Origin of spurious multiple bands in the amplification of microsatellite sequences.

Multiple band artifacts are seen commonly in the analyses of short repetitive sequences, also known as microsatellites, using the polymerase chain reaction (PCR). In this study, the conditions of PCR were examined for five microsatellite loci (D2S119, D2S123, D5S409, D11S904, and interferon alpha) in an attempt to eliminate this artifact. In addition, and a possible mechanism for the formation of the multiple band artifact in non-denaturing polyacrylamide gel electrophoresis was also explored. The intensity of multiple bands increased when the numbers of PCR cycles were increased. The multiple bands were abolished simply by reducing PCR cycle numbers and were reproduced from single specific PCR products undergoing alternate denaturation and reassociation without primer extension. This finding suggests that formation of multiple bands in non-denaturing gel electrophoresis is a result of improper annealing of PCR fragments, rather than being the result of polymerase slippage and 3' non-template extension, as has been reported previously.

Microsatellite instability and/or loss of heterozygosity in young gastric cancer patients in Italy.

Gastric cancers are rarely diagnosed before the age of 40 years and the incidence reaches a peak during the 7th decade in the general population. A molecular mechanism of early tumor onset may be determined by comparing microsatellite instability (MSI), indicative of error-prone mismatch repair, and loss of heterozygosity (LOH) between gastric cancers in patients < or = 40 years of age and those of older ages. Three to 5 chromosomal loci, where MSI and/or LOH are commonly found in gastric cancers in the general population, were examined in formalin-fixed, paraffin-embedded samples from 102 patients < or = 40 years of age using a polymerase chain reaction-based non-radioactive screening method. MSI and/or LOH at a minimum of 1 locus were detected in 11/102 patients. The frequency of MSI and/or LOH at the D11S904 locus was significantly higher than that at the D2S119, D2S123, D5S409 and IFNA regions. No preferential genetic changes at the D11S904 locus were observed in elderly patients. Among several clinicopathological variables, a statistically significant association with MSI and/or LOH was observed only for tumors located at the cardia, compared with tumors at the antrum and the corpus. Our findings suggest that a unique mechanism may be involved in increasing the susceptibility of the D11S904 locus for either MSI or LOH, especially for cardia tumors in young patients. Early onset of gastric cancers in patients < or = 40 years of age is associated with genetic changes at preferential chromosomal loci, including D11S904.

p53 alterations but no human papillomavirus infection in preinvasive and advanced squamous esophageal cancer in Italy.

Geographic differences in exposure to suspected carcinogens have been identified in esophageal carcinogenesis, and both p53 alterations and human papillomavirus (HPV) infection have been reported in esophageal squamous carcinoma (ESC) from high-risk areas, including China and South Africa. The status of p53 alterations and HPV infection in ESC has not been determined in northern Italy, where the incidence of ESC is low. Formalin-fixed paraffin-embedded esophageal samples containing normal, dysplastic, and carcinomatous tissue from 18 patients were examined for p53 protein accumulation with immunohistochemistry, p53 mutation (exons 5-8) with PCR-single-strand conformation polymorphism analysis and DNA sequencing, and HPV infection with PCR using general primers to amplify the L1 gene. Accumulation of p53 protein was observed in both precancerous and carcinomatous lesions. p53 mutations were rare in dysplastic lesions but were detected in 9 of 18 carcinomas, a finding consistent with reports from other geographic areas. Examination of the p53 mutation spectrum revealed no hot spot mutation. In contrast, HPV was not found in any of these 18 cases. This is consistent with the findings from other low ESC risk areas in which HPV infection may not play a crucial role in esophageal oncogenesis, whereas the high risk of ESC in China and South Africa may be attributed to frequent HPV infection.