Cacilda Casartelli

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.

Chromosomes in the genesis and progression of ependymomas

Cytogenetic analysis was performed on cultures of primary ependymal tumors with different degrees of malignancy (I-IV) obtained from four patients, none of whom had received therapy before karyotypic evaluation. The most common abnormalities were monosomy 17 and 22 in four cases and losses of sex chromosomes in three cases. Structural rearrangements of chromosome 2 were a finding for all cases and involved loss of material at 2q32-34. Other structural chromosome abnormalities detected involved chromosomes 4, 6, 10, 11, 12, and X. We also reviewed data on 22 cases previously reported.

Analysis of p73 gene in meningiomas with deletion at 1p

The p73 gene has been mapped to 1p36.33, a chromosome region that is frequently deleted in a wide variety of neoplasms including meningiomas. The protein encoded by p73 shows structural and functional similarities to p53 and may thus represent a candidate tumor suppressor gene. To determine whether p73 is involved in the development of meningiomas, we examined 30 meningioma samples with proven 1p deletion for mutations of p73. Sequence analysis of the entire coding region of the p73 gene revealed previously reported polymorphisms in eight cases. A tumor-specific missense mutation as a result of an A-to-G transition with an Asn204Ser change was found in one meningioma that nevertheless retained the normal allele. These results suggest that if p73 plays a role in meningioma carcinogenesis, it must be in a manner different from the Knudson two-hit model.

Osteoid osteomas with chromosome alterations involving 22q

Cytogenetic analysis was performed in two osteoid osteomas. In both, the modal chromosome number was 46. One of the cases presented a del(22)(q13.1) as the sole clonal chromosome alteration. The other had clonal monosomies of chromosomes 3, 6, 9, 17, 19, and 21, as well as a +del(22)(q13.1) was detected as a non-clonal chromosome alteration. There is only one osteoid osteoma reported so far showing clonal karyotypic alterations. The cytogenetic behavior of osteoid osteomas described here was different from that of the osteoid osteoma of the literature. Numerical alterations of chromosomes 3, 6, 9, 17, 19, 21 and 22 have been described in several neoplasias including bone tumors. The breakpoint of chromosome 22 involves a region where important genes for the regulation of the cell cycle have been mapped.

Karyotypic evolution of human meningioma: Progression through malignancy☆

Cytogenetic analysis of an untreated sarcomatous meningioma from a patient submitted to two surgeries separated by 1 year are reported. The material from the first surgery was mostly hypodiploid, with a modal chromosome number of 42. Alterations of chromosome 22 were found in 80.6% of the cells. Four chromosome markers were found involving chromosomes 1, 2, 6, and 22, and numerical alterations involving chromosomes 8, 9, 10, 13, 14, 15, 18, 19, 20, 21, and Y. Although the modal chromosome number of the material from the second surgery was 45 (17.9% of the cells), 43.1% of its cells were hyperdiploid, 73% of these being in the triploid-tetraploid range. Dicentric and ring chromosomes were very frequent. Alterations involving chromosome 22 were still present. There was a recurrent trisomy of chromosome 3. To our knowledge, this is the first cytogenetic description, with banding techniques, of a malignant meningioma.

Aneurysmal bone cyst with chromosomal changes involving 7q and 16p

An aneurysmal bone cyst was submitted to cytogenetic analysis. The modal chromosome number was 46. The composite karyotype was 40 approximately 48,XY,-Y[4],-6[3],del(7)(q32)[3],-9[3],+12[2],+13[2], inv(16)(p13.1q24)[4],-17[3],-19[4],-20[3][cp13]. The clonal structural changes detected were del(7)(q32) and inv(16)(p13.1q24). The breakpoints involved affected areas to which important genes for cell cycle regulation have been mapped. There is only one report in the literature of three aneurysmal bone cysts presenting clonal karyotypic alterations. The cytogenetic study of the aneurysmal bone cyst reported here showed different results when compared to those previously described in the literature.

Allelic status of 1p and 19q in oligodendrogliomas and glioblastomas: multiplex ligation-dependent probe amplification versus loss of heterozygosity.

Identification of the 1p/19q allelic status in gliomas, primarily those with a major oligodendroglial component, has become an excellent molecular complement to tumor histology in order to identify those cases sensitive to chemotherapy. In addition to loss of heterozygosity (LOH), fluorescence in situ hybridization (FISH), or comparative genomic hybridization (CGH), multiplex ligation-dependent probe amplification (MLPA) has been shown to be an alternative methodology to identify deletions of those chromosome arms. We used MLPA to explore the 1p and 19q allelic constitution in a series of 76 gliomas: 41 tumors with a major oligodendroglial component, 34 glioblastomas, and one low-grade astrocytoma. We compared the MLPA findings of the oligodendroglial cases with those previously obtained using LOH in the same samples. Thirty-eight of 41 oligodendrogliomas displayed identical findings by both LOH and MLPA, and losses at either 1p and/or 19q were identified in 12 of 35 (34%) astrocytic tumors. These findings agree with data previously reported comparing MLPA versus FISH or CGH in gliomas and suggest that MLPA can be used in the identification of the 1p/19q allelic deletions on these brain neoplasms.

