A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth

<p>Abstract</p> <p>Background</p> <p>Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of <it>KRAS </it>are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS), which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells.</p> <p>Methods</p> <p>DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for <it>KRAS </it>amplification by quantitative PCR, and investigated <it>KRAS </it>amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of <it>KRAS </it>knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively.</p> <p>Results</p> <p>DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the <it>KRAS </it>gene locus. Amplification of the <it>KRAS </it>locus was detected in 15% (3/20) of gastric cancer cell lines (8–18-fold amplification) and 4.7% (4/86) of primary gastric tumors (8–50-fold amplification). <it>KRAS </it>mutations were identified in two of the three cell lines in which <it>KRAS </it>was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased <it>KRAS </it>copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type <it>KRAS</it>, but not in cells with amplified mutant <it>KRAS</it>. Knock-down of <it>KRAS </it>in gastric cancer cells that carried amplified wild-type <it>KRAS </it>resulted in the inhibition of cell growth and suppression of p44/42 MAP kinase and AKT activity.</p> <p>Conclusion</p> <p>Our study highlights the utility of DGS for identification of copy-number alterations. Using DGS, we identified <it>KRAS </it>as a gene that is amplified in human gastric cancer. We demonstrated that gene amplification likely forms the molecular basis of overactivation of KRAS in gastric cancer. Additional studies using a larger cohort of gastric cancer specimens are required to determine the diagnostic and therapeutic implications of <it>KRAS </it>amplification and overexpression.</p

Septic shock-related acute esophageal necrosis and stenosis : three cases of acquired esophageal stenosis presenting a similar clinical course

We present a case of acute esophageal necrosis (AEN) and two acquired esophageal stenosis cases which showed a similar clinical course after an episode of septic shock. Extensive stenosis of the distal esophagus developed in all cases, which were refractory to dilation therapy and required surgical intervention. The etiology of the latter two cases was deduced from the stenosis after septic shock-induced AEN. Since the diagnosis of AEN is based on its characteristic endoscopic findings which can only be confirmed at the onset of the disease, we therefore called these cases "septic shock-related esophageal stenosis" or abbreviated to "septic esophageal stenosis". Further study of similar cases is required for understanding the etiology and management of AEN and the relevant disorder which may cause esophageal stenosis