other yet-unidentified genetic alterations might contribute to tumorigenesis in GC. Furthermore, although GC might be a monoclonal process, the presence of different tumor clones cannot be ruled out.     

脑肿瘤中P53基因突变与突变型P53蛋白表达的研究

采用聚合酶链反应－单链构象多态性（ＰＣＲ－ＳＳＣＰ）法对４１例脑肿瘤进行Ｐ５３基因突变的检测，并与抗突变型Ｐ５３蛋白单抗的免疫组化染色结果相对照，结果显示脑肿瘤中Ｐ５３基因突变率为３４．１％（１４／４１），胶质瘤为２８％（１０／３６）。Ｐ５３基因突变与突变型Ｐ５３蛋白表达具有高度一致性，且二者均与脑肿瘤的分化程度有关。Ｐ５３基因突变及蛋白的表达不仅出现在高恶度胶质瘤及转移癌，而且可出现在低恶度胶质瘤中，表明Ｐ５３基因的突变在脑瘤的发生、发展过程中起重要作用。     

The MTHFR C677T polymorphism modifies age at onset in Parkinson’s disease

Hyperhomocysteinemia is a risk factor for Parkinson’s disease (PD) and may result from genetic mutations or/and environmental factors. 5,10-methylenetetrahydrofolate reductase (MTHFR) is a folate-dependent enzyme that catalyzed remethylation of homocysteine (Hcy) and the MTHFR C677T polymorphism makes the MTHFR enzyme thermolabile causing hyperhomocysteinemia. In this study we analyzed whether two functional polymorphisms of MTHFR gene, A1298C and C677T, affect age of onset in PD. We enrolled 120 patients with sporadic PD. Patients were divided into three groups based on MTHFR C677T polymorphisms: (a) homozygotes wild type (CC) (b) heterozygotes (CT) and (c) homozygotes carriers of mutation (TT). MTHFR SNPs were analyzed using High-Resolution Melt analysis and ANOVA was performed to assess whether polymorphisms of MTHFR gene could influence age of onset. The MTHFR A1298C polymorphism had no effect on PD age at onset (p = 1.0) while there was a significant association with MTHFR C677T (p = 0.019 Bonferroni-adjusted post hoc) showing an earlier onset in CC as compared with TT. (p = 0.024). No differences were found for vascular load assessed with magnetic resonance imaging, pharmacological therapy and cognitive state for two MTHFR SNPs. Our results suggest a possible association of MTHFR C677T with age at onset of PD and may have important implications regarding the role of MTHFR.     

AKT1E17K mutations cluster with meningothelial and transitional meningiomas and can be detected by SFRP1 immunohistochemistry

The activating E17K mutation in the AKT1 gene has been detected in several tumor entities. Currently several clinical studies with specific AKT1 inhibitors are under way. To determine whether AKT1 mutations are involved in human tumors of the nervous system, we examined a series of 1,437 tumors including 391 primary intracranial brain tumors and 1,046 tumors of the coverings of the central and peripheral nervous system. AKT1E17K mutations were exclusively seen in meningiomas and occurred in 65 of 958 of these tumors. A strong preponderance was seen in the variant of meningothelial meningioma WHO grade I of basal and spinal localization. In contrast, AKT1E17K mutations were rare in WHO grade II and absent in WHO grade III meningiomas. In order to more effectively detect this mutation, we tested for immunohistochemical markers associated with this alteration. We observed strong up-regulation of SFRP1 expression in all meningiomas with AKT1E17K mutation and in HEK293 cells after transfection with mutant AKT1E17K, but not in meningiomas and HEK293 cells lacking this mutation.     

Analysis of NF1 somatic mutations in cutaneous neurofibromas from patients with high tumor burden

Neurofibromatosis type 1, (NF1) is a complex, autosomal dominant disorder characterized by benign and malignant tumors which result from NF1 gene mutations. The molecular mechanisms that underlie NF1 tumorigenesis are still poorly understood although inactivation of other modifying loci in conjunction with NF1 mutations is postulated to be involved. These modifying loci may include deficiencies in mismatch repair genes and elements involved in cell cycle regulation (TP53, RB1, and CDKN2A). We have analyzed the somatic mutations in 89 cutaneous neurofibromas derived from three unrelated NF1 patients with high tumor burden, by loss of heterozygosity (LOH) analysis of the NF1, TP53, RB1, and CDKN2A genes, by assessing microsatellite instability (MSI), by direct sequencing of the NF1, TP53, and several mismatch repair (MMR) genes and by multiplex ligation-dependent probe amplification of the NF1 and TP53 genes. The aim was both to assess the possible clonality of these tumors and also to assess the involvement of other potential genetic loci in the development of these neurofibromas. Somatic NF1 mutations were identified in 57 (64%) of neurofibroma samples. Each mutation was distinct demonstrating the independent origin of each tumor. While somatic LOH of the TP53 gene was identified in four tumors, no specific deletions or sequence variations were identified. LOH of markers flanking the RB1 gene was also found in one tumor but no CDKN2A mutations were detected. Although evidence of MSI was seen in 21 tumors, no MMR gene alterations were identified. The identification of LOH involving TP53 and RB1 loci is a novel finding in benign cutaneous neurofibromas possibly demonstrating an alternative underlying molecular mechanism associated with the development of these benign tumors from this cohort of patients.