Integrated genomic study of quadruple-WT GIST (KIT/PDGFRA/SDH/RAS pathway wild-type GIST)

BACKGROUND: About 10-15% of adult gastrointestinal stromal tumors (GIST) and the vast majority of pediatric GIST do not harbour KIT or platelet-derived growth factor receptor alpha (PDGFRA) mutations (J Clin Oncol 22:3813-3825, 2004; Hematol Oncol Clin North Am 23:15-34, 2009). The molecular biology of these GIST, originally defined as KIT/PDGFRA wild-type (WT), is complex due to the existence of different subgroups with distinct molecular hallmarks, including defects in the succinate dehydrogenase (SDH) complex and mutations of neurofibromatosis type 1 (NF1), BRAF, or KRAS genes (RAS-pathway or RAS-P).In this extremely heterogeneous landscape, the clinical profile and molecular abnormalities of the small subgroup of WT GIST suitably referred to as quadruple wild-type GIST (quadrupleWT or KITWT/PDGFRAWT/SDHWT/RAS-PWT) remains undefined. The aim of this study is to investigate the genomic profile of KITWT/PDGFRAWT/SDHWT/RAS-PWT GIST, by using a massively parallel sequencing and microarray approach, and compare it with the genomic profile of other GIST subtypes. METHODS: We performed a whole genome analysis using a massively parallel sequencing approach on a total of 16 GIST cases (2 KITWT/PDGFRAWT/SDHWT and SDHBIHC+/SDHAIHC+, 2 KITWT/PDGFRAWT/SDHAmut and SDHBIHC-/SDHAIHC- and 12 cases of KITmut or PDGFRAmut GIST). To confirm and extend the results, whole-genome gene expression analysis by microarray was performed on 9 out 16 patients analyzed by RNAseq and an additional 20 GIST patients (1 KITWT/PDGFRAWTSDHAmut GIST and 19 KITmut or PDGFRAmut GIST). The most impressive data were validated by quantitave PCR and Western Blot analysis. RESULTS: We found that both cases of quadrupleWT GIST had a genomic profile profoundly different from both either KIT/PDGFRA mutated or SDHA-mutated GIST. In particular, the quadrupleWT GIST tumors are characterized by the overexpression of molecular markers (CALCRL and COL22A1) and of specific oncogenes including tyrosine and cