Chromosome 7q allelic losses in pancreatic carcinoma.

During our DNA fingerprinting studies of paired normal and pancreatic cancer tissues using arbitrarily primed PCR, we noticed a band showing an apparent homozygous deletion in a pancreatic cancer cell line and a decreased intensity in a number of primary cancers. That band was assigned to chromosome 7. Such information led us to analyze chromosome 7 loss of heterozygosity (LOH) in a panel of 12 cryostat-enriched primary pancreatic cancers and 2 pancreatic cancer cell lines, despite the reportedly low frequency of chromosome 7 LOH in xenograft-enriched pancreatic cancers. Seventeen PCR-amplified CA-microsatellite polymorphic sites were analyzed. One of the two cell lines and eight common-type cancers (including all five poorly differentiated and three of five moderately differentiated cancers) showed chromosome 7q LOH, whereas the two uncommon types of ductal cancer (one adenosquamous and one mucinous noncystic) scored negative. Our data suggest that chromosome 7q LOH is a frequent event (80%) in cryostat-enrichable common pancreatic ductal carcinomas, that is, those primarily of high cellularity. The chromosome 7q smallest common deleted region described by our cases was between 7q31.1 and 7q32.

Cancers of the papilla of vater: mutator phenotype is associated with good prognosis.

Cancer of the papilla (ampulla) of Vater is an uncommon disease that kills 60% of affected patients. There is general agreement that local spread of the tumor (T stage) is the only significant and independent prognostic factor for this cancer, whereas the predictive value of tumor grade and lymph node metastases is controversial. The genetic anomalies involved in this process have the potential to serve as additional prognostic markers. We explored 25 ampullary cancers for the occurrence of instability at simple repeat DNA sequences (microsatellites) of the type seen in replication error phenotype (RER-positive) cancers. Ten microsatellites from five different chromosomes were amplified by PCR from both normal and cancer tissue DNA of the same patients. A tumor was defined as RER-positive when microsatellite instability was found in the majority (>/=6) of the loci analyzed. Five cancers (20%) showed a RER phenotype and were associated with long survival of patients (32-96 months), whereas RER-negative cancers had a significantly poorer prognosis (Mantel-Cox test; P = 0.0084), with a median actuarial survival of 17 months. We also report that three (12%) patients belonged to cancer-prone families and four (16%) were cancer-prone individuals.

Immunoreactivity for latent membrane protein 1 of Epstein-Barr virus in nevi and melanomas is not related to the viral infection.

Epstein-Barr virus (EBV) is a human herpes virus with oncogenic potential, associated with several malignancies. The EBV-encoded latent membrane protein 1 (LMP1) is one of nine proteins regularly expressed in virally infected and immortalised B lymphocytes. We now document the consistent immunoreactivity for LMP1 in 90% of 65 nevi and melanomas, using the monoclonal antibody cocktail CS1-4. The immunocytochemical findings, however, were not confirmed using reverse-transcription polymerase chain reaction (RT-PCR) experiments, which failed to demonstrate any actual expression of LMP1 mRNA. In situ hybridisation for EBV-encoded RNAs (EBERs 1 and 2) and PCR amplification of EBV genomic sequences also failed to document any viral infection. Several normal and neoplastic human tissues have also been immunostained for LMP1, without any positive staining, with the exception of a minor percentage of skin melanocytes and of normal blasts of the myeloid and erythroid lineages. We conclude that the vast majority of nevi and melanomas express a still uncharacterised molecule, cross-reacting with anti-LMP1 (CS1-4) antibodies, which may be considered a consistent marker of melanocytic proliferations. The immunoreactivity of normal and neoplastic human tissues for the anti-LMP1 reagent should not be taken as evidence of EBV infection.

Comparison of heteroduplex and single-strand conformation analyses, followed by ethidium fluorescence visualization, for the detection of mutations in four human genes.

Non-isotopic DNA single-strand conformation analysis and heteroduplex analysis by ethidium bromide fluorescence visualization (SSCAE and HAE, respectively) were compared for the detection of 15 different naturally occurring mutations in 15 different DNA samples. The mutations included single nucleotide transitions, transversions and deletions, in CFTR (cystic fibrosis transmembrane conductance regulator), COL4A5 (collagen type IV alpha 5 chain), HEXB (hexosaminidase B), and COL1A2 (collagen type 1 alpha 2 chain) genes, responsible for diseases of medical interest. Genomic DNA from peripheral blood leukocytes or cDNA from reverse-transcribed fibroblast mRNA were amplified by polymerase chain reaction (PCR), and then analysed by two SSCAE and one HAE protocol. Fourteen out of 15 mutations (93%) were detected with one or the other method. HAE was more sensitive than SSCAE for the larger products (257-426 bp). The only undetected mutation was then identified with the use of a different primer, located farther from the mutation was then identified with the use of a different primer, located farther from the mutation site, thus increasing the combined efficiency of the two methods to 100%. We believe that combined use of SSCAE and HAE is a good, cheap and safe approach for mutation screening in a human gene.

A novel missense mutation in exon 3 of the COL4A5 gene associated with late-onset Alport syndrome.

We have identified a novel missense transition (362G-->A) in exon 3 of the COL4A5 gene in a male patient with late-onset Alport syndrome. We used non-isotopic single strand conformation polymorphism, heteroduplex analysis, and automated DNA sequencing. The mutation changes a conserved glycine at codon 54 for an aspartic acid (Gly54Asp), which abolishes a BstNI site. Using restriction analysis, we identified the heterozygous carrier status in the two daughters of the proband. Our findings are in keeping with the hypothesis that slower progressive forms of Alport syndrome are more often associated with missense mutations rather than large deletions or frameshifts. This is the first mutation described in the N-terminus triple helical 7S domain of the COL4A5 gene in an Alport syndrome patient.

APC gene mutations and allelic losses in sporadic ampullary tumours: evidence of genetic difference from tumours associated with familial adenomatous polyposis.

We explored APC gene mutations and chromosome 5q21 allelic losses (5qLOH) in 18 neoplasms of the papilla of Vater, including 6 early-stage tumours (3 adenomas, 3 carcinomas) and 12 advanced-stage cancers. Eleven PCR-amplified polymorphic sequences were used to analyse 5qLOH. APC mutations were investigated both by an in vitro APC-protein truncation test and by single-strand conformation polymorphism analysis. Mutations in the Ki-ras, N-ras and p53 genes were also assessed. We found: 5qLOH in 8 of 16 cases (50%), including 1 adenoma, 3 early- and 4 advanced-stage cancers; APC mutations in 2 adenomas and 1 advanced-stage carcinoma; Ki- or N-ras mutations in 3 adenomas and 3 advanced-stage cancers; p53 mutations in 2 early-stage and 7 advanced-stage adenocarcinomas. Our results suggest that 5qLOH, APC mutations and ras mutations are present at early stages, whereas p53 inactivation is associated with progression of malignancy in a large proportion of cases. These data indicate that sporadic ampullary tumours differ from those occurring in familial adenomatous polyposis in the frequency (17% vs. 64%) as well as in the site of APC somatic mutations, suggesting a different molecular pathogenesis in the 2 conditions.