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.

Aberrant methylation of multiple genes in neuroblastic tumours. relationship with MYCN amplification and allelic status at 1p.

Aberrant hypermethylation occurs in tumour cell CpG islands and is an important pathway for the repression of gene transcription in cancers. We investigated aberrant hypermethylation of 11 genes by methylation-specific polymerase chain reaction (PCR), after treatment of the DNA with bisulphite, and correlated the findings with MYCN amplification and allelic status at 1p in a series of 44 neuroblastic tumours. This tumour series includes five ganglioneuromas (G), one ganglioneuroblastoma (GN) and 38 neuroblastomas (six stage 1 tumours; five stage 2 tumours; six stage 3 cases; 19 stage 4 tumours, and two stage 4S cases). Aberrant methylation of at least one of the 11 genes studied was detected in 95% (42 of 44) of the cases. The frequencies of aberrant methylation were: 64% for thrombospondin-1 (THBS1); 30% for tissue inhibitor of metalloproteinase 3 (TIMP-3); 27% for O6-methylguanine-DNA methyltransferase (MGMT); 25% for p73; 18% for RB1; 14% for death-associated protein kinase (DAPK), p14ARF, p16INK4a and caspase 8, and 0% for TP53 and glutathione S-transferase P1 (GSTP1). No aberrant methylation was observed in four control normal tissue samples (brain and adrenal medulla). MYCN amplification was found in 11 cases (all stage 4 neuroblastomas), whereas allelic loss at 1p was identified in 16 samples (13 stage 4 and two stage 3 neuroblastomas, and one ganglioneuroma). All but one case with caspase 8 methylation also displayed MYCN amplification. Our results suggest that promoter hypermethylation is a frequent epigenetic event in the tumorigenesis of neuroblastic tumours, but no specific pattern of hypermethylated genes could be demonstrated.

Six novel mutations in the NF2 tumor suppressor gene.

Six novel mutations were identified in the NF2 tumor suppressor gene in a panel of meningiomas and neurinomas. Screening was performed using a combination of single-strand conformation polymorphism and heteroduplex analyses on polymerase chain reaction-amplified DNA from tumors and matched peripheral blood lymphocytes. Mutations involved exons 2, 7, 11 and 12, and corresponded to three frameshift, one nonsense, one missense and one polymorphism.

Absence of mutation of the p73 gene in astrocytic neoplasms.

In subgroups of astrocytic neoplasms, including glioblastoma (GBM), mutations of the p53 tumour suppressor gene lead to loss of growth-suppressive properties. A p53-related gene termed p73 has recently been identified; its gene product shows structural and functional similarities to p53. After being mapped to chromosome region 1p36, p73 was proposed to act as a tumour suppressor gene, as this region is frequently deleted in a variety of human cancers, including astrocytic tumours. To determine whether p73 is involved in astrocytoma/GBM development, we analysed 10 pilocytic astrocytomas, 15 WHO grade II astrocytomas, 15 WHO grade III anaplastic astrocytomas, and 20 GBM for p73 gene alterations. In parallel, we used six polymorphic markers to determine the allelic status of region 1p36 in this tumour series. Although loss of heterozygosity was evidenced in 12 of 60 cases (20% of samples), PCR-SSCP and direct sequencing failed to detect any gene mutation in the entire coding region and intronic sequences of p73. Eight tumours displayed five distinct polymorphic nucleotide changes, also present in the corresponding normal DNA. These variations consisted of T-->C variation, with no change in Thr173; C-->T transition, with no change in His197; exon 9 simultaneous double change C-->T and T-->C , with no variations in Ala336 and His349, respectively, and C-->T change at exon 9/-24 position of intron 8. These results suggest that, in astrocytic gliomas, p73 may not play a major role as a tumour suppressor, but the relatively high incidence of LOH confirms the presence at 1p36 of an as yet unidentified gene of this category, with a key function in astrocytoma/GBM progression.

Involvement of 9p in metastatic ovarian adenocarcinomas.

By a direct method and after in vitro culture, we cytogenetically analyzed five ovarian adenocarcinomas, using six samples of ascitic fluid. The structural aberrations included rearrangements of chromosomes 1 and 3, in agreement with the results of other researchers. The long arm of chromosome 6 was rearranged in three samples, but no translocation t(6;14) was found. Rearrangements involving 9p were present in all cases, with breakpoints at p13 or p22-23.

Acute nonlymphocytic leukemia with severe hypodiploidy and one double minute chromosome.

We present a case of acute nonlymphocytic leukemia with severe hypodiploidy (38 chromosomes), complex karyotype, and one double minute chromosome. These unusual chromosome findings could be related to the rapid course of the patient's disease.

Incidence and origin of dicentric chromosomes in cultured meningiomas.