Methylation status of ANAPC1, CDKN2A and TP53 promoter genes in individuals with gastric cancer

Gastric cancer is the forth most frequent malignancy and the second most common cause of cancer death worldwide. DNA methylation is the most studied epigenetic alteration, occurring through a methyl radical addition to the cytosine base adjacent to guanine. Many tumor genes are inactivated by DNA methylation in gastric cancer. We evaluated the DNA methylation status of ANAPC1, CDKN2A and TP53 by methylation-specific PCR in 20 diffuse- and 26 intestinal-type gastric cancer samples and 20 normal gastric mucosa in individuals from Northern Brazil. All gastric cancer samples were advanced stage adenocarcinomas. Gastric samples were surgically obtained at the João de Barros Barreto University Hospital, State of Pará, and were stored at -80 degrees C before DNA extraction. Patients had never been submitted to chemotherapy or radiotherapy, nor did they have any other diagnosed cancer. None of the gastric cancer samples presented methylated DNA sequences for ANAPC1 and TP53. CDKN2A methylation was not detected in any normal gastric mucosa; however, the CDKN2A promoter was methylated in 30.4% of gastric cancer samples, with 35% methylation in diffuse-type and 26.9% in intestinal-type cancers. CDKN2A methylation was associated with the carcinogenesis process for ~30% diffuse-type and intestinal-type compared to non-neoplastic samples. Thus, ANAPC1 and TP53 methylation was probably not implicated in gastric carcinogenesis in our samples. CDKN2A can be implicated in the carcinogenesis process of only a subset of gastric neoplasias.

Conventional cytogenetic characterization of a new cell line, ACP01, established from a primary human gastric tumor

Gastric cancer is the second most frequent type of neoplasia and also the second most important cause of death in the world. Virtually all the established cell lines of gastric neoplasia were developed in Asian countries, and western countries have contributed very little to this area. In the present study we describe the establishment of the cell line ACP01 and characterize it cytogenetically by means of in vitro immortalization. Cells were transformed from an intestinal-type gastric adenocarcinoma (T4N2M0) originating from a 48-year-old male patient. This is the first gastric adenocarcinoma cell line established in Brazil. The most powerful application of the cell line ACP01 is in the assessment of cytotoxicity. Solid tumor cell lines from different origins have been treated with several conventional and investigational anticancer drugs. The ACP01 cell line is triploid, grows as a single, non-organized layer, similar to fibroblasts, with focus formation, heterogeneous division, and a cell cycle of approximately 40 h. Chromosome 8 trisomy, present in 60% of the cells, was the most frequent cytogenetic alteration. These data lead us to propose a multifactorial triggering of gastric cancer which evolves over multiple stages involving progressive genetic changes and clonal expansion.

Analysis of the polymorphisms XRCC1Arg194Trp and XRCC1Arg399Gln in gliomas.

XRCC genes (X-ray cross-complementing group) were discovered mainly for their roles in protecting mammalian cells against damage caused by ionizing radiation. Studies determined that these genes are important in the genetic stability of DNA. Although the loss of some of these genes does not necessarily confer high levels of sensitivity to radiation, they have been found to represent important components of various pathways of DNA repair. To ensure the integrity of the genome, a complex system of DNA repair was developed. Base excision repair is the first defense mechanism of cells against DNA damage and a major event in preventing mutagenesis. Repair genes may play an important role in maintaining genomic stability through different pathways that are mediated by base excision. In the present study, we examined XRCC1Arg194Trp and XRCC1Arg399Gln polymorphism using PCR-RFLP in 80 astrocytoma and glioblastoma samples. Patients who had the allele Trp of the XRCC1Arg194Trp polymorphism had an increased risk of tumor development (OR = 8.80; confidence interval at 95% (95%CI) = 4.37-17.70; P < 0.001), as did the allele Gln of XRCC1Arg399Gln (OR = 1.01; 95%CI = 0.53-1.93; P = 0.971). Comparison of overall survival of patients did not show significant differences. We suggest that XRCC1Arg194Trp and XRCC1Arg399Gln polymorphisms are involved in susceptibility for developing astrocytomas and glioblastomas.