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。     

100例脑肿瘤中p53基因突变与P53蛋白积聚的研究

目的:研究不同类型脑肿瘤中的p53基因突变与P53蛋白积聚及其相关性。方法:采用聚合酶链反应-单链构象多态性(PCR-SSCP)分析及免疫组化法检测100例脑肿瘤p53基因突变及蛋白表达。结果:p53基因突变率为11%(11/100),其中高恶度胶质瘤为37.5%(6/16),低恶度胶质瘤4.3%(1/23),脑膜瘤6.9%(2/29),转移瘤40.0%(2/5)。P53蛋白表达阳性率为22%(22/100),其中高恶度胶质瘤为62.5%(10/16),低恶度胶质瘤为26.1%(6/23),脑膜瘤10.3%(3/29),转移瘤60%(3/5);其他肿瘤均未发现p53基因突变或蛋白表达。P53蛋白表达阳性的22例中伴有p53基因突变者11例,多见于高恶度肿瘤。结论:p53基因失活在脑肿瘤恶性进展过程中起重要作用。p53基因突变与P53蛋白积聚相关,但并非唯一因素。     

NF2 tumor suppressor gene: a comprehensive and efficient detection of somatic mutations by denaturing HPLC and microarray-CGH

The NF2 tumor suppressor gene, located in chromosome 22q12, is involved in the development of multiple tumors of the nervous system, either associated with neurofibromatosis 2 or sporadic ones, mainly schwannomas and meningiomas. In order to evaluate the role of the NF2 gene in sporadic central nervous system (CNS) tumors, we analyzed NF2 mutations in 26 specimens: 14 meningiomas, 4 schwannomas, 4 metastases, and 4 other histopathological types of neoplasms. Denaturing high performance liquid chromatography (denaturing HPLC) and comparative genomic hybridization on a DNA microarray (microarray- CGH) were used as scanning methods for small mutations and gross rearrangements respectively. Small mutations were identified in six out of seventeen meningiomas and schwannomas, one mutation was novel. Large deletions were detected in six meningiomas. All mutations were predicted to result in truncated protein or in the absence of a large protein domain. No NF2 mutations were found in other histopathological types of CNS tumors. These results provide additional evidence that mutations in the NF2 gene play an important role in the development of sporadic meningiomas and schwannomas. Denaturing HPLC analysis of small mutations and microarray-CGH of large deletions are complementary, fast, and efficient methods for the detection of mutations in tumor tissues.     

Apoptosis and apoptosis-related gene products in primary non-Hodgkin’s lymphoma of the central nervous system

The incidence of primary lymphomas of the central nervous system (CNS) has significantly increased over the last years. However, the pathogenesis of this serious and fatal disease is still largely unknown. The aim of the present study was to investigate whether impairment of apoptosis is involved in the pathogenesis of primary CNS lymphomas. A series of 35 primary CNS lymphomas was investigated for the presence of apoptotic cells and the expression of apoptosis-inhibiting and proapoptotic gene products of the bcl family by application of the terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) technique and immunohistochemistry. The majority (23/35) of the tumors contained no or less than 10% of apoptotic cells. All tumors were MIB-1 positive, and 53% of them showed a high proliferative activity with more than 20% MIB-1-positive cells. The bcl-2 gene was expressed in 54% of the tumors (19/35), whereas bcl-x and bax gene products were present in only a low fraction of these lymphomas (4/35). In contrast, bak and the tumor suppressor gene p53 product were not detectable. These findings indicate that apoptosis is inhibited in the majority of this series of primary CNS lymphomas. Since there was no statistical correlation between the degree of apoptosis and the expression of proteins of the bcl gene family, other apoptosis-inhibiting factors may be involved in the pathogenesis of primary CNS lymphomas. Received: 13 January 1998 / Accepted: 13 March 1998     

Clinicopathologic Characteristics of Brain Tumors are Associated with the Presence and Patterns of TP53 Mutations: Evidence from the IARC TP53 Database

