Multiple hyperplastic polyps in the stomach: evidence for clonality and neoplastic potential

The origin and neoplastic potential of gastric epithelial polyps remains an area of great interest, and treatment choices are a topic of controversy. This report describes a patient diagnosed with three concurrent hyperplastic gastric polyps that were studied for genetic alterations. The polyps were investigated for alterations in the K-ras oncogene and the p53 tumor suppressor gene and for p21WAF1/Cip1 and MDM2 protein overexpression. In addition, loss of heterozygosity at several loci that are frequently involved in human cancer was analyzed, microsatellite instability, a hallmark of the "mutator" phenotype, was determined, and Epstein-Barr virus infection was investigated. All separate areas from the three independent polyps harbored the same activating point mutation in codon 12 of the K-ras oncogene, indicating a clonal origin. DNA sequence alterations in p53 were not found, although high p53 protein levels could be shown by immunohistochemistry in areas of carcinoma within the largest polyp. No alterations in any of the other molecular markers were observed. The results strongly favor a clonal origin of the three independent gastric polyps and support the notion that these hyperplastic polyps may carry a risk for malignanc

MLPA diagnostics of complex microbial communities. Relative quantification of bacterial species in oral biofilms.

A multitude of molecular methods are currently used for identification and characterization of oral biofilms or for community profiling. However, multiplex PCR techniques that are able to routinely identify several species in a single assay are not available. Multiplex Ligation-dependent Probe Amplification (MLPA) identifies up to 45 unique fragments in a single tube PCR. Here we report a novel use of MLPA in the relative quantification of targeted microorganisms in a community of oral microbiota. We designed 9 species specific probes for: Actinomyces gerencseriae, Actinomyces naeslundii, Actinomyces odontolyticus, Candida albicans, Lactobacillus acidophilus, Rothia dentocariosa, Streptococcus mutans, Streptococcus sanguinis and Veillonella parvula; and genus specific probes for selected oral Streptococci and Lactobacilli based on their 16S rDNA sequences. MLPA analysis of DNA pooled from the strains showed the expected specific MLPA products. Relative quantification of a serial dilution of equimolar DNA showed that as little as 10 pg templates can be detected with clearly discernible signals. Moreover, a 2 to 7% divergence in relative signal ratio of amplified probes observed from normalized peak area values suggests MLPA can be a cheaper alternative to using qPCR for quantification. We observed 2 to 6 fold fluctuations in signal intensities of MLPA products in DNAs isolated from multispecies biofilms grown in various media for various culture times. Furthermore, MLPA analyses of DNA isolated from saliva obtained from different donors gave a varying number and intensity of signals. This clearly shows the usefulness of MLPA in a quantitative description of microbial shifts