Mutational study of the 1p located genes p18ink4c, Patched-2, RIZ1 and KIF1B in oligodendrogliomas.

Loss of 1p heterozygosity is one of the most characteristic events in oligodendrogliomas. Several genes located in this region have been previously studied to find the target gene implicated in the development of this tumor without success. Patched-2, RIZ1 and KIF1B are novel oncosuppressor genes located at 1p and involved in different kinds of tumors. We have studied these genes and p18(ink4c) using PCR/SSCP methods to detect sequence variations in a series of 40 oligodendrogliomas in which the allelic status at 1p was analyzed. Polymorphisms or no sequence changes were detected in all four genes analyzed. None of the genes analyzed seem to be the target-gene mapped at 1p involved by mutation in oligodendroglioma development.

Molecular analysis of the erbB gene family calmodulin-binding and calmodulin-like domains in astrocytic gliomas.

Primarily involved in cell proliferation and differentiation processes, the plasma membrane-bound ErbB tyrosine kinase receptor family is formed by four members: erbB1/EGFR, erbB2/HER2/Neu, erbB3/HER3 and erbB4/HER4. Calmodulin (CaM) is a Ca2+-binding protein involved in the regulation of multiple intracellular processes that binds directly to EGFR in the presence of Ca2+, inhibiting its tyrosine kinase activity. Two main regions in the receptor have been implicated in this relationship: the calmodulin-binding domain (CaM-BD) and the calmodulin-like domain (CaM-LD); their sequences are highly conserved in other members of this family of receptors. The presence of mutations, amplification and/or overexpression and genomic rearrangement of these domains was investigated for all four erbB family genes in a series of 89 glial tumors, including 44 WHO grade IV glioblastomas, 21 WHO grade III anaplastic astrocytomas, and 24 WHO grade II astrocytomas. Gene alterations were only found in the regions of interest in EGFR. One glioblastoma showed an in frame tandem duplication of the intracellular region including CaM-LD (exons 18-25). CaM-BD gene overdose was evidenced in 18 tumors that showed EGFR amplification in other domains. Over-expression of CaM-BD and CaM-LD was detected in 6 and 17 cases, respectively, of the 19 tumors in which this study was performed. The other three genes coding for the ErbB receptors did not present point mutations, or rearrangements, and only a very low amplification rate was found for erbB2 (1 case) and erbB3 (4 cases). No overexpression of erbB2, erbB3 or erbB4 was detected. These findings suggest that EGFR is the main erbB gene family member non-randomly involved in malignant glioma development, and that the two domains under study, due to their high conservation and wide separation in the EGFR sequence, are good marker regions for evaluating EGFR/erbB1 gene amplification, as well as for analysing the presence of transcripts corresponding to truncated cytosolic forms of the receptor in these tumors.

Methylation status of TP73 in meningiomas.

Deletions at 1p are frequent in meningioma and represent a genetic marker associated with the genesis of atypical WHO grade II forms. Previous mutational analysis of TP73, a structurally and functionally TP53 homologous gene located at 1p36.33, failed to demonstrate a significant rate of sequence variations linked to gene inactivation in meningiomas with 1p loss. As an alternative, TP73 may be inactivated through aberrant 5' CpG island methylation, a primary mechanism participating in the inactivation of tumor suppressor genes during tumorigenesis. We determined the methylation status of the TP73 gene in a series of 60 meningiomas (33 grade I, 24 grade II, and 3 grade III samples), including tumors with deletion at 1p (n=30) and with intact 1p (n=30). Aberrant methylation was detected in 10 cases (33%) with 1p deletion and in 3 tumors (10%) with retention of alleles at this chromosome arm. The distribution of the 13 cases of methylation according to malignancy grade was 7 grade I, 5 grade II, and 1 grade III tumor. Accordingly, although TP73 aberrant methylation was more frequent in meningiomas with 1p deletion (P<0.05), no association with the grade of malignancy could be established. These findings, together with the previously reported increased TP73 expression in malignant meningiomas suggest that opposing functions of this gene may characterize distinct subsets of tumors: suppressed or reduced expression as a result of CpG methylation in some grade I-grade II tumors, and enhanced expression in some more malignant forms.

