DNA methylation of multiple genes in vestibular schwannoma: Relationship with clinical and radiological findings.

HYPOTHESIS: The purpose of this study was to examine the DNA methylation profile of several genes in a series of vestibular schwannomas, and to analyze its relationship with clinical and radiological features. BACKGROUND: Aberrant methylation of promoter regions is a major mechanism for silencing of tumor suppressor genes in several tumors. There is limited information about methylation status in vestibular schwannoma, with no clinical or radiological implications described to date. METHODS: The methylation status of 16 tumor-related genes including RASSF1A, RAR-B, VHL, PTEN, HMLH1, RB1, TP16, CASP8, ER, TIMP3, MGMT, DAPK, TP73, GSTP1, TP14, and THBS1 was examined in a series of 22 vestibular schwannomas.The bisulfite modification of genomic DNA was performed. Clinical and radiological features were compared with the methylation results. RESULTS: Methylation values from 9% to 27% were found in 12 of 16 genes tested, including RASSF1A, VHL, PTEN, TP16, CASP8, TIMP3, MGMT, DAPK, THBS1, HMLH1, TP73, and GSTP1. A significant association was found between CASP8 and RASSF1A methylation. Methylation of CASP8 was associated with the patient's age and the tumor size. Methylation of TP73 was associated with hearing loss. RASSF1A methylation was inversely correlated with the clinical growth index. CONCLUSION: Aberrant methylation of tumor-related genes may play a role in the development of vestibular schwannomas. Our results may provide useful clues to the development of prognostic assays for these tumors.

Promoter CpG methylation of multiple genes in pituitary adenomas: frequent involvement of caspase-8.

The epigenetic changes in pituitary adenomas were identified by evaluating the methylation status of nine genes (RB1, p14(ARF), p16(INK4a), p73, TIMP-3, MGMT, DAPK, THBS1 and caspase-8) in a series of 35 tumours using methylation-specific PCR analysis plus sequencing. The series included non-functional adenomas (n=23), prolactinomas (n=6), prolactinoma plus thyroid-stimulating hormone adenoma (n=1), growth hormone adenomas (n=4), and adrenocorticotropic adenoma (n=1). All of the tumours had methylation of at least one of these genes and 40% of samples (14 of 35) displayed concurrent methylation of at least three genes. The frequencies of aberrant methylation were: 20% for RB1, 17% for p14(ARF), 34% for p16(INK4a), 29% for p73, 11% for TIMP-3, 23% for MGMT, 6% for DAPK, 43% for THBS1 and 54% for caspase-8. No aberrant methylation was observed in two non-malignant pituitary samples from healthy controls. Although some differences in the frequency of gene methylation between functional and non-functional adenomas were detected, these differences did not reach statistical significance. Our results suggest that promoter methylation is a frequent event in pituitary adenoma tumourigenesis, a process in which inactivation of apoptosis-related genes (DAPK, caspase-8) might play a key role.

EGFR intragenic loss and gene amplification in astrocytic gliomas.

We have studied EGFR gene amplification and allelic status of chromosome 7 in 68 tumors consisting of 34 WHO grade IV glioblastomas (26 primary and 8 secondary), 14 WHO grade III anaplastic astrocytomas, and 20 WHO grade II astrocytomas, by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), quantitative PCR, and microsatellite analysis. EGFR gene amplification was present in 27 of these tumors (40%), and we identified allelic losses at 7p11 approximately p14 in 38 of the 68 cases (56%), including 17 tumors displaying loss for EGFR intragenic markers. The positive correlation (P < 0.05, chi(2)) between tumors with EGFR intragenic loss and EGFR gene amplification, frequently displaying the EGFR vIII form, suggests that EGFR gene rearrangement leading to intragenic loss is a molecular event that participates in the amplification process of this gene.

Real-time quantitative PCR analysis of regions involved in gene amplification reveals gene overdose in low-grade astrocytic gliomas.

