Molecular genetic study of a metastatic oligodendroglioma.

Extracranial spread of neuroectodermal tumors is an unusual event, most frequently expected from glioblastomas and medulloblastomas. Single cases of metastatic oligodendrogliomas have been described, but no genetic data are reported. Oligodendrogliomas are characterized by distinct genetic alterations, i.e. loss of heterozygosity (LOH) of 1p and 19q; therefore, molecular genetic analysis of metastatic cases is of considerable interest. It may be instrumental in defining the distant tumor as metastatic oligodendroglioma and give clues to the genetic events associated with the highly malignant transformation. We present the case of a patient with multiple bone metastases from a cerebral oligodendroglioma. Oligodendroglioma grade II was the diagnosis both at original and second operation, performed 7 and 1 years before the extracranial dissemination. The extraneural spread presented before the local intracranial recurrence. The patient received procarbazine, lomustine, vincristine chemotherapy and radiotherapy after the second surgery. The computed tomography-guided biopsy of the bone lesions revealed tumor cells positive for GFAP, S-100 and Leu-7 and negative for cytokeratin, LCA and EMA. The genetic analysis of DNA from the original tumor, the bone metastasis and the autoptic brain tumor showed LOH of 1p; heterozygous deletion of CDKN2A/p 16 was detected as additional alteration in the metastasis and in the intracranial tumor at autopsy. TP53, MDM2 and CDKN2A/p14ARF genes were unchanged. Repeated brain surgery and extended survival may have acted as promoter of extraneural dissemination. Loss of CDKN2A most probably played an important role in the malignant progression: its involvement in metastatic potential remains to be clarified. Our data confirm that malignant transformation of oliogodendrogliomas may be undetected by histology and underscore the importance of genetic analysis. Coincidentally with intensive anticancer therapy, chemotherapy included, employed in patients with oligodendroglioma and the ensuing long survival, the frequency of metastatic oliogodendrogliomas may increase.

[Sex chromosome abnormality: report of three clinical cases of X pentasomy]. / Anormalità dei cromosomi sessuali: segnalazione di tre casi con pentasomia X.

Pentasomy X cases are very few. In this study we describe three clinical cases (two newborn children and a girl in puberal age) of females showing a 49, XXXXX cariotype. The salient phenotypic characteristics of these cases (heart defects, growth deficiency, craniofacial and hand abnormalities) are compared to the clinico-pathological data described in literature.

Mutational analysis of the NM23.H1 gene in human breast cancer.

NM23.H1 is a protein connected with tumor progression. Loss of heterozygosity and reduced expression of the gene have been associated with poor prognosis and increased incidence of metastases in many epithelial tumors. The aim of this study was to detect the presence of NM23.H1 point mutations or small deletions in human breast carcinomas by using the single-strand-conformation polymorphism (SSCP) technique. Mutational analysis was performed on 76 breast tumors, 10 of which had allelic deletion of the gene. The NM23.H1 mRNA content also was evaluated in each sample. Only a C-to-A transversion leading to a stop codon was found in the 5' untranslated region of exon 1. A polymorphic SSCP pattern was identified in exon 1; direct sequencing showed a C-to-T transition 30 nucleotides upstream from the 5' splice site flanking exon 1. None of the tumors analyzed presented both alleles inactivated. Our results suggest that NM23.H1 is rarely inactivated by point mutations.

Microsatellite alterations and K-ras, TGFbetaRII, IGFRII and bax mutations in sporadic cancers of the gastrointestinal tract.

DNA was analyzed from 57 sporadic gastrointestinal tumors (34 pancreatic cancers, 23 colon tumors) and cognate normal tissues to verify whether mutations at coding sequences were associated with microsatellite instability (MSI). Genomic instability was present in 41% (14/34) of pancreatic samples and in 26% (6/23) of colon cancers previously tested by six microsatellite markers. The tumors included 37 cases showing no MSI; 15 cases with MSI at only 1 locus and 5 cases with MSI at 2 or more loci. All the samples were screened for mutations in genes containing repeated tracts in their coding sequences (TGFbetaRII, IGFRII and bax) and in codon 12 of the K-ras oncogene. Furthermore, loss of heterozygosity (LOH) at NM23.H1 locus was tested, 17/34 (50%) pancreatic tumors and 6/23 (26%) colon cancers showed mutations in codon 12 of K-ras; allelic loss of NM23. H1 locus was found in 6/18 (33%) informative colon tumors and in no pancreatic cancers. The TGFbetaRII, IGFRII and bax genes were altered in 3 (13%), 1 (4%) and 3 (13%) out of 23 colon tumors respectively, but no mutation was detected in pancreatic cancers. Mutations in the repeated nucleotide stretches within the coding sequences of TGFbetaRII, IGFRII and bax genes were found only in colon tumors with a high unstable phenotype (more than 3 microsatellite loci altered).