Deletion and aberrant CpG island methylation of Caspase 8 gene in medulloblastoma.

Aberrant methylation of promoter CpG islands in human genes is an alternative genetic inactivation mechanism that contributes to the development of human tumors. Nevertheless, few studies have analyzed methylation in medulloblastomas. We determined the frequency of aberrant CpG island methylation for Caspase 8 (CASP8) in a group of 24 medulloblastomas arising in 8 adult and 16 pediatric patients. Complete methylation of CASP8 was found in 15 tumors (62%) and one case displayed hemimethylation. Three samples amplified neither of the two primer sets for methylated or unmethylated alleles, suggesting that genomic deletion occurred in the 5' flanking region of CASP8. Our findings suggest that methylation commonly contributes to CASP8 silencing in medulloblastomas and that homozygous deletion or severe sequence changes involving the promoter region may be another mechanism leading to CASP8 inactivation in this neoplasm.

Promoter methylation status of multiple genes in brain metastases of solid tumors.

The aberrant methylation of the CpG island promoter regions acquired by tumor cells is one mechanism for loss of gene function. The high methylation rate for RB1 and death-associated protein-kinase gene (DAP-kinase) (60 and 90%, respectively) previously found in brain metastases suggests this mechanism could be non-randomly associated to tumor progression and metastasis. Thus, in addition to these two genes, we determined the methylation status of the genes p16INK4a, glutathione S-transferase P1 (GSTP1), O6-methylguanine DNA methyltransferase (MGMT), thrombospondin-1 (THBS1), p14ARF, TP53, p73, and tissue inhibitor of metalloproteinase 3 (TIMP-3), in 18 brain metastases of solid tumors, with methylation specific PCR. The metastases were derived from malignant melanoma (three cases), lung carcinoma (six cases), breast carcinoma (three cases), ovarian carcinoma (two cases) and one each from colon, kidney, bladder and undifferentiated carcinoma. We detected methylation levels in the tumor samples of 83% in p16INK4a, 72% in DAP-kinase, 56% in THBS1, 50% in RB1, 39% in MGMT, 33% in GSTP1 and p14ARF each, 22% in p73 and TIMP-3 each, and 11% in TP53. The methylation index (number of genes methylated/number of genes tested) varied between 0.1 and 0.6, with an average of 0.42, indicating that a high grade of gene methylation accumulates parallel to the tumor metastasis process. Our data suggest an important role for gene methylation in the development of brain metastases, primarily involving epigenetic silencing of DAP-kinase, THBS1 and the cell-cycle regulators RB1/p16INK4a.

Hypermethylation of the DNA repair gene MGMT: association with TP53 G:C to A:T transitions in a series of 469 nervous system tumors.

O6-methylguanine-DNA methyltransferase (MGMT) plays a major role in repairing DNA damage from alkylating agents. By removing alkyl groups from the O6-position in guanine, MGMT can prevent G:C to A:T transition mutations, a type of variation frequently involving TP53 mutations in brain tumors. Promoter hypermethylation of CpG islands in tumor-related genes can lead to their transcriptional inactivation, and this epigenetic mechanism has been shown to participate in MGMT silencing in some cancers, including those affecting the nervous system. Accordingly, a link between both genetic and epigenetic anomalies may exist in these neoplasms. To determine the relevance of defective MGMT function due to aberrant methylation in relation to the presence of TP53 mutations, we studied 469 nervous system tumors (including all major histological subtypes) for MGMT promoter methylation and TP53 mutations at exons 5-8. Overall, aberrant methylation occurred in 38% of the samples (180/469), with values higher than 50% in the more malignant forms such as glioblastomas and anaplastic gliomas including those with astrocytic, oligodendroglial and ependymal differentiation. In contrast, the non-glial tumors displayed an overall aberrant MGMT promoter methylation of 26%, even though this group includes highly malignant tumors such as neuroblastomas, medulloblastomas and brain metastases. Overall, TP53 mutations were found in 25% of the methylated MGMT tumors (45/180), whereas only 10% of the unmethylated MGMT tumors (30/289) showed TP53 changes (P < 0.001). G:C to A:T changes occurred at CpG sites in 9% of methylated tumors, and in 0.7% of the unmethylated samples. This type of transition at non-CpG dinucleotides was also more frequent in the tumors with aberrant MGMT methylation (5%) than the unmethylated tumors (0.7%). These data suggest that MGMT silencing as a result of promoter hypermethylation may lead to G:C to A:T transition mutations in the TP53 gene of some histological nervous system tumor subtypes.

