Juan A. Rey

NF2 gene mutations and allelic status of 1p, 14q and 22q in sporadic meningiomas

Formation of meningiomas and their progression to malignancy may be a multi-step process, implying accumulation of genetic mutations at specific loci. To determine the relationship between early NF2 gene inactivation and the molecular mechanisms that may contribute to meningioma tumor progression, we have performed deletion mapping analysis at chromosomes 1, 14 and 22 in a series of 81 sporadic meningiomas (54 grade I (typical), 25 grade II (atypical) and two grade III (anaplastic)), which were also studied for NF2 gene mutations. Single-strand conformational polymorphism analysis was used to identify 11 mutations in five of the eight exons of the NF2 gene studied. All 11 tumors displayed loss of heterozygosity (LOH) for chromosome 22 markers; this anomaly was also detected in 33 additional tumors. Twenty-nine and 23 cases were characterized by LOH at 1p and 14q, respectively, mostly corresponding to aggressive tumors that also generally displayed LOH 22. All three alterations were detected in association in seven grade II and two grade III meningiomas, corroborating the hypothesis that the formation of aggressive meningiomas follows a multi-step tumor progression model.

Chromosomal patterns in human malignant astrocytomas

Cytogenetic analysis by direct and/or in vitro preparations was performed on 34 malignant astrocytomas. Thirty tumors showed near-diploid chromosome numbers, whereas, tritetraploid chromosome complements were present in four tumors. The most frequent chromosomal changes implied numerical deviations by a gain of chromosomes #7, #19, and #20, and by losses of #10, #22, and Y. Structural rearrangements were present in stem- or side lines of 24 tumors. Although no common chromosomal rearrangement seems to exist among those tumors, chromosomes #1, #6, #7, and #9 were predominantly involved. Polysomy and structural rearrangements of chromosome #7 could be related to the overexpression of epidermal growth factor gene, previously observed in some malignant gliomas.

Chromosomal composition of a series of 22 human low-grade gliomas

G-banded chromosomal analysis was performed on direct and/or in vitro cultures of 22 low-grade gliomas, including nine grade I-II astrocytomas, nine oligodendrogliomas, one mixed tumor oligodendroglioma-astrocytoma, and three ependymomas. Normal diploid stem lines were present in most astrocytomas and oligodendrogliomas, whereas, all three ependymomas displayed polyploid modal numbers. However, secondary cell lines showed the presence of clonal recurrent numerical abnormalities, mainly polysomy 7, monosomy 10 and 22, and loss of the Y chromosome. Clonal structural rearrangements were present with a low incidence; they mainly involved chromosomes #1 and #7. These patterns of chromosome involvement seem to correlate with the scarce previous cytogenetic banding data available from low-grade gliomas. They are also similar to the chromosome alterations found in high-grade gliomas.

Chromosomal involvement secondary to −22 in human meningiomas☆

Cytogenetic analyses have been performed on cultures in vitro from 32 human meningiomas, seeking chromosomal anomalies in addition to characteristic monosomy 22. Eight cases showed stem lines with normal karyotype, whereas, monosomy 22 as the only chromosomal deviation characterized the stem line of ten tumors. In 14 samples stem lines or modal numbers displaying numerical deviations (other than -22) and/or structural rearrangements were found. A hyperdiploid modal number was present in three, whereas, it was hypodiploid in the remainder. Numerical deviations in these tumors involved mainly #14 by losses, and also #22; recurrent structural rearrangements involving 1p and 11p were also characteristic features. Thus, these results could imply that involvement of #14, 1p, and 11p would be a form of clonal evolution secondary to monosomy 22 in certain meningiomas.

Allelic status of chromosome 1 in neoplasms of the nervous system

By using five highly polymorphic markers, the allelic status of chromosome 1 was established in a series of 236 tumors of the nervous system, including all major histologic subtypes: gliomas, meningiomas, neurinomas, neuroblastomas, medulloblastomas, etc. Loss of alleles at 1p was observed at significant frequencies in neuroblastomas (26% of cases), meningiomas (32%), and malignant gliomas (37%) (primarily oligodendrogliomas [94%]). This anomaly was also detected in two of 23 neurinomas, two of three neurofibrosarcomas, one primary lymphoma, and two metastatic tumors of the brain. The analysis of tumors displaying partial 1p deletions suggests the existence of two distinct regions, 1p36 and 1p35-p32, in which loci nonrandomly involved in the development of neurogenic neoplasms might be located.

Abnormalities of chromosome 22 in human brain tumors determined by combined cytogenetic and molecular genetic approaches.

Southern blot hybridization studies were performed on a panel of 130 blood/tumor samples from brain neoplasms including all major histologic subtypes: 50 meningiomas, 18 neurinomas, 56 gliomas, and six others. To detect abnormalities involving chromosome 22, polymorphic probes were used to analyze eight loci located in this chromosome: D22S9, IGLV, D22S20, D22S32, MB, PDGF-B, D22S80, and D22S171. Loss of heterozygosity (LOH) was observed in 40 cases including monosomy, terminal, and interstitial deletions, which suggest the location of recessive tumor genes in certain chromosome 22 subregions (22q11.3-q12 in neurinomas and meningiomas, and 22q13 in malignant gliomas). Cytogenetic studies were performed in parallel on the same tumors, in most instances corroborating the presence of abnormalities for chromosome 22. Nevertheless, discrepancies between the cytogenetic and molecular findings were observed in several cases, suggesting that the use of both methodologies in combination might provide key information on the incidence and extent of the abnormalities involving chromosome 22 in human brain tumors.

