Ones current Nutlin -Mission

This lead to a list of five The most important and vital AZD6482 -Match events. For all 5 events we manually inspected the read pileups in IGV to look for possible artifacts. Of the 5 only one had no obvious defects. The other 4 had either strand bias issues and or position bias. Targeted exon resequencing The mutational status of SDHA was assessed by direct Sanger sequencing of genomic DNA. Protocols and primers are available on request. Sequence analysis was performed with Applied Biosystems Se quence ScannerTM v1. 0. Western blotting Western blotting was performed to assess the expression of SDHA in WT GIST with and without SDHA muta tion. Frozen tissue from seven WT GIST were homogenized in RIPA buffer supplemented with prote ase and phosphatase inhibitors.

Electrophoresis and im munoblotting were performed on the protein extracts using 30 ug of protein per sample and the anti SDHA rabbit polyclonal antibody was diluted according to the manu facturers recommendations. Following hybridization with the secondary anti rabbit antibody, the blots were incubated with Immun Star horseradish peroxidase luminal enhancer and exposed onto Kodak Biomax MR Film. Immunohistochemistry Immunohistochemistry was applied on all 17 WT GIST cases tested using SDHA and SDHB, according to manufacturers recommendations. The results of the SDHA and SDHB immunohistochem istry were recorded blindly to the KIT, PDGFRA or SDHA genotyping. One KIT exon 11 mutant GIST from a young adult patient was used as control. The immu noreactivity was scored as negative if the tumor cells were negative but the entrapped normal tissues stained positive.

Conversely, a positive result was interpreted if the tumor cells showed the same intensity staining as the internal positive control cells. If the tumor showed a weak intensity of staining, significantly lower than the normal tissue, the result was interpreted as partial loss of expression. Results Mutation of SDHA is a recurrent event in young adults with KIT and PDGFRA WT GIST Massive parallel next generation sequencing of six cases of WT GIST revealed that the GIST from the young adult patient carried a C to T transition at nucleotide 206 in SDHA exon 2, a nonsense mutation resulting in the replacement of ar ginine with a stop codon at residue 31 of SDHA, causing truncation of the peptide chain at residue 30.

This result was then validated by targeted SDHA exon 2 Sanger sequencing from the DNA isolated from both tumor and normal tis sue, in keeping with a germline mutation. Of note, the SDHA sequence electropherogram of the normal DNA revealed equivalent proportion of the wild type and the mutated allele, whereas tumor DNA con tained predominantly the mutated allele, indicat ing relative loss of the wild type SDHA allele. This patient is alive with disease 66 months after the ini tial diagnosis, and was treated with multiple kinase inhibitors with marginal responses, including imatinib, sunitinib, sorafenib and sirolimus, and being presently on regorafenib.