Genetic and epigenetic alteration of the NF2 gene in sporadic meningiomas.

The role of the NF2 gene in the development of meningiomas has recently been documented; inactivating mutations plus allelic loss at 22q, the site of this gene (at 22q12), have been identified in both sporadic and neurofibromatosis type 2-associated tumors. Although epigenetic inactivation through aberrant CpG island methylation of the NF2 5' flanking region has been documented in schwannoma (another NF2-associated neoplasm), data on participation of this epigenetic modification in meningiomas are not yet widely available. Using methylation-specific PCR (MSP) plus sequencing, we assessed the presence of aberrant promoter NF2 methylation in a series of 88 meningiomas (61 grade I, 24 grade II, and 3 grade III), in which the allelic constitution at 22q and the NF2 mutational status also were determined by RFLP/microsatellite and PCR-SSCP analyses. Chromosome 22 allelic loss, NF2 gene mutation, and aberrant NF2 promoter methylation were detected in 49%, 24%, and 26% of cases, respectively. Aberrant NF2 methylation with loss of heterozygosity (LOH) at 22q was found in five cases, and aberrant methylation with NF2 mutation in another; LOH 22q and the mutation were found in 16 samples. The aberrant methylation of the NF2 gene also was the sole alteration in 15 samples, most of which were from grade I tumors. These results indicate that aberrant NF2 hypermethylation may participate in the development of a significant proportion of sporadic meningiomas, primarily those of grade I.

DNA methylation of multiple promoter-associated CpG islands in meningiomas: relationship with the allelic status at 1p and 22q.

The purpose of this research was to examine the DNA methylation profile of meningiomas. Accordingly, we examined the DNA methylation status of ten tumor-related genes (RB1, p16(INK4a), p73, MGMT, ER, DAPK, TIMP-3, p14(ARF), THBS1, and Caspase-8) in 98 meningiomas (68 grade I; 27 grade II; and 3 grade III samples) using methylation-specific PCR and sequencing. The most frequently methylated genes were THBS1 (30%), TIMP-3 (24%), p16(INK4a) (17%), MGMT (16%), p73 (15%), ER (15%), and p14(ARF) (13%), whereas methylation was relatively rare in the other genes (<10%). Methylation occurred in at least one gene in 77.5% of the cases and in three or more genes in 25.5%. Methylation was tumor specific since it was absent in the controls: two non-neoplastic meningeal samples and two non-neoplastic brain samples. The frequency of aberrant gene methylation in grade I versus grade II-III tumors showed some differences for TIMP-3, THBS1, MGMT, p16(INK4a) and p73; these differences reached statistical significance for TIMP-3: 18% in grade I versus 40% in grade II-III (P < 0.02). Our previous loss of heterozygosity studies provided the allelic constitution at 1p and 22q for 60 of the 98 meningiomas included in this report. The level of aberrant promoter methylation increased in tumors (30 samples) displaying 1p loss (either isolated or as concurrent deletion at 1p/22q; P = 0.014). These meningiomas primarily accumulated the epigenetic changes of THBS1 (14/30; 47%; P < 0.005), TIMP-3 (12/30; 40%; P < 0.05), p73 (10/30; 26%; P < 0.02) and p14(ARF) /p16(INK4a)(7/30 each one; 23%; not significant). Our findings indicate that aberrant DNA methylation of promoter-associated CpG islands in meningiomas contributes to the development of these tumors.