Biological diversity in the development and progression of brain tumors may be based on the consequences of the nature of the TP53 mutation in the cancer sample. This study was designed to estimate the possible impact of the presence and spectrum of TP53 mutations on clinical variability of brain tumors using the IARC TP53 Database (R17). Somatic and germline mutation patterns differ in brain tumor carriers. The most frequent mutation in sporadic brain tumors is mutation R273C, which is relatively rare in grade 4 tumors compared with lower-grade tumors (p = 1.2 × 10^?5, OR 0.43, 95 % CI 0.29–0.63). Mutations at all hot spots, DNA contact mutations, and mutations in the conserved regions of the TP53 gene are also more common in grade 1–3 tumors than in grade 4 tumors. The frequencies of missense mutations at hotspot codons and DNA contact mutations gradually decrease in all three age groups studied, indicating the role of these mutations in early-onset tumors. The role of TP53 somatic mutations in the development of brain tumors has been elucidated in the individual-participant meta-analysis that provided, for the first time, strong evidence that mean age at the onset of sporadic brain tumor is significantly lower in patients with mutated compared with wild-type TP53 in all groups stratified by tumor grade. The presence and patterns of TP53 mutations are associated mainly with the age at the onset and with the development of less malignant brain tumors. Malignant degeneration of brain tumors may depend on other genetic determinants.     

Analysis of p53 gene mutations in low- and high-grade astrocytomas by polymerase chain reaction-assisted single-strand conformation polymorphism and immunohistochemistry

Using polymerase chain reaction-assisted single-strand conformation polymorphism (PCR-SSCP) and immunohistochemical analyses, mutations in the p53 tumor suppressor gene were examined in 19 low-and high-grade gliomas. By PCR-SSCP and nucleotide analyses, p53 gene mutation was seen in 7 gliomas. Out of the 7 mutations, 3 were located at the CpG site of the previously proposed hot-spot codons 248 and 273, 2 were at codons 171 and 214 and the other 2 were in intron 5, 1 at the splice acceptor site and the other in the vicinity of the splice donor site. The latter 4 mutations have not, or only rarely, been observed in gliomas or in other tumors. However, their effect on the structural and functional alteration of the p53 protein was suggested by positive intranuclear p53 immunostaining in neoplastic cells in 3 mutations including the 1 at the splice acceptor site. In connection with glioma grading, the p53 gene mutation was shown to have occurred in both low- and high-grade gliomas, often in most of the neoplastic cells, as suggested by lack of distinct normal bands and ladders in SSCP and direct sequencing, respectively. The absence of recurrence and malignant transformation over a considerably long postoperative time in our low-grade glioma cases suggested that the p53 gene mutation might not be sufficient for the progression from low- to high-grade gliomas. The frequency of detection of mutation was 7/19(37%) by PCR-SSCP, 8/19(42%) by immunohistochemistry and 10/19(53%) by both methods. The results of PCR-SSCP and immunohistochemistry were consistent in 14 cases(73.7%), but not in 5 cases(26.3%). Thus, the use of both methods was recommended to survey the occurrence of p53 gene mutation more accurately.Supported in part by the Fujisawa Foundation     

Accumulation of wild-type p53 in astrocytomas is associated with increased p21 expression

Approximately one quarter of human astrocytomas show immunohistochemical positivity for p53 protein but lack p53 gene mutations, which could reflect either an accumulation of wild-type p53 protein or an inadequate sensitivity of mutation detection. Since wild-type p53 up-regulates p21 expression, increased p21 expression in those astrocytomas with p53 accumulation in the absence of mutations would argue that the protein was wild type in these tumors. We therefore compared p21 expression with p53 gene and protein status in 48 primary human astrocytomas. Single-strand conformation polymorphism analysis and direct sequencing of the p53 gene showed mutations in 11 tumors (22.9%), while immunohistochemistry revealed positive staining in 19 cases (39.6%). Those tumors with p53 immunopositivity in the absence of p53 mutation had significantly increased p21 expression when compared to either mutant p53 or p53-immunonegative cases. Neither p53 nor p21 status correlated with proliferation indices, as assessed by Ki-67 immunohistochemistry. These results support the hypotheses that functionally wild-type p53 accumulates in some astrocytomas, and that alternative cell cycle checkpoints (such as the p16 pathway) may be more important than p21 in regulating proliferation in astrocytomas. Received: 11 October 1996 / Revised, accepted: 17 January 1997     

Expression of oncogenic molecules in primary central nervous system lymphomas in immunocompetent patients