Microsatellite instability and mismatch repair gene inactivation in sporadic pancreatic and colon tumours.

Genomic instability has been proposed as a new mechanism of carcinogenesis involved in hereditary non-polyposis colorectal cancer (HNPCC) and in a large number of sporadic cancers like pancreatic and colon tumours. Mutations in human mismatch repair genes have been found in HNPCC patients, but their involvement in sporadic cancer has not been clarified yet. In this study we screened 21 pancreatic and 23 colorectal sporadic cancers for microsatellite instability by ten and six different microsatellite markers respectively. Microsatellite alterations were observed at one or more loci in 66.6% (14/21) of pancreatic cancers and in 26% (6/23) colon tumours, but all the pancreatic and half of the colon samples showed a low rate of microsatellite instability. All the unstable samples were further analysed for mutations in the hMLH1 and hMSH2 genes and for hypermethylation of the hMLH1 promoter region. Alterations in the hMLH1 gene were found only in colorectal tumours with a large presence of microsatellite instability. None of the pancreatic tumours showed any alteration in the two genes analysed. Our results demonstrate that microsatellite instability is unlikely to play a role in the tumorigenesis of sporadic pancreatic cancers and confirm the presence of mismatch repair gene alterations only in sporadic colon tumours with a highly unstable phenotype.

Microsatellite instability and allelic losses in neuroendocrine tumors of the gastro-entero-pancreatic system.

Carcinoids are well differentiated tumors, able to secrete a variety of bioactive and hormonal products. Neuroendocrine tumors occur either sporadically or as part of familial syndromes (MEN1, MEN2). Defective DNA mismatch repair is implicated in a variety of gastrointestinal and other cancers; however, its role in the tumorigenesis of carcinoids has not been assessed. Formalin-fixed, paraffin-embedded archivial pathology tissues from 16 neuroendocrine tumors and 9 related metastases were studied by microdissection and microsatellite analysis of extracted DNA to evaluate the degree of microsatellite instability, a marker of defective mismatch repair. The carcinoid tumors analyzed display no microsatellite instability, but, interestingly, show a number of allelic deletions scattered throughout the genome. Particularly, the vast majority of pancreatic derived tumors display loss of heterozygosity on the short arm of chromosome 8. These results suggest that genomic instability is probably not involved in neuroendocrine carcinogenesis and a tumor suppressor gene involved in pancreatic carcinoid tumorigenesis could probably be localized on chromosome 8p12-22.

Microsatellite instability is co-selectable with gene amplification in a mammalian mutator phenotype.

The effect of an unbalanced nucleotide pool on the stability of dinucleotide (CA)n microsatellite sequences was investigated in the mutator phenotype CSA7 clone isolated from Chinese hamster CHEF18 cell line. A series of clones isolated from CHEF18 and CSA7 cells and resistant to 6-thioguanine (TG) were shown to be stable at the three examined microsatellite loci. Furthermore, the clones isolated from CHEF18 cells and resistant to N-phosphonacetyl-L-aspartate (PALA) were stable, whereas those isolated from CSA7 clone were unstable. At the biological level the clones with instability did not show tolerance of methylnitrosourea-induced damage, thus excluding the presence of a defective mismatch repair. On the contrary, the molecular characterization showed that the instability, measured as extension or contraction of (CA)n repeats, involved numerous repeats resembling the amplification rather than mutation of the microsatellite loci. The results therefore indicate that the unbalanced nucleotide pool of CSA7 clone influences the overall rate of gene amplification and support that the unstable microsatellites are coselectable with other gene amplification events.

Microsatellite alterations and p53, TGFbetaRII, IGFIIR and BAX mutations in sporadic non-small-cell lung cancer.

Fifty-two sporadic primary non-small-cell lung carcinomas (NSCLC) were examined for microsatellite instability. Six different microsatellite markers localized on chromosomes 2, 5, 8, 10, 11 and 17 were used. Genomic instability was observed in 35% (18/52) of NSCLC at single or multiple loci. The tumors were also analyzed for p53-gene mutations by PCR-SSCP analysis. Polynucleotide stretch frameshift mutations of TGFbetaRII (transforming-growth-factor-beta receptor II), IGFIIR (insuline growth-factor II receptor) and BAX genes were also analyzed. RER+ (replication-error-positive) tumors appear not to be affected by a higher rate of point mutations in coding sequences: no correlation was found between microsatellite instability and point mutations in the p53 gene, and the RER+ tumors showed no alterations in stretches of nucleotide inside TGFbetaRII, BAX or IGFIIR.

