Second line therapy for patients that progressed or were intolerant to imatinib was sunitinib. This study was approved by the institutional review board and inhibitor Imatinib was performed in accordance with national regulations. KIT and PDGFRA mutation screening DNA isolation from formalin fixed, paraffin embedded tumor samples and from physically disaggregated fresh frozen tissue fragments was performed using an adapta tion of the technique described by Lungu and colleagues or a salting out chloroform mixed methodology, respectively. Using the DNA extracted from each sample, KIT and PDGFRA target sequences were amplified by poly merase chain reaction on a standard termocycler. Primers and conditions were as described in the literature.
Direct sequencing was performed on an ABI PRISM 310 automatic Inhibitors,Modulators,Libraries sequencer using the Big Dye Ter minator Chemistry, according to the manufacturers recommenda tions. All results were confirmed with a second indepen dent analysis. Comparative genomic hybridization Fresh frozen tumor samples from 29 patients were ana lyzed by CGH following the procedure of Kallioniemi et al, with modifications previously described. Samples were analyzed with a Cohu 4900 CCD camera using an automated filter wheel coupled to a Zeiss Axio plan fluorescence microscope and a Citovysion system version 3. 9. For each sample, data from 10 cells were combined to generate average ratio profiles with 99% confidence intervals and aberrations were scored whenever the sample profile Inhibitors,Modulators,Libraries and the stan dard reference profile at 99% did not overlap.
Description of the CGH copy number changes followed the guidelines suggested by the International System for Human Cytogenetic Nomenclature 2005. Statistical analysis Relevant clinico pathological and genetic variables were cross tabulated and analyzed Inhibitors,Modulators,Libraries using the chi square or Fishers exact test. The number of chromosomal aber rations was compared within Inhibitors,Modulators,Libraries groups of samples with dif ferent mutation genotypes using the non parametric Mann Whitney U test. Kaplan Meyer survival curves using log rank test were computed for relevant clinical and genetic events. Inhibitors,Modulators,Libraries For statistical purposes, patients with recurrent or metastatic lesions were included in the high risk group. A P value lower than 0. 05 was considered sta tistically significant. All analysis was performed using the Statistical Package for Social Sciences software, version 15.
Results Clinicopathologic characteristics of the patients A total of 80 patients diagnosed with GIST www.selleckchem.com/products/DAPT-GSI-IX.html were enrolled in this study. Tumor location was obtainable in 79 cases, of which 67 corresponded to primary lesions Additional file 1. Twelve recurrent or metastatic lesions were ana lyzed due to lack of the primary sample. For four of the patients, a second sample collected after disease progres sion could additionally be assessed, increasing the num ber of lesions submitted to sequencing analysis to 84.