It is possible, therefore, selleck chem Sunitinib that these drugs may have elicited the observed clinical benefit for reasons unrelated to our hypothesis. However, this analysis did provide clinically useful information and provided the rationale for a therapeutic regime that, Inhibitors,Modulators,Libraries whilst not cura tive, did establish stable disease for several months. We propose that complete genetic characterization in this manner represents a tractable methodology for the study of rare cancer types and can aid in the determina tion of relevant therapeutic approaches in the absence of established interventions. Furthermore, the establish ment of repositories containing the genomic and tran scriptomic information of individual cancers coupled with their clinical responses to therapeutic intervention will be a key factor in furthering the utility of this approach.
We envisage that as sequencing costs con tinue to decline, whole genome characterization will become a routine part of cancer pathology. Materials and methods For detailed methodology Inhibitors,Modulators,Libraries see Additional file 1. A sum mary of the sites used for genomic and transcriptomic analyses is shown in Figure S6 in Additional file 1. Gen ome sequence data have been deposited at the European Genome Phenome Archive, which is hosted by the European Bioinformatics Institute, under the accession number. Sample preparation Tumor DNA was extracted from formalin fixed, paraf fin embedded lymph node sections using the Qiagen DNeasy Blood and Tissue Kit. Normal DNA was prepared from leukocytes using the Gentra PureGene blood kit as per the manufacturers instructions.
Genome DNA library construction and sequencing were carried out using the Genome Analyzer II as per the manufacturers instructions. Tumor RNA was derived Inhibitors,Modulators,Libraries from fine needle aspirates of lung metastases and normal RNA was extracted from leuko cytes using Trizol Inhibitors,Modulators,Libraries and the processing for transcriptome analysis was con ducted as previously described. The relapse sample was obtained by Inhibitors,Modulators,Libraries surgical excision of the skin metastasis under local anesthetic 5 days after cessation with sorafenib sulindac treatment. DNA was extracted using the Gentra PureGene Tissue kit and RNA was extracted using the Invitrogen Trizol kit, and the geno mic library and transcriptome library were constructed as previously described. Mutation detection and copy number analysis DNA sequences were aligned to the human reference, HG18, using MAQ version 0.
7. 1. To identify muta tions and quantify transcript levels, WTSS data were aligned to the genome and a database of exon junctions. SNPs from the tumor tissue whole genome shot gun sequencing and WTSS were detected using MAQ SNP filter parameters of consensus quality 30 and depth 8 and minimum mapping quality 60. All other parameters were left as the default settings. truly Addi tional filters to reduce false positive variant calls included the base quality score of a variant had to be 20.