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Schwannomas are tumors that develop from Schwann cells in the peripheral nerves and commonly arise from the vestibular nerve. Vestibular schwannomas can present unilaterally and sporadically or bilaterally when the tumor is associated with neurofibromatosis Type 2 (NF2) syndrome. The molecular hallmark of the disease is biallelic inactivation of the NF2 gene. The epigenetic signature of schwannomas remains poorly understood and is mostly limited to DNA methylation of the NF2 gene, whose altered expression due to epigenetic factors in this tumor is controversial. In this study, we tested the genomewide DNA methylation pattern of schwannomas to shed light on this epigenetic alteration in these particular tumors. The methodology used includes Infinium Human Methylation 450K BeadChip microarrays in a series of 36 vestibular schwannomas, 4 nonvestibular schwannomas, and 5 healthy nerves. Our results show a trend toward hypomethylation in schwannomas. Furthermore, homeobox (HOX) genes, located at four clusters in the genome, displayed hypomethylation in several CpG sites in the vestibular schwannomas but not in the nonvestibular schwannomas. Several microRNA (miRNA) and protein-coding genes were also found to be hypomethylated at promoter regions and were confirmed as upregulated by expression analysis; including miRNA-21, Met Proto-Oncogene (MET), and PMEPA1. We also detected methylation patterns that might be involved in alternative transcripts of several genes such as NRXN1 or MBP, which would increase the complexity of the methylation and expression patterns. Overall, our results show specific epigenetic signatures in several coding genes and miRNAs that could potentially be used as therapeutic targets.

Schwannomas and grade I meningiomas are non-metastatic neoplasms that share the common mutation of gene NF2. They usually appear in neurofibromatosis type 2 patients. Currently, there is no drug treatment available for both tumors, thus the use of wide expression technologies is crucial to identify therapeutic targets. Affymetrix Human Gene 1.0 ST was used to test global gene expression in 22 meningiomas, 31 schwannomas and, as non-tumoral controls, 3 healthy meningeal tissues, 8 non-tumoral nerves and 1 primary Schwann cell culture. A non-stringent P-value cut-off and fold change were used to establish deregulated genes. We identified a subset of genes that were upregulated in meningiomas and schwannomas when compared to their respectively healthy tissues, including PDGFD, CDH1 and SLIT2. Thus, these genes should be thoroughly studied as targets in a possible combined treatment.

Clinical response to Gefitinib (Iressa, ZD1839) has been found to be associated with somatic mutations, primarily of exons 18-21, of the epidermal growth factor receptor gene (EGFR) in non-small cell lung cancer (NSCLC). Evidence of a positive response was also reported recently on a patient with brain metastasis from NSCLC. On the other hand, amplification of EGFR appears to be associated with a poor prognosis. To determine whether EGFR mutations and amplification are involved in the tumorigenesis of brain metastases, we performed polymerase chain reaction/single-strand conformation polymorphism to examine exons 1, 2, and 7-26 of EGFR in a series of 18 brain metastases. The metastases 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. In addition to several sequence polymorphisms, we identified two mutations on E19 consisting of 18-base pair (bp) deletions: 2423-24440del and 2426-2443del. These mutations presented in lesions derived from kidney carcinoma and lung adenocarcinoma. By real-time quantitaive polymerase chain reaction technique, we determined the amplification/overdose status of EGFR by analyzing exons 11 and 25. Amplification (5- to 100-fold) was identified in three tumors, and overdose (low-level gene amplification corresponding to increases of 1- to 5-fold) presented in four additional metastases. These findings suggest that EGFR mutations and polymorphisms are not exclusively present in metastases derived from lung carcinoma. Accordingly, targeting of EGFR to determine molecular alterations of this gene may be useful in the management of patients with brain metastases.

Clear cell renal cell carcinoma (ccRCC) is the most common malignant neoplasm of the kidney. The majority of hereditary and sporadic ccRCC cases are associated with germline and somatic mutations in the Von Hippel-Lindau gene (VHL), respectively. Gross deletions at the VHL locus can result either in ccRCC or in a mild clinical phenotype, with the absence of ccRCC development. Our goal in this study was to identify the molecular basis responsible for these differences in the clinical behavior in order to predict patients' phenotype. Using multiplex ligation-dependent amplification (MLPA), we identified and characterized gross VHL deletions in Spanish VHL families. A candidate gene related to this clinical association, HSPC300, was identified and depleted by RNA interference. It was possible to narrow the susceptibility region related to the mild clinical phenotype down to ∼14 kb that included HSPC300 (C3orf10), a regulator of actin dynamics and cytoskeleton organization. Whereas 9 out of 10 families with ccRCC retained HSPC300 in the germline, loss of the HSPC300 locus was associated with mild clinical presentation of the disease in 6 out of 8 families. In fact, genetic depletion of HSPC300 resulted in cytoskeleton abnormalities and cytokinesis arrest in several tumor cell lines including ccRCC cells, suggesting that tumor cell proliferation was compromised in the absence of HSPC300. These clinical and functional data indicate a relevant function of HSPC300 in tumor cell progression, and suggest future therapeutic strategies based upon the inhibition of HSPC300 in renal cell carcinoma and possibly on other cancers. Hum Mutat 28(6), 613–621, 2007. © 2007 Wiley-Liss, Inc.

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 (P0.001). 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.

