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Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid—locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation. [ABSTRACT FROM AUTHOR]

Summary: We report on a case of mucinous carcinoma of the thyroid in an 82-year-old Japanese woman. The 3 × 2 × 2-cm thyroid tumor located in the patient''s right lateral lobe was soft and yellowish gray on the cut section. Microscopically, the tumor was composed entirely of strands or solid clusters accompanied by extensive extracellular mucin. Extracellular mucin was positive with Alcian blue stain and negative with periodic acid–Schiff stain. Immunohistochemically, the tumor cells were positive for thyroglobulin, thyroid-specific transcription factor 1, and low–molecular-weight cytokeratin but negative for carcinoembryonic antigen, calcitonin, and high–molecular-weight cytokeratin. The MIB-1 labeling index was rather high (11%), and immunoexpression of p53 was detectable in the nuclei of carcinoma cells. From these findings, we classified the present tumor as a mucinous carcinoma of the thyroid (poorly differentiated thyroid carcinoma producing massive extracellular mucin). The patient died of multiple metastases 4 years after the initial operation. [Copyright &y& Elsevier]

Recently, we have reported that the protein 4.1B immunolocalization occurred only in matured columnar epithelial cells of normal rat intestines. This finding suggested that protein 4.1B expression could be examined for a possible change during neoplastic transformation of the intestinal mucosa. In the present study, we first present the distribution of mouse protein 4.1B in normal intestinal epithelial cells and tumor cells using theadenomatous polyposis coli(Apc) mutant mouse model. A low level of protein 4.1B expression coincided with the phenotypic transition to carcinoma. To examine the protein 4.1B expression in human intestinal mucosa, we used another antibody against an isoform of the human protein 4.1B, DAL-1 (differentially expressed adenocarcinoma of the lung). Human DAL-1 was also expressed in matured epithelial cells in human colons, with a definite expression gradient along the crypt axis. In human colorectal cancer cells, however, DAL-1 expression was not detected. These results suggest that mouse protein 4.1B and human DAL-1 might have a striking analogy of functions, which may be integrally involved in epithelial proliferation. We propose that loss of protein 4.1B/DAL-1 expression might be a marker of intestinal tumors, indicative of a tumor suppressor function in the intestinal mucosa. [ABSTRACT FROM AUTHOR]