We have studied gene amplification of genes located in 1q32 (GAC1, ELF3, MDM4, and ren1), 4q11 (PDGFR-alpha), and in 12q13-14 (MDM2 and CDK4) using quantitative real-time PCR in a group of 86 tumors consisting of 44 WHO grade IV glioblastomas (GBM) (34 primary and 10 secondary tumors), 21 WHO grade III anaplastic astrocytomas (AA), and 21 WHO grade II astrocytomas (AII). Gene amplification was present in 56 of the 86 samples (65%) in at least 1 gene in our series. GAC1 (51%) and MDM4 (27%) were the most frequently amplified genes within the 1q32 amplicon, and their higher amplification frequency was statistically significant (P<0.05, chi) in the low-grade astrocytomas. Concordant co-amplification was determined for ELF3 and ren1 or ren1 and MDM4 in the grade III-IV tumors. MDM2 amplification was significantly more frequent in primary GBM (16%) than was in secondary GBM (0%). The present study shows that gene amplification in the studied regions is already present in low-grade astrocytic tumors and that amplification of some genes may represent another molecular marker to differentiate primary from secondary GBM.

Mutational analysis of the DAL-1/4.1B tumour-suppressor gene locus in meningiomas.

The DAL-1/41B gene (differentially expressed in adenocarcinoma of the lung), located in the chromosome 18p11.3 region, belongs to the protein family 4.1 (membrane-associated proteins), which includes the product of the NF2 gene (merlin), and the proteins, ezrin, radixin, and moesin. DAL-1/4.1B is normally expressed at high levels in the brain, with lower levels in the kidney, intestine, and testis. DAL-1/4.1B is known to suppress growth in meningiomas and can be lost in about 60% of sporadic meningiomas as an early event in tumorigenesis; it is a critical growth regulator in the pathogenesis of neoplastic transformation. The similarity between the DAL-1/4.1B protein and merlin, with their high levels of expression in the brain and their recurrent loss in meningiomas, and the lack of previous DAL-1/4.1B mutational analysis reports initiated this mutational study of DAL-1/4.1B in a series of 83 meningiomas. We found the following sequence variations; Ala555Thr (G1663A in exon 13) and Thr950Lys (C2849A in exon 19) in two cases each, and one case with a 5pb deletion (del taaaa) in intron 18. A polymorphism in exon 14 (C2112T/Thr704Thr, also known as C2166T) was also identified; the tumoral allelic constitutions were heterozygous C/T in 15, homo- or hemizygous C in 67 and hemizygous T in one tumour. The low mutational frequency in our study discounts sequence variations in DAL-1/4.1B as the main mechanism underlying participation of this gene in the neoplastic transformation of meningiomas, and suggests that other inactivating mechanisms, such as epigenetic changes, may participate in DAL1/4.1B silencing.

No evidence of INI1hSNF5 (SMARCB1) and PARVG point mutations in oligodendroglial neoplasms.

Allelic losses of chromosome 22 found in oligodendrogliomas suggest that at least one tumor suppressor gene on chromosome 22 is inactivated during the multistep process of tumorigenesis in this glial tumor. INI1hSNF5 (HUGO symbol: SMARCB1), located at 22q11, encodes a component of the ATP-dependent chromatin remodeling hSWI-SNF complex; it is a tumor suppressor gene that is mutated in several malignant tumors. The PARVG gene, located at 22q13, has been found to exhibit reduced expression in some cancer lines. Both genes are thus candidate tumor suppressors, potentially involved in the pathogenesis of gliomas. We performed mutation analyses of INI1hSNF5 and PARVG in a series of 40 oligodendrogliomas, but only sequence polymorphic variations were identified. Accordingly, INI1hSNF5 and PARVG do not seem to be the tumor suppressor genes involved in oligodendroglioma development and progression.

Real-time quantitative PCR analysis of gene dosages reveals gene amplification in low-grade oligodendrogliomas.

Proto-oncogene amplification is an important alteration that is present in about 45% to 50% of high-grade human gliomas. We studied this mechanism in 8 genes (cyclin-dependent kinase-4 [CDK4], MDM2, MDM4, renin-angiotensin system-1, ELF3, GAC1, human epidermal growth factor receptor-2, and platelet-derived growth factor receptor-A gene) in a series of 40 oligodendrogliomas (World Health Organization (WHO) grade II, 21; WHO grade III, 13; and WHO grade II-III oligoastrocytomas, 6) using real-time quantitative polymerase chain reaction. Amplification of at least 1 of these genes was detected in 58% of samples (23/40). By histopathologic grade, 67% of grade II oligodendrogliomas (14/21), 46% of grade III anaplastic oligodendrogliomas (6/13), and 50% of mixed oligoastrocytomas (3/6) were positive for amplification of at least 1 gene. CDK4, MDM2, and GAC1 were the most frequently involved genes (12/40 [30%], 12/40 [30%], and 13/40 [33%], respectively). Our findings demonstrate gene amplification in low-grade samples indicating that it is an important alteration in the early steps of oligodendroglioma development and, therefore, might be considered a molecular mechanism leading to malignant progression toward anaplastic forms.