We studied overexpression of p53, Bcl-2, Bcl-6, c-Myc and Mdm2 proteins by immunohistochemistry for a total of 27 primary central nervous system B cell lymphomas (CNS lymphomas) in immunocompetent patients and one CNS lymphoma in an AIDS patient. The expression of Epstein-Barr (EB) virus-encoded small RNA-1 (EBER-1) was also analysed using in situ hybridisation. Overexpression (more than 20% of cells stained) of p53 protein was detected in 8 of 27 immunocompetent cases (30%); 6 cases showed a nuclear stain and 2 cases showed cytoplasmic stain (nuclear exclusion). Strong Bcl-2 or Bcl-6 immunoreactivity suggestive of overexpression was seen, respectively, in 5 (19%) and 6 (22%) cases; 2 cases were positive for both immunoreactivities. Interestingly, overexpression of Bcl-2 or Bcl-6 was not seen in the cases which showed p53 overexpression (P < 0.03; chi-square test). EBER-1 expression was not detected in any of the 27 immunocompetent cases, but was found in the AIDS-related CNS lymphoma, which also showed an overexpression of Bcl-6, but not Bcl-2. None of the cases showed c-Myc or Mdm2 overexpression. Taken together, it is suggested that CNS lymphoma in immunocompetent hosts is a distinct disease that has a different molecular profile from those of systemic lymphoma and/or AIDS-related CNS lymphoma. Received: 17 July 1997 / Revised: 1 September 1997 / Revised, accepted: 10 November 1997     

Monoclonal antibody to human midkine reveals increased midkine expression in human brain tumors.

We produced a rat IgG2a monoclonal antibody against the carboxyl terminal region of human midkine (MK), a novel growth factor. This monoclonal antibody was used in immunohistochemical studies to compare the expression of MK, proliferating cell nuclear antigen (PCNA) and p53 protein in 133 primary brain tumors and 21 carcinoma metastases to the central nervous system. Approximately half of the glioblastomas multiforme (GBMs) (19/32), medulloblastomas (8/14), primitive neuroectodermal tumors (PNETs) (5/11), breast carcinoma metastases (Br-Mts) (6/10) and lung carcinoma metastases (L-Mts) (5/11) as well as some astrocytomas (2/14) had tumor cells that expressed MK; however, oligodendrogliomas, ependymomas, schwannomas, meningiomas, and pituitary adenomas did not express MK. The values of the PCNA-labeling index were statistically higher in GBMs, medulloblastomas, PNETs, Br-Mts, and L-Mts that expressed MK than in those that did not (Wilcoxon rank-sum test, p < 0.05). There was no correlation between MK and p53 protein in all tumor types. Normal and non-neoplastic brain tissues were negative for MK, PCNA, and p53 protein. We conclude that primary and metastatic tumors of the brain express MK and that the MK expression in brain tumors may depend, in part, on the proliferating potential.     

Central nervous system lymphoma in the acquired immunodeficiency syndrome

Primary central nervous system (CNS) lymphomas were studied in fifteen autopsied patients with the acquired immunodeficiency syndrome (AIDS). Using the working formulation for non-Hodgkin's lymphomas, the tumors were classified as large cell (7 patients), mixed large and small cell (6 patients), small cleaved cell (1 patient), and unclassifiable (1 patient). The mixed lymphomas displayed unusual features characterized by a high mitotic rate and the presence of numerous medium-sized cells (5 to 10 mus), not classifiable using the working formulation. Focal T cell and lymphoplasmacytoid B cell infiltrates accompanied lymphoma cells at the periphery of and remote from solid tumor masses in 9 cases. Immunohistochemical analysis of the lymphomas suggested B cell neoplasms. All of these patients had concurrent CNS and systemic cytomegalovirus (CMV) infections. The CNS infections were of both viral (CMV, human immunodeficiency virus (HIV), varicella zoster virus (VZV), progressive multifocal leukoencephalopathy (PML) and non-viral (toxoplasmosis, candidiasis) etiology. In the general AIDS population at our institution, the autopsy incidence of CNS infections and systemic CMV was 63% and 60%, respectively. In contrast, the incidence for both these entities was 0% in otherwise healthy, non-AIDS patients with CNS lymphoma supports the hypothesis that viral infection plays a role in the pathogenesis of CNS lymphoma in the immunocompromised. Polyclonal lymphoplasmacytoid B and T cell infiltrates accompanying lymphoma may produce diagnostic difficulties on surgical biopsy. As these infiltrates were a frequent feature in this study, we caution that their recognition does not argue against the presence of CNS lymphoma.     