Genetic instability, p53 and nm23 mutation and clinicopathological features in rectal carcinoma.

Defective DNA mismatch repair proteins fail to correct replication errors (RERs). These defects may lead to secondary, mutation of oncogenes and tumor suppressor genes. Microsatellite instability might be a marker of such replication errors. Eighteen rectal tumors were examined to evaluate genetic instability, in sporadic rectal cancer by PCR. RERs were observed in 27.8% of the cases. No significant difference was noticed between RER+ and RER- patients as far as prognosis, clinicopathological features and p53 gene mutation are concerned. The incidence of nm23 gene mutation was the only statistically significant difference between the 2 groups. Three patients with only one altered microsatellite showed advanced tumor and nm23 gene mutation. Two cases with 5 altered microsatellites and nm23 gene mutated are disease-free: in one of them the p53 gene was also mutated. Probably more than one altered microsatellite is necessary to protect from the effects of secondary mutations.

A region on the long arm of chromosome 16 is frequently deleted in metastatic node-negative breast cancer.

The aim of the present study was to define which region of chromosome 16q is most relevant for evaluation of the risk of metastatic recurrence in human breast cancer cases that are lymph node-negative at the time of diagnosis. For this purpose we examined 36 cases of sporadic breast carcinoma subdivided into 3 groups: the first group: no metastatic progression after an average follow-up time of 15 years; including patients with and without lymph node metastases at the time of diagnosis; the second group: N+ (node-positive) patients only, developed metastasis in five years from surgical excision. The last group was composed of patients who developed metastasis but were N0 (node-negative) at diagnosis. A statistically significant association was found between LOH (loss of heterozygosity) at 16q and metastatic progression of the neoplastic disease. 16q LOH was identified as a new independent molecular marker of progression for tumor N0 at diagnosis.

BRCA1 germline mutational spectrum in Italian families from Tuscany: a high frequency of novel mutations.

BRCA1 germline mutations confer susceptibility to familial breast and ovarian cancer. Mutational hot spots have never been detected in BRCA1 cDNA. Some mutations have been reported several times whereas some others appear to be population-related. In this study a group of 36 Italian families were analysed for BRCA1 germline mutations. All of them were screened by allele-specific oligonucleotide hybridization (ASO) for three recurrent mutations (185delAG, 5382insC, nt332-T>G). Twenty families, selected because of their high risk of carrying BRCA1 mutations, were subjected to analysis of the entire coding sequence of the gene. A total of eight mutations were found. ASO screening demonstrated only one known mutation in one patient, whereas cycle sequencing revealed five new mutations. Three of these new mutations were frameshifts: one occurred in exon 11 (1499insA), one in exon 16 (4873delCA) and one in the splice site of exon 3 (252delAAgt). Two were missense mutations (Cys64Arg; Asn158Tyr). The same frameshift mutation, 1499insA, was detected in three unrelated families. Haplotype analysis supported the hypothesis that two of these families may have had common ancestors, whereas in the third family the analysis was uninformative. BRCA1 germline mutations were found in one out of two families with ovarian cancer, in five out of eight families with breast-ovarian cancer, and in two out of 11 families with breast cancer. All three families with 1499insA mutations included at least one case of ovarian cancer. The majority of the ovarian cancers (4/5) associated with detectable BRCA1 germline mutations were of serous histotype.

A deletion map of chromosome 17q in sporadic human mammary carcinomas.

The long arm of chromosome 17 is a frequent target of allelic losses at multiple sites during breast cancer formation and progression. Several genes linked to breast carcinomas have been mapped on this chromosome such as BRCA1, NME and erbB2 genes. The aim of this work was to delineate a deletion map on chromosome 17q and to examine the role of loss of heterozygosity (LOH) during breast tumor development and progression looking for correlation between LOH on 17q and various histopathological parameters. A series of 71 human mammary carcinomas and the corresponding peripheral blood lymphocytes has been studied for loss of heterozygosity at 6 different polymorphic loci on chromosome 17q. 46 out of 71 (65%) tumors showed LOH on 17q. A positive correlation was found between allelic loss for BRCA1 flanking markers and young age at diagnosis. The absence of estrogen receptors was more frequently observed in tumors with deleted BRCA1 flanking markers.