Molecular analysis of genomic abnormalities in human gliomas.

A series of 57 malignant gliomas, including 27 astrocytomas grade III-IV (glioblastoma multiforme), 15 astrocytomas grade I-II, and 15 tumors with major oligodendroglial component, was examined to detect molecular abnormalities of loci at specific chromosome regions. At the cytogenetic level, these regions have been shown to be nonrandomly involved in neoplastic development of these histologic subtypes of tumor. We used a panel of 24 polymorphic DNA probes to analyze loss of heterozygosity (LOH) at loci on chromosomes 7, 9, 10, 13, 17p, and 22q. In addition, the retinoblastoma (RB1) oncosuppressor gene, the platelet-derived growth factor A (PDGFA) gene, and the epidermal growth factor receptor (EGFR) gene were analyzed directly. Loss of genetic information on the short arm of chromosome 17 was observed in both low- and high-grade astrocytomas, whereas no oligodendroglial tumor was characterized by this type of aberration. LOH for chromosome 10, mainly compatible with loss of the entire chromosome, was primarily evidenced in the more malignant forms and in isolated cases diagnosed as low-grade astrocytomas. Again, no oligodendroglial tumor displayed losses of chromosome 10. In contrast, four tumors with major oligodendroglial component showed losses involving 9p markers, primarily interferon A and B (IFNA, IFNB); this feature was also observed in two low-grade astrocytomas and in 11 high-grade tumors. Isolated cases displayed LOH for markers on chromosomes 13 and 22, whereas EGFR amplification was almost exclusively evidenced in the more malignant forms which, in most instances, also presented LOH for chromosome 10. In general, the samples with lower malignancy stage displayed a lesser grade of abnormalities, mainly restricted to losses at 17p and chromosome 10 in astrocytomas grade I-II and at 9p in oligodendrogliomas. In contrast, about 50% of the high-grade tumor samples analyzed included abnormalities at two or more loci, with a recurrent association of EGFR amplification and LOH for chromosome 10; this association was evident in 26% of the high-grade astrocytomas.

Regulation of sonic hedgehog-GLI1 downstream target genes PTCH1, Cyclin D2, Plakoglobin, PAX6 and NKX2.2 and their epigenetic status in medulloblastoma and astrocytoma

BackgroundThe Sonic hedgehog (Shh) signaling pathway is critical for cell growth and differentiation. Impairment of this pathway can result in both birth defects and cancer. Despite its importance in cancer development, the Shh pathway has not been thoroughly investigated in tumorigenesis of brain tumors. In this study, we sought to understand the regulatory roles of GLI1, the immediate downstream activator of the Shh signaling pathway on its downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6 in medulloblastoma and astrocytic tumors.MethodsWe silenced GLI1 expression in medulloblastoma and astrocytic cell lines by transfection of siRNA against GLI1. Subsequently, we performed RT-PCR and quantitative real time RT-PCR (qRT-PCR) to assay the expression of downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6. We also attempted to correlate the pattern of expression of GLI1 and its regulated genes in 14 cell lines and 41 primary medulloblastoma and astrocytoma tumor samples. We also assessed the methylation status of the Cyclin D2 and PTCH1 promoters in these 14 cell lines and 58 primary tumor samples.ResultsSilencing expression of GLI1 resulted up-regulation of all target genes in the medulloblastoma cell line, while only PTCH1 was up-regulated in astrocytoma. We also observed methylation of the cyclin D2 promoter in a significant number of astrocytoma cell lines (63%) and primary astrocytoma tumor samples (32%), but not at all in any medulloblastoma samples. PTCH1 promoter methylation was less frequently observed than Cyclin D2 promoter methylation in astrocytomas, and not at all in medulloblastomas.ConclusionsOur results demonstrate different regulatory mechanisms of Shh-GLI1 signaling. These differences vary according to the downstream target gene affected, the origin of the tissue, as well as epigenetic regulation of some of these genes.

Search for mutations of the hRAD54 gene in sporadic meningiomas with deletion at 1p32.

The hRAD54 gene is related to a family of genes involved in DNA recombination and repair and encodes a protein with DNA helicase activity. hRAD54 has been mapped to 1p32, a region frequently involved in deletions in a variety of tumor types, including atypical and anaplastic meningiomas. To determine whether alterations of hRAD54 are a common event in meningeal tumors, by means of polymerase chain reaction–single‐stranded conformation analysis we examined 29 tumor samples characterized by 1p deletions for hRAD54 mutations. Although 18 tumors displayed allelic loss at the gene region (1p32) as determined by microsatellite marker analysis, the sole coding‐sequence alteration detected corresponded to a T → C transition, with no amino‐acid change. The genotype distribution was 10.34% TT, 44.8% TC, and 44.8% CC, whereas in the normal controls it was 3.77% TT, 13.2% TC, and 83.01% CC, and most meningiomas with 1p32 deletion retained allele C. Another polymorphism due to a T → C change was evidenced at nt 3008, in the 3′ untranslated region. This change was evidenced in all cases we sequenced. These results appear to exclude the involvement of the hRAD54 gene in the pathogenesis of the nontypical meningiomas, although a detrimental effect of the hRAD54 polymorphisms cannot be ruled out. Mol. Carcinog. 24:300–304, 1999.

Deletion 3p in two lung adenocarcinomas metastatic to the brain

Cytogenetic analysis by direct and in vitro preparation of brain metastatic lesions from two lung adenocarcinomas have shown the presence of a recurrent chromosomal abnormality, del(3p). These results add new evidence about the presence of 3p- marker chromosomes in adenocarcinoma of the lung.