脑胶质瘤中c—myc基因扩增,H—ras及p53基因突变

研究脑胶质瘤中癌基因c-myc的扩增、H-ras的突变及抑癌基因p53的5～8外显子的突变情况以及与胶质瘤的恶性程度的关系.采用差异性PCR(DPCR)及PCR-SSCP、PCR-RFLP等方法检测22例脑胶质瘤的基因突变情况.22例脑胶质瘤中c-myc扩增率为63.6%(14/22),H-ras的突变率为36.4%(8/22),p53的突变率45.5%(10/22).提示:癌基因c-myc的扩增及抑癌基因p53的突变与脑胶质瘤的恶性程度有关(P<0.05),而癌基因H-ras的突变则与脑胶质瘤的发生有关,与脑胶质瘤的恶性程度无关(P>0.05).各类型脑胶质瘤基因突变未发现不同(P>0.05),可能与标本量少有关.     

Activating mutations of the GNAQ gene: a frequent event in primary melanocytic neoplasms of the central nervous system

Primary melanocytic neoplasms of the central nervous system (CNS) are uncommon neoplasms derived from melanocytes that normally can be found in the leptomeninges. They cover a spectrum of malignancy grades ranging from low-grade melanocytomas to lesions of intermediate malignancy and overtly malignant melanomas. Characteristic genetic alterations in this group of neoplasms have not yet been identified. Using direct sequencing, we investigated 19 primary melanocytic lesions of the CNS (12 melanocytomas, 3 intermediate-grade melanocytomas, and 4 melanomas) for hotspot oncogenic mutations commonly found in melanocytic tumors of the skin (BRAF, NRAS, and HRAS genes) and uvea (GNAQ gene). Somatic mutations in the GNAQ gene at codon 209, resulting in constitutive activation of GNAQ, were detected in 7/19 (37%) tumors, including 6/12 melanocytomas, 0/3 intermediate-grade melanocytomas, and 1/4 melanomas. These GNAQ-mutated tumors were predominantly located around the spinal cord (6/7). One melanoma carried a BRAF point mutation that is frequently found in cutaneous melanomas (c.1799 T>A, p.V600E), raising the question whether this is a metastatic rather than a primary tumor. No HRAS or NRAS mutations were detected. We conclude that somatic mutations in the GNAQ gene at codon 209 are a frequent event in primary melanocytic neoplasms of the CNS. This finding provides new insight in the pathogenesis of these lesions and suggests that GNAQ-dependent mitogen-activated kinase signaling is a promising therapeutic target in these tumors. The prognostic and predictive value of GNAQ mutations in primary melanocytic lesions of the CNS needs to be determined in future studies.     

Humoral immune response to p53 in malignant glioma

p53 immunoreactivity and humoral immune response to p53 were examined in 14 patients with malignant glioma, including 4 patients with leptomeningeal glioma cell dissemination. Twelve patients expressed p53 protein within the tumour tissue. p53 antibodies were detected in the serum in 2 of 14 patients but never in the cerebrospinal fluid (CSF). Soluble p53 protein was detected neither in serum nor in CSF of the glioma patients. CSF levels of the immunosuppressive cytokine, transforming growth factor (TGF)-β, were elevated in the glioma patients, including those with a humoral response to p53. These preliminary findings raise the possibility of systemic humoral immune responses to antigens, including mutant p53, expressed by glioma cells in the central nervous system. Received: 7 August 1997 Received in revised form: 29 October 1997 Accepted: 20 November 1997     