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.

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.

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.

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.

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.

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.

Aberrant CpG island methylation of multiple genes in ependymal tumors.

Aberrant methylation of promoter CpG islands in human genes represents an alternative mechanism for genetic inactivation, and contributes to the development of human tumors. Nevertheless, thus far, few reports have analyzed methylation in ependymomas. We determined the frequency of aberrant CpG island methylation of several tumor-associated genes: p16(INK4a), RB1, MGMT, DAPK, TIMP3, THBS1, TP73, NF2 and Caspase 8 in a group of 27 ependymomas, consisting of 22 WHO grade II samples and five anaplastic WHO grade III tumors. The respective methylation indices (number of genes methylated/total genes analyzed) for both tumor groups was 0.195 and 0.198. Overall methylation rates greater than 20% were detected in MGMT, TIMP3, THBS1 and TP73. NF2 and Caspase 8 each presented hypermethylation in less than 10% of cases, and the cell-cycle regulators RB1/p16(INK4a) were hypermethylated in 4% and 18% of the samples, respectively, mostly affecting the low-grade forms. Our findings suggest that methylation commonly contributes to the inactivation of cancer-related genes in ependymomas.

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.

[Metastasis tumors of the central nervous system: molecular biology]. / Metástasis en el sistema nervioso central: biología molecular.

Metastases in the nervous system represent an important and growing problem in the clinical practice, being the cause of a great mortality in the developed countries. This article reviews the few data available on the molecular mechanisms involved in the pathogenesis of these tumours, leading to oncogene activation, inactivation of tumour suppressor genes, not only by the classical mechanisms, but also by the tumour cell epigenetic balance alteration. We conclude that all this knowledge will lead in the future to a better diagnosis, treatment and clinic evolution of these patients.

CpG island methylation in sporadic and neurofibromatis type 2-associated schwannomas.

PURPOSE: The purpose of this research was to examine the DNA methylation profile of schwannomas. EXPERIMENTAL DESIGN: We examined the DNA methylation status of 12 tumor-related genes (NF2, RB1, p14(ARF), p16(INK4a), p73, TIMP-3, MGMT, DAPK, THBS1, caspase-8, TP53, and GSTP1) in 44 sporadic and/or NF2-associated schwannomas using methylation-specific PCR. RESULTS: The most frequently methylated genes were THBS1 (36%), p73 (27%), MGMT (20%), NF2 (18%), and TIMP-3 (18%). The RB1/p16INK4a gene pair displayed aberrant methylayed alleles in 15% of cases, whereas methylation was relatively rare in the other genes (<5%). Methylation was tumor specific because it was absent in two nonneoplastic nerve sheath samples and two nonneoplastic brain samples studied as controls. CONCLUSIONS: Our findings indicate that aberrant methylation seems to be a mechanism for NF2 gene inactivation, considered an early step in schwannoma tumorigenesis, and as well, aberrant hypermethylation of other tumor-related genes might represent secondary events that also contribute to the development of these tumors.

Aberrant CpG island methylation in neurofibromas and neurofibrosarcomas.

Aberrant methylation of the promoter CpG island of human genes is an alternative gene inactivation mechanism that contributes to the carcinogenesis of human tumours. We have determined the methylation status of the CpG island of 11 tumour-related genes (RB1, p14ARF, p16INK4a, p73, TIMP-3, MGMT, DAPK, THBS1, caspase 8, TP53 and GSTP1) in 18 neurofibromas (including one plexiform neurofibroma) and three neurofibrosarcomas, as well as two non-neoplastic peripheral nerve sheath samples, using methylation-specific polymerase chain reaction. The series included sporadic and neurofibromatosis type 1-associated tumours. The incidence of aberrant methylation in the tumour samples was 52% for THBS1, 43% for MGMT, 33% for TIMP-3, 19% each for p16INK4a and p73, 14% for RB1, 5% for p14ARF, and 0% for DAPK, caspase 8, TP53 and GSTP1. No methylation of these genes was detected in the two samples of non-neoplastic peripheral nerve sheath. All but three samples in the study displayed aberrant methylation in at least one of the studied genes, and there was no correlation between methylation status and the patients' clinical parameters. These findings suggest that methylation of some tumour-related genes may play a significant role in the tumourigenesis of neurofibromas/neurofibrosarcomas.