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Pleomorphic xanthoastrocytoma (PXA) is a tumor of astrocytic lineage that may have anaplastic features; such a phenotype usually correlates with a less favorable outcome. The molecular profile of PXA differs from other astrocytic tumors, but the molecular mechanisms of its formation and progression are still undefined. We analyzed a loss of heterozygosity and mutations of the SMARCB1 (also known as SNF5 or INI1) and TP53 genes in four cases of PXA. In just one case a TP53 mutation (Cys238Tyr) was readily detectable at the mRNA level, but it was almost undetectable during DNA sequencing. This case was also the only one exhibiting anaplastic features and TP53 overexpression as defined by immunohistochemistry. This observation supports our earlier findings on discrepancies in TP53 status in glioblastomas. We suggest that the incidence of TP53 mutations in pleomorphic xanthoastrocytoma may be underestimated and that molecular approaches should be used for greater diagnostic precision.

Background: Recently published data showed discrepancies between P53 cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers., Methods: To this end, we analyzed 23 colorectal cancers for P53 mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry., Results: We found P53 gene mutations in 16 cases (15 missense and 1 nonsense). Two of the 15 cases with missense mutations showed alterations based only on cDNA, and not DNA sequencing. Moreover, in 6 of the 15 cases with a cDNA mutation those mutations were difficult to detect in the DNA sequencing, so the results of DNA analysis alone could be misinterpreted if the cDNA sequencing results had not also been available. In all those 15 cases, we observed a higher ratio of the mutated to the wild type template by cDNA analysis, but not by the DNA analysis. Interestingly, a similar overexpression of P53 mRNA was present in samples with and without P53 mutations., Conclusion: In terms of colorectal cancer, those discrepancies might be explained under three conditions: 1, overexpression of mutated P53 mRNA in cancer cells as compared with normal cells; 2, a higher content of cells without P53 mutation (normal cells and cells showing K-RAS and/or APC but not P53 mutation) in samples presenting P53 mutation; 3, heterozygous or hemizygous mutations of P53 gene. Additionally, for heterozygous mutations unknown mechanism(s) causing selective overproduction of mutated allele should also be considered. Our data offer new clues for studying discrepancy in P53 cDNA and DNA sequencing analysis.

O(6)-methylguanine DNA methyltransferase (MGMT) reduces cytotoxicity of therapeutic or environmental alkylating agents. MGMT promoter methylation has been associated with TP53 G: C to A:T transition mutations in various types of cancers, and with poor prognosis in patients who did not receive chemotherapy. Mutations of TP53 are more frequent in secondary than in primary glioblastoma, thus the expected MGMT promoter methylation was low in primary glioblastoma. Glioblastoma patients with MGMT promoter methylation showed better response to chemotherapy based on alkylating agents and longer survival than patients without MGMT methylation. We examined 32 primary glioblastomas, treated with radiotherapy and surgery, for TP53 mutation by direct sequencing and MGMT promoter methylation by methylation-specific PCR. MGMT promoter methylation and TP53 mutations were detected in 72% and 31% of primary glioblastoma, respectively. Although not statistically significant, the frequency of TP53 G:C to A:T mutations were higher in cases with (26%) than without (11%) MGMT promoter methylation (p=0.376). MGMT promoter methylation had no impact on patient survival. Our data indicate that MGMT promoter methylation occurs frequently in primary glioblastoma, but does not lead to G:C to A:T TP53 mutations, has no independent prognostic value and is not a predictive marker unless glioblastoma patients are treated with chemotherapy.

We screened 50 glioblastomas for P53 mutations. Five glioblastomas showed heterozygous mutations, while three were putatively heterozygous. Six of these eight glioblastomas showed elimination of wild-type P53 mRNA. These results strongly suggest that some sort of mechanism(s) favouring mutated over wild-type P53 mRNA exists in glioblastoma cells with heterozygous mutations of this gene.

We report here the mutational analysis of hSNF5/INI1 and TP53 genes performed on 11 specimens of choroid plexus carcinomas (CPC) in which a large number of abnormalities has been detected by molecular biology techniques. Loss of heterozygosity (LOH) analysis performed on six tumors revealed losses on chromosomes 1, 3, 5, 9, 10, 13, 16, 18, and 22. However, there were no abnormalities on 17p and mutations of the TP53 gene have been observed for two tumors comprising exons 5 and 7, respectively. Exon 4 of hSNF5/INI1 was mutated in one tumor with LOH restricted to the hSNF5/INI1 locus. There was no coexistence of mutations in both analyzed genes. Our analysis confirms the presence of the hSNF5/INI1 mutations and proves involvement of TP53 mutations in sporadic cases of CPC.

In the present study, the expression of P53 and MDM2 proteins were examined in 94 soft-tissue sarcomas (35 malignant fibrohistiocytomas, 15 neurosarcomas, 14 liposarcomas, 13 leiomyosarcomas, 11 fibrosarcomas and 6 dermatofibrosarcomas) by immunohistochemistry. The immunohistochemical findings were correlated with P53 mutation analysis using PCR-SSCP, PCR-HDF and direct sequencing, and MDM2 amplification studies by differential PCR. P53 immunopositivity was found in 25 out of 94 (26.6%) cases. Alterations of the P53 gene were detected in 12 (12.8%) tumors; eight of these tumors revealed P53 immunoreactivity. A high number of P53 positive and P53 mutated tumors were histologically defined as poorly differentiated G3 (64.0% and 75.0%, respectively). MDM2 immunopositivity was revealed in 36 out of 94 (38.3%) cases. MDM2 amplification occurred in 17 tumors (18.1%); only nine of these tumors exhibited MDM2 immunoreactivity. Overall, MDM2 positivity was not associated with MDM2 amplification in 27 out of 94 tumors (28.7%). There was no significant correlation between MDM2 overexpression and histological grade. However, when the samples were stratified by immunophenotype, the majority of tumors (52.5%) with isolated MDM2 overexpression (dissociated from P53 positivity) were defined histologically as low grade (G1 + G2). These results support the notion that besides P53 alterations, MDM2 gene deregulation seems to be an important event in sarcomas evolution. Additionally, the mechanism of MDM2-mediated degradation of P53 protein, without involving stabilization and inactivation of P53 gene, should be considered for better understanding of all features of tumor progression processes.