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BackgroundIt is mandatory to confirm the absence of mutations in the KRAS gene before treating metastatic colorectal cancers with epidermal growth factor receptor inhibitors, and similar regulations are being considered for non-small cell lung carcinomas (NSCLC) and other tumor types. Routine diagnosis of KRAS mutations in NSCLC is challenging because of compromised quantity and quality of biological material. Although there are several methods available for detecting mutations in KRAS, there is little comparative data regarding their analytical performance, economic merits, and workflow parameters.MethodsWe compared the specificity, sensitivity, cost, and working time of five methods using 131 frozen NSCLC tissue samples. We extracted genomic DNA from the samples and compared the performance of Sanger cycle sequencing, Pyrosequencing, High-resolution melting analysis (HRM), and the Conformité Européenne (CE)-marked TheraScreen DxS and K-ras StripAssay kits.Results and conclusionsOur results demonstrate that TheraScreen DxS and the StripAssay, in that order, were most effective at diagnosing mutations in KRAS. However, there were still unsatisfactory disagreements between them for 6.1% of all samples tested. Despite this, our findings are likely to assist molecular biologists in making rational decisions when selecting a reliable, efficient, and cost-effective method for detecting KRAS mutations in heterogeneous clinical tumor samples.

Key words: CD133/Nestin/Non-small cell lung cancer (NSCLC)/Cancer stem cell (CSC)/ImmunofluorescenceAims. No effective treatment for lung cancer exists currently. One reason for this, is the development of drug resistance, assumed to be associated with cancer stem cell (CSCs) emergence within the tumour. This pilot study aimed to identify CSCs in 121 non-small cell lung cancer (NSCLC) patient samples via detection of the expression of stem cell markers -CD133 and nestin.Material and methods. Archived paraffin blocks of 121 patient samples were prepared as Tissue Microarrays (TMA). Indirect immunohistochemical staining was used to determine the level of expression of CD133 and nestin. Double immunofluorescence staining was used to investigate the co-expression of these two markers. To determine the correlation between expression of nestin and CD133 with the length of asymptomatic period and overall patient survival we used the Kaplan-Meyer analysis.Results. CD133 expression was detected in 22 (19%), nestin in the epithelium in 74 (66%) and vasculature in 78 (70%) of patients. Co-expression of these two markers was found in 21 (17%) patients in less than 1% of positive cells without impact on disease free or overall survival.Conclusions. We identified CD133 + /nestin + cells as novel potential markers of lung cancer CSCs.

Sphingosine 1-phosphate (S1P) is a bioactive lipid metabolite associated with cancer cell proliferation, survival, migration and regulation of tumor angiogenesis in various cellular and animal models. Sphingosine kinase-1 (SphK1) and S1P lyase are the main enzymes that respectively control the synthesis and degradation of S1P. The present study analyzed the prognostic and predictive value of SphK1 and S1P lyase expression in patients with non-small cell lung cancer (NSCLC), treated with either surgery alone or in combination with adjuvant carboplatin and navelbine. Formalin-fixed, paraffin-embedded tissue samples from 176 patients with NSCLC were stained immunohistochemically using antibodies against SphK1 and S1P lyase, and their expression was correlated with all available clinicopathological factors. Increased expression of SphK1 was significantly associated with shorter overall and disease free survival in patients treated with adjuvant platinum-based chemotherapy. No prognostic relevance for S1P lyase expression was observed. Collectively, the results suggest that the immunohistochemical detection of SphK1 may be a promising predictive marker in NSCLC patients treated with adjuvant platinum-based chemotherapy.

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