No abstract

Loss of function of the p53 tumor suppressor gene due to mutation occurs early in astrocytoma tumorigenesis in about 30-40% of cases. This is believed to confer a growth advantage to the cells, allowing them to clonally expand due to loss of the p53-controlled G1 checkpoint and apoptosis. Genetic instability due to the impaired ability of p53 to mediate DNA damage repair further facilitates the acquisition of new genetic abnormalities, leading to malignant progression of an astrocytoma into anaplastic astrocytoma. This is reflected by a high rate of p53 mutation (60-70%) in anaplastic astrocytomas. The cell cycle control gets further compromised in astrocytoma by alterations in one of the G1/S transition control genes, either loss of the p16/CDKN2 or RB genes or amplification of the cyclin D gene. The final progression process leading to glioblastoma multiforme seems to need additional genetic abnormalities in the long arm of chromosome 10; one of which is deletion and/or functional loss of the PTEN/MMAC1 gene. Glioblastomas also occur as primary (de novo) lesions in patients of older age, without p53 gene loss but with amplification of the epidermal growth factor receptor (EGFR) genes. In contrast to the secondary glioblastomas that evolve from astrocytoma cells with p53 mutations in younger patients, primary glioblastomas seem to be resistant to radiation therapy and thus show a poorer prognosis. The evaluation and design of therapeutic modalities aimed at preventing malignant progression of astrocytomas and glioblastomas should now be based on stratifying patients with astrocytic tumors according to their genetic diagnosis.

While it is established that p53 mutation plays a critical role in the carcinogenesis of astrocytic brain tumors, its role remains to be clarified for other types of tumors in the central nervous system (CNS). Using a yeast-based assay which tests the ability of human p53 to activate transcription, we analyzed p53 mutations in 85 non-astrocytic CNS tumors, including 4 benign neuronal tumors (3 central neurocytomas and 1 pineocytoma), 12 primitive neuroectodermal tumors, 14 germ cell tumors (7 germinomas, 7 non-germinomatous tumors), 4 craniopharyngiomas, 14 ependymomas, 22 schwannomas, 10 primary brain lymphomas in immunocompetent patients, and 5 bone tumors of the skull. The only tumors found to contain p53 mutations were 3 malignant lymphomas. The presence of mutations in these cases was confirmed by DNA sequencing. Given the high accuracy and sensitivity of the yeast assay and previous negative results using conventional techniques, this indicates that p53 mutation is a rare event in non-astrocytic CNS tumor types examined here.

We previously reported clonal expansion of p53 mutations in malignant astrocytic tumors detected with a yeast p53 functional assay that measures mutant p53 alleles quantitatively and loss of p53 transcriptional competence qualitatively (Tada et al., Int J Cancer 67:447-450, 1996). This method selectively detects inactivating mutations and is relatively insensitive to contamination of tumor samples with normal tissue. To determine whether the mutation frequency and spectrum detected in this way differ from those seen with conventional techniques, 54 malignant astrocytomas were tested with the yeast assay, and the abnormalities detected were characterized by DNA sequencing. Inactivating p53 mutations were found in 67% of anaplastic astrocytomas and 41% of glioblastomas. Overall, mutations were found in 48% of tumors, compared with only 29% in previous studies (P < 0.005), a difference that probably reflects the greater sensitivity of the yeast assay than of conventional techniques. The frequency of mutations in anaplastic astrocytomas (in our study plus published studies) was significantly higher than in glioblastomas (39% vs 29%; P < 0.05). This suggests that acquisition of p53 mutations is not rate limiting for progression to glioblastoma and that many glioblastomas develop by p53-independent pathways. Sequencing of mutant p53 cDNAs rescued from yeast showed that the mutation spectrum for functionally inactive mutants was nearly identical to the spectra from previous studies on structural mutants, indicating that transcriptional activity is the critical biological target of p53 mutation in malignant astrocytomas.

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.

The authors describe the case of a 69-year-old man with an intracerebral hemorrhage due to rupture of a nontraumatic aneurysm of the middle meningeal artery (MMA). The ipsilateral posterior cerebral artery (PCA) was occluded, and dural anastomoses developed as the main collateral pathway between the MMA and the cortical branch of the PCA, on which the aneurysm was located. It is considered that increased hemodynamic stress to the collateral pathway contributed to the formation of the aneurysm.

The patient is free of disease at the age of 2 years and 6 months.

We have developed a new yeast-based assay for the detection of PTEN nonsense mutation, and applied it to a total of 42 astrocytic tumors. The assay utilizes homologous recombination of PCR-ampli®ed PTEN cDNA samples to a yeast vector which expresses an inframe PTEN::ADE2 chimera protein. An allele of nonsense mutation in the sample PTEN mRNA gives a truncated chimera protein in a yeast cell, resulting in the formation of a red colony. The assay and subsequent sequence analysis demonstrated nonsense mutations as red colonies of more than 10% in one of 10 anaplastic astrocytomas and six of 18 glioblastomas, but none in six pilocytic astrocytomas or in eight astrocytomas. Sequence analysis of white colonies showed one missense mutation in a glioblastoma. Interestingly, four of seven nonsense mutations were frame-shifts due to exon skipping. In addition, pink colonies were found in one of six pilocytic astrocytomas, three of eight astrocytomas, two of 10 anaplastic astrocytomas, and 10 of 18 glioblastomas. Sequence analysis of the pink colonies revealed a sequence similar to those reported as cPTEN/PTH2. By testing mRNA and genomic DNA, it was found to be a processed pseudogene which was transcribed. The cPTEN expression was complementary to PTEN mutation, for 14 of 18 glioblastomas showed either PTEN mutation or cPTEN expression and only one case showed both PTEN mutation and cPTEN expression (P50.046), suggesting a pathological role of cPTEN expression as an alternative to PTEN mutation in glioblastomas.