Based on the cytogenetic findings in 32 human meningiomas, an analysis of dicentric chromosomes, usually present in cultures from meningiomas, has been performed. The incidence and origin of such markers have been analyzed and the chromosomal composition of the stem line in the corresponding sample established (i.e., normal karyotype, -22 as the sole chromosomal deviation, or complex karyotypes in addition to #22 abnormalities). More than 10 dicentric chromosomes were found in 12 of 32 meningiomas (37.5%). Sixty-eight of the markers could be identified individually or as belonging to a chromosome group. Fifty-three percent of the meningiomas characterized by a complex stem line karyotype also displayed dicentric chromosomes in variant cells, whereas only 12.5% of meningiomas with a normal diploid stem line showed such chromosomal aberrations. Chromosomes of groups C and D participated most frequently in the genesis of dicentrics; however, chromosomes 19, 20, 3, 6, and 13 were the most frequently involved. Thus, the existence of a nonrandom pattern of involvement supports the fact that dicentrics might play a biologic role in the progression of human meningiomas.

Cytogenetic findings in an effusion secondary from pleural mesothelioma.

Cytogenetic analysis was performed on cells from pleural effusion secondary to an epithelial mesothelioma. Although chromosomal findings (pseudodiploid stemline, including marker chromosomes involving chromosomes #1, #3, #5, and #6) suggested the neoplastic origin of the effusion, routine cytologic diagnosis was negative.

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.

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.

Cytogenetic findings in a human malignant melanoma metastatic to the brain.

Cytogenetic analysis by G-banding of direct and preparations of a malignant melanoma metastatic to the brain in vitro showed a pseudodiploid modal chromosome number, including five marker chromosomes, one of which was an i(6p). These results agree with those recently reported about the preferential involvement of chromosome #6 in malignant melanoma.

Cytogenetic follow-up from direct preparation to advanced in vitro passages of a human malignant glioma.

In order to evaluate which cytogenetic evolutionary patterns take place during the in vitro establishment of a permanent glioma cell line, cytogenetic follow-up was performed on direct preparations and primary cultures from a malignant astrocytoma and on serial passages for more than 2 years of in vitro propagation. Sixteen passages were studied, and the presence of a marker chromosome in direct preparations and after in vitro growth permitted us to identify the clonal evolution of the resulting permanent cell line. Near-triploid cells present in the direct study became the main cell population in vitro; chromosomal losses and the acquisition of a few new marker chromosomes were also characteristic features, leading to a stable modal number of around 60 chromosomes in the last passages analyzed. The results provide new evidence of the existence of more than one pattern of chromosomal evolution during the in vitro establishment of human gliomas.

Involvement of 22q12 in a neurofibrosarcoma in neurofibromatosis type 1.

We describe the cytogenetic and molecular genetic findings in a neurofibrosarcoma arising in a patient affected by neurofibromatosis type 1. Multiple chromosomal rearrangements were found but only a few of them were identified as clonal abnormalities, including a deletion of chromosome 22, which at the molecular level proved to be interstitial, mainly involving the 22q12 region. Loss of heterozygosity for markers D22S32 and MB was observed. These findings are in agreement with previous data which suggest a possible involvement of a gene located at 22q11-q13.1 during the neoplastic development of some neurofibromatosis type 1-associated tumors.

Serial cytogenetic study of a human glioma cell line.

Chromosome studies were performed on a human glioma cell line. Nineteen passages were studied, and a heterogeneous chromosome complement with variable modal numbers was found. The main range varied from tetraploidy to triploidy. The presence of identical markers (9p-, 11p+, an increase in the copies of chromosome No. 7) in different passages suggests a clonal evolution.

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.

Chromosome studies in two human brain tumors.

The cytogenetic findings based on G- and C-banding in two human brain tumors (a meningioma and an astrocytoma) are reported. Both tumors were characterized by hypodiploid modal numbers (45 and 40 chromosomes, respectively), chromosome 22 abnormalities, and the presence of several markers. This observation supports the hypothesis of the association of No. 22 chromosome abnormalities with tumors of the brain.

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.

Cytogenetic clones in a recurrent neurofibroma.

Chromosome studies were performed on a plexiform neurofibroma arising in a probable von Recklinghausen's disease patient, who also showed a de novo constitutional reciprocal translocation, t(1;22)(p32;q11). Banding analysis of the metaphases obtained from two primary cultures in vitro showed the presence of five cytogenetic clones, characterized by different chromosomal rearrangements. In addition to t(1;22), marker chromosomes involved pairs 1, 2, 3, 5, 8, 9, 10, 12, 16, and X. These findings suggest a possible polyclonal evolution in this neurofibroma.

A case of pituitary adenoma with 58 chromosomes.

Chromosome analysis was performed on a growth hormone secreting pituitary adenoma. G-banding analysis showed a stem line with 58 chromosomes including numerical deviations of pairs #3, #5, #7, #9, #11, #12, #13, #17, #19, #20, and gonosomes was found. These findings add new evidence of the nonrandom involvement of C and F group chromosomes in numerical deviations in this tumor type.

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.