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 (

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 hypermethylation represents a primary mechanism in the inactivation of tumor suppressor genes during tumorigenesis. To determine the frequency and timing of hypermethylation during carcinogenesis of nonastrocytic tumors, we analyzed promoter methylation status of 10 tumor-associated genes in a series of 41 oligodendrogliomas (22 World Health Organization [WHO] grade II; 13 WHO grade III; 6 WHO grade II–III oligoastrocytomas) and 7 WHO grade II–III ependymomas, as well as 2 nonneoplastic brain samples, by a methylation-specific polymerase chain reaction. Aberrant CpG island methylation was detected in 9 of 10 genes analyzed, and all but one sample displayed anomalies in at least one gene. The frequencies of hypermethylation for the 10 genes were as follows, in oligodendrogliomas and ependymomas, respectively: 80% and 28% for MGMT ; 70% and 28% for GSTP1 ; 66% and 57% for DAPK ; 44% and 28% for TP14 ARF ; 39% and 0% for THBS1 ; 24% and 28% for TIMP3 ; 24% and 14% for TP73 ; 22% and 0% for TP16 INK4A ; 3% and 14% for RB1 ; and 0% in both neoplasms for TP53 . No methylation of these genes was detected in normal brain tissue samples. We conclude that a high frequency of aberrant methylation of the 5′ CpG island of the MGMT , GSTP1 , TP14 ARF , THBS1 , TIMP3 , and TP73 genes is observed in nonastrocytic neoplasms. This aberration seems to occur early in the carcinogenesis process (it is already present in the low-grade forms), although in some instances ( DAPK , THBS1 , and TP73 ) it appears also associated with the genesis of anaplastic forms.

We have determined the promoter CpG island methylation status of O(6)-methylguanine-DNA methyltransferase (MGMT), glutathione-S-transferase P1 (GSTP1), death-associated protein kinase (DAPK), p14(ARF), thrombospondin-1 (THBS1), tissue inhibitor of metalloproteinase-3 gene (TIMP-3), p73, p16(INK4A), RB1, and TP53 genes in three primary central nervous system lymphomas (PCNSL). Five genes (GSTP1, DAPK, TIMP-3, p16(INK4A), and RB1) were hypermethylated in two samples, whereas MGMT, THBS1, and p73 were aberrantly methylated in only one sample. No case presented CpG island methylation for the p14(ARF) and TP53 genes. These findings concur with previous data suggesting a frequent inactivation of p16(INK4A) and very limited involvement of TP53 in PCNSL and also provide insights into the epigenetic molecular involvement of other tumor-related genes in this neoplasm.

Allelic losses of chromosome 22 are commonly found in ependymomas and oligodendrogliomas, suggesting that at least one tumor suppressor gene on chromosome 22 must be inactivated during the multistep process of tumorigenesis in these glial tumors. The neurofibromatosis 2 gene (NF2) located at 22q12, is a candidate tumor suppressor gene potentially involved in the pathogenesis of gliomas. Because there have been only a few studies of the NF2 gene in glial tumors other than astrocytoma, we screened the entire 17 NF2 exons for mutations in a series of 47 nonastrocytic tumors, including 40 oligodendrogliomas and 7 ependymomas. Only one mutation was detected, a 59-base pair insertion in exon 3 from a spinal anaplastic ependymoma. These results concur with previous findings proposing preferential inactivation of the NF2 gene in a subgroup of ependymomas, and suggest that the NF2 gene is not the target of chromosome 22 aberrations in oligodendrogliomas.

Chromosome studies were performed after short-term in vitro culture of 39 samples from neurinomas and two samples from malignant schwannomas. Clonal abnormalities involving chromosome 22 were observed in 23 cases, as the sole chromosomal deviation in 12 of them. In 11 samples, other clonal numerical and/or structural aberrations were detected in addition to loss of chromosome 22, either in the same cells or in cells other than those having monosomy 22. Within the group of neurinomas with no involvement of chromosome 22, there were again two cytogenetically distinctive subgroups: one with an abnormal karyotype, and the second with a normal chromosome complement. Our findings confirm that monosomy 22 is a characteristic feature of this type of neoplasm, but also suggest the existence of different cytogenetic subgroups of neurinomas. © 1993 Wiley-Liss, Inc.

Loss of constitutional heterozygosity as determined through the analysis of restriction-fragment-length polymorphism (RFLP) on tumoral and constitutional DNA has proven to be helpful to delimit the location of tumor-suppressor genes in the human genome. In malignant gliomas this approach indicates that chromosomes 9p, 10, 17p, and 22 may contain genes of this category involved in its origin and/or progression. Regarding chromosome 22, the data so far provided by molecular studies confirmed those previously reported by cytogenetic studies, suggesting the existence of a sub-group of malignant gliomas characterized by monosomy of this chromosome. However, the precise location of the putative glioma suppressor gene on chromosome 22 remains ambiguous. We have perormed a combined cytogenetic and RFLP study on a series of 31 gliomas, looking for structural abnormalities of this chromosome. In 3 instances, terminal deletions of the long arm of chromosome 22 were observed by both methodologies, suggesting that the band q13 region distal to the D22S80 marker might be the critical domain non-randomly involved in tumor suppression of gliomas., Comunidad Autonoma de Madrid, Grant. Grant Number: C267/90 from PB 88-0079.

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