Analysis of KRAS Mutations of Exon 2 Codons 12 and 13 by SNaPshot Analysis in Comparison to Common DNA Sequencing

Due to the call for fast KRAS mutation status analysis for treatment of patients with monoclonal antibodies for metastatic colorectal cancer, sensitive, economic, and easily feasible methods are required. Under this aspect, the sensitivity and specificity of the SNaPshot analysis in comparison to the commonly used DNA sequencing was checked. We examined KRAS mutations in exon 2 codons 12 and 13 with DNA sequencing and SNaPshot analysis in 100 formalin-fixed paraffin-embedded tumor tissue samples of pancreatic carcinoma, colorectal carcinoma, and nonsmall cell lung cancer specimens of the primary tumor or metastases. 40% of these samples demonstrated mutated KRAS genes using sequencing and SNaPshot-analysis; additional five samples (45/100) were identified only with the SNaPshot. KRAS mutation detection is feasible with the reliable SNaPshot analysis method. The more frequent mutation detection by the SNaPshot analysis shows that this method has a high probability of accuracy in the detection of KRAS mutations compared to sequencing

Environmental Isocyanate-Induced Asthma: Morphologic and Pathogenetic Aspects of an Increasing Occupational Disease

Occupational diseases affect more and more people every year. According to the International Labour Organization (ILO), in 2000 an estimated amount of at least 160 million people became ill as a result of occupational-related hazards or injuries. Globally, occupational deaths, diseases and injuries account for an estimated loss of 4% of the Gross Domestic Product. Important substances that are related to occupational diseases are isocyanates and their products. These substances, which are used in a lot of different industrial processes, are not only toxic and irritant, but also allergenic. Although the exposure to higher concentrations could be monitored and restricted by technical means, very low concentrations are difficult to monitor and may, over time, lead to allergic reactions in some workers, ending in an occupational disease. In order to prevent the people from sickening, the mechanisms underlying the disease, by patho-physiological and genetical means, have to be known and understood so that high risk groups and early signs in the development of an allergic reaction could be detected before the exposure to isocyanates leads to an occupational disease. Therefore, this paper reviews the so far known facts concerning the patho-physiologic appearance and mechanisms of isocyanate-associated toxic reactions and possible genetic involvement that might trigger the allergic reactions

Differential miRNA-Expression as an Adjunctive Diagnostic Tool in Neuroendocrine Tumors of the Lung

Pulmonary malignancies with neuroendocrine differentiation represent a rare subclass of lung carcinomas, which vary in the extent of differentiation and grade of biological aggressiveness. In particular, neuroendocrine tumors are classified into well differentiated typical and atypical carcinoids as well as poorly differentiated large cell neuroendocrine and small cell lung carcinomas. Tiny MicroRNAs have been identified as reliable classifiers in distinct cancer types and seem to play important roles in cellular processes like regulation of cell growth, differentiation and apoptosis. In the present study, two different microRNAs (miR-21 and miR-34a) were explored for their involvements in pathogenesis of subtypes and finally in differential diagnosis of pulmonary neuroendocrine tumors. miR-21 was upregulated in poorly differentiated neuroendocrine tumors (mean rank: 26.8; 28.75) as compared to carcinoids (mean rank: 12.33; 12.07) with a significance of 0.00033. High-expression levels of miR-34a were associated with atypical carcinoids (p = 0.010). A close association is implicated between the elevated miR-21 values in high-grade and miR-34a patterns in low-grade atypical neuroendocrine lung carcinomas, which could potentially be exploited as practical supportive markers for differential lung cancer diagnosis in routine. However, some additional extended research and validation studies are needed to utilize them as routine markers or potential molecular targets for personalized medicine

Real-Time PCR Data Processing Shown by the Analysis of Colorectal Specific Candidate Genes, ERCC1, RRM1 and TS in Relation to β2M as Endogenous Control

Currently, quantitative real-time PCR (Q-PCR) of archival formalin-fixed, paraffin embedded (FFPE) tissue is a critical tool for research and is not well established in routine diagnostics. Therefore, continuous improvement in mathematics and statistics associated with interpreting final accurate and reproducible results are fundamental. This project describes and discusses specificity and sensitivity with respect to intra- and inter-assay variances by use of a commercial Human Reference RNA and individual RNA derived from colorectal cancer patients (n = 25). All patients were treated with 5-fluoruracil (5-FU) and a concomitant pelvic radiotherapy (50.4 Gy). Quality assessment of target tissue samples was evaluated by clinicopathological findings and optical density (OD) measurements. We analyzed the steady state messenger RNA (mRNA) expression level of a small panel of cancer relevant genes, excision repair cross-complementing group 1 (ERCC1), ribonucleoside-diphosphate reductase subunit M1 (RRM1), thymidylate synthase (TYMS) and ß-2microglobulin (ß-2M) as endogenous control. The mRNA of a Human Reference RNA, tumor and non-neoplastic material was reverse transcribed into its complementary DNA (cDNA). cDNA was amplified based on dual-labeled TaqMan real-time fluorescence measurements. The real-time efficiency and therefore the output data can be influenced through the kind of calibrator used, the amount and quality of used RNA and by the degree of individual assay variability. Each sample presents an individual amplification curve. Thus, confirmation of primer specificity, one or more invariant endogenous controls, RNA and cDNA quality, as well as real-time PCR amplification efficiencies and linearity calculations from individual slopes or R2-values must be included in each study

Basement Membrane in Middle Ear Cholesteatoma Immunohistochemical and Ultrastructural Observations

Bujía J, Holly A, Sudhoff H, et al. Basement Membrane in Middle Ear Cholesteatoma Immunohistochemical and Ultrastructural Observations. Annals of Otology, Rhinology & Laryngology. 1996;105(10):804-810.We investigated the distribution of basement membrane zone (BMZ) components collagen type IV, collagen type VII, and fibronectin in human middle ear cholesteatoma, auditory meatal skin, and middle ear mucosa using both immunohistochemical and ultrastructural methods. Collagen type IV inununoreactivity of skin and middle ear mucosa is continuous in the BMZ, whereas cholesteatoma frequently showed absent immunoreactivity or focal discontinuities. Collagen type VII inununoreactivity is detected similarly within the BMZ of cholesteatoma and skin. Fibronectin immunoreactivity is observed within the dermoepithelial junction of skin and middle ear mucosa. In cholesteatoma, however, fibronectin immunoreactivity is markedly increased within the extrinsic BMZ and the subepithelial connective tissue. The ultrastructural arrangement of the BMZ of cholesteatoma is like that of skin; however, it exhibits distinct alterations of the lamina fibroreticularis and lamina densa. Our results outline cholesteatoma as a disease with disturbed cell matrix interactions analogous to those of wound reepithelialization