In addition, frozen tumor tissues of the following patients were excluded from the study: patients who had received preoperative neoadjuvant treatments, patients with double primary lung cancer, and patients who had undergone incomplete resections or who had not been subjected to mediastinal lymph node dissections.The selected frozen tumor tissues were used for the microdissection.Briefly, frozen tissues were lightly stained with HE to improve visualization and the necrotic tumor tissues and intervening normal tissues were removed.The quantity and quality of RNA were analyzed using a spectrometer, respectively.Frozen samples of resected lung tumors were obtained within min of resection and subdivided into mg samples and snap frozen at C.Each specimen was associated with an immediately adjacent sample embedded for histology in OCT medium and stored at C.Frozen sections of embedded samples stained with HE was used to confirm the postoperative pathologic diagnosis and to estimate the cellular composition of adjacent samples.All specimens underwent pathologic review by two pathologists.One hundred nine tumors obtained during the same period were excluded because they did not meet one or more of the eligibility criteria.We reasoned that translocations in the ALK gene would result in disparate levels of expression between exons and of the breakpoint, with the expression higher in the end. After performing array normalization and background correction for all probes, we restricted our analysis to the probes uniquely mapping to the ALK gene. To correct for differences in probe response Diphenhydramine hydrochloride characteristics across the gene, for every sample, we divided each probe intensity value by the average probe intensity across the other wildtype specimens.For each cell line, we computed the location of the most likely breakpoint as the probe that gives the maximum deviation between average expression of and probe subsets.The resulting PCR products were analyzed using agarose gel electrophoresis.Genotyping for KRAS, epidermal growth factor receptor, HER, BRAF, and PIKCA was done using either R.In the screen of lung cancer cell lines, exon arrays showed that H and H cell lines had significantly higher signal for ALK probes to corresponding to exons to of ALK compared with other cell lines.Probes were assigned into three categories based on their Amiodarone hydrochloride labeling intensity: nonresponsive probes. Only highintensity probes were used in breakpoint detection.B, reverse transcriptionPCR detection of EMLALK fusion in NSCLC cell lines and tumors.Primer set amplifies EMLALK fusion genes from H, H, and DFCI cell lines but not from A line.Bacterial artificial chromosome DNA was labeled with either spectrum red dUTP or spectrum greendUTP by nick translation using manufacturers recommended conditions.Slides for metaphase fluorescence in situ hybridization from cell lines were prepared using standard cytogenetic methodologies.Cells were plated at a to the medium after h, and the cells were incubated for another h, after which the cells were analyzed as described previously. Percent apoptosis was estimated from the subG cell fraction.Mice were randomized to four treatment groups once the mean tumor volume reached to mm: vehicle. The experiment was terminated when the mean size of either the treated or control groups reached, mm.Using reverse transcriptionPCR, we were able to confirm the presence of the EMLALK fusion gene product in both H and H but not in any other of the cell lines.

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