The p53 gene and its role in human brain tumors

Mutation of the p53 gene is among the most common lesions in a variety of human tumors, including those of the central nervous system. In most instances, mutation of one p53 allele is followed by loss of the remaining wild-type allele, resulting in cells with a complete absence of functional wild-type p53 protein. However, in some situations, such as at initiation of spontaneously arising gliomas or as the germline configuration of patients with the Li-Fraumeni syndrome, cells clearly carry both wild-type and mutant p53 alleles. These observations lead to the hypothesis that p53 mutations can give rise to loss of tumor suppressor functions as well as to gain of oncogenic transformation capabilities. In this review, we define the types of mutations that occur in the p53 gene in various glial tumors, contrast that with the spectra described in other human tumor types, and discuss the biochemistry and physiology of the p53 protein and its ability to regulate and be regulated by other gene products. We use this information to propose roles for p53 in the initiation and progression of human gliomas. © 1995 Wiley-Liss, Inc.     

bcl-2 protein expression in astrocytomas in relation to patient survival and p53 gene status

bcl-2 protein expression was characterized in a series of 58 astrocytomas from 21 pediatric and 37 adult patients. As part of a continuing attempt to define relevant prognostic factors which may predict clinical outcome, we have determined the impact of bcl-2 accumulation in malignant astrocytes on the length of patient survival. Aberrant overexpression of bcl-2 protein in tumor cells was detected in 57% (12 of 21) of pediatric and 73% (27 of 37) of the adult cases. Among pediatric patients, the median survival in months showed no relationship with the incidence of bcl-2-positive tumors. Among the adult patients, a favorable prognostic indicator was low-tumor grade (P = 0.05). bcl-2-positive tumors occurred with similar frequencies in WHO grades III and IV of malignancy. When bcl-2 expression in tumor cells was tested as a variable to predict for patient survival, the 6 patients without bcl-2 expression among 23 adult patients with grade IV tumors had a shorter median survival. The same 58 tumors had been previously analyzed for alterations of p53: 4 pediatric and 16 adult tumors had p53 gene mutations. There was no significant difference in median survival related to p53 gene status. There was no relationship between bcl-2 expression and p53 gene status: approximately equal numbers of tumors with either wild-type or mutant p53 were bcl-2 negative or bcl-2 positive. bcl-2 expression is high (40–100%) among other tumors of the central nervous system which also show low malignant potential. Up-regulation of bcl-2 in malignant astrocytes or constitutive expression in some tumor types may be a factor leading to a more favorable clinical outcome. Received: 27 January 1997 / Revised: 21 March 1997 / Accepted: 28 March 1997     

Secretory meningiomas are defined by combined KLF4 K409Q and TRAF7 mutations

Meningiomas are among the most frequent intracranial tumors. The secretory variant of meningioma is characterized by glandular differentiation, formation of intracellular lumina and pseudopsammoma bodies, expression of a distinct pattern of cytokeratins and clinically by pronounced perifocal brain edema. Here we describe whole-exome sequencing analysis of DNA from 16 secretory meningiomas and corresponding constitutional tissues. All secretory meningiomas invariably harbored a mutation in both KLF4 and TRAF7. Validation in an independent cohort of 14 secretory meningiomas by Sanger sequencing or derived cleaved amplified polymorphic sequence (dCAPS) assay detected the same pattern, with KLF4 mutations observed in a total of 30/30 and TRAF7 mutations in 29/30 of these tumors. All KLF4 mutations were identical, affected codon 409 and resulted in a lysine to glutamine exchange (K409Q). KLF4 mutations were not found in 89 non-secretory meningiomas, 267 other intracranial tumors including gliomas, glioneuronal tumors, pituitary adenomas and metastases, 59 peripheral nerve sheath tumors and 52 pancreatic tumors. TRAF7 mutations were restricted to the WD40 domains. While KLF4 mutations were exclusively seen in secretory meningiomas, TRAF7 mutations were also observed in 7/89 (8 %) of non-secretory meningiomas. KLF4 and TRAF7 mutations were mutually exclusive with NF2 mutations. In conclusion, our findings suggest an essential contribution of combined KLF4 K409Q and TRAF7 mutations in the genesis of secretory meningioma and demonstrate a role for TRAF7 alterations in other non-NF2 meningiomas.     

Molecular analysis for p53 and mdm2 in intracranial germ cell tumors

Intracranial germ cell tumors (ICGTs) are uncommon neoplasms. The histological appearance of ICGTs is indistinguishable from that of the usual testicular germ cell tumors (TGTs). Recently, several reports have associated molecular abnormalities of p53 and mdm2 in TGTs with their malignancies. However, whether ICGTs are associated with molecular abnormalities is still unknown. We analyzed a series of 16 ICGTs for mutations in the TP53 gene by single-strand conformation polymorphisms, and for amplification of the MDM2 gene using differential PCR. In addition, the same 16 tumors were examined for p53 and mdm2 protein overexpression using antibodies directed against p53 [monoclonal antibodies (mAb) 1801 and DO7] and mdm2 (IF2), respectively. Twelve (75%) and 2 (13%) of the 16 ICGTs reacted with DO7 and PAb1801, respectively, and 1 (6%) carried a TP53 gene mutation. Thirteen (81%) of the 16 ICGTs reacted with IF2, and 3 (19%) carried MDM2 gene amplification. The less frequent TP53 gene mutation compared with MDM2 gene amplification, and the frequently expressed p53 and mdm2 protein, are similar to the case for TGTs. It is tempting to speculate that ICGTs might have the same cellular origins as TGTs with abnormalities in p53 and mdm2, which could play an important role of tumorigenesis. Received: 9 March 1999 / Revised, accepted: 10 May 1999     

Étude immunohistochimique de 16 lymphomes primitifs du système nerveux central

Immunohistochemical study in 16 cases of primary lymphoma of the central nervous systemSixteen primary lymphomas of the central nervous system (CNS) have been studied with an immunoperoxidase technique (PAP) for the demonstration of intracytoplasmic immunoglobulins. The material was obtained by biopsy (10 cases) and at autopsy (6 cases). For comparison, ten other tumors (glial tumors and secondary lymphomas involving the nervous tissue) were simultaneously investigated. In the 16 primary lymphomas, 14 contained intracellular immunoglobulins that were considered “monoclonal” in 9 cases, “probably monoclonal” in 4 cases but “uninterpretable” in the last one.According to the Kiel classification, the 13 malignant lymphomas with intracellular immunoglobulins were classified, morphologically, as immunoblastic sarcomas (9 cases) or immunocytomas (4 cases). No immunoglobulins were detected in 2 cases: 1 lymphoblastic lymphoma and 1 centrocytic lymphoma. Various amounts of intracytoplasmic immunoglobulins were detected in inflammatory cells and glial cells (either reactive or tumoral) but the pattern of staining was consistent with current concepts of polyclonality. Therefore, a diagnosis based on the Kiel classification of lymphomas and the PAP technique will allow a more accurate prognosis on the evolution of primary lymphomas of the CNS with aspect of “high grade” and “low grade” malignancy.     

Characterisation of molecular alterations in microdissected archival gliomas

Classification of gliomas according to their molecular characteristics may be important in future histopathological diagnosis. However, gliomas frequently display heterogeneity at the histological, biological and molecular level. In this study of archival diagnostic gliomas, precision microdissection was used to enrich samples in the most malignant cells or to investigate intratumoural histological heterogeneity. Analysis of tumour samples microdissected from the most aggressive regions, representative of the histopathological diagnosis, revealed PTEN mutations in 4/14 anaplastic astrocytomas, 4/13 glioblastomas and 1 gliosarcoma, but not in 19 low-grade gliomas. Using a novel PCR procedure and direct sequence analysis of the entire coding sequence, TP53 mutations were detected in 1/3 pilocytic astrocytomas, 3/13 astrocytomas, 4/14 anaplastic astrocytomas, 5/13 glioblastomas and 1 gliosarcoma. All but one of the tumours with TP53 mutation showed p53 immunopositivity, but 5 low-grade and 10 high-grade gliomas had p53 protein nuclear accumulation in the absence of detectable mutation. p53 status was unrelated to p21 expression. Neither PTEN nor TP53 mutations influenced the proliferative index or microvessel density of high-grade astrocytomas. Unusual findings include: TP53 mutation in a juvenile pilocytic astrocytoma; TP53 and PTEN mutations in a de novo glioblastoma, a gliosarcoma with identical mutations in gliomatous and sarcomatous components, and an infratentorial anaplastic astrocytoma with an earlier supratentorial grade II astrocytoma bearing the same TP53 mutation but not the PTEN mutation or loss of heterozygosity (LOH) of 10q23. Similarly, the transition to high-grade histology was associated with acquisition of PTEN mutations and 10q23.3 LOH in two de novo high-grade tumours with regions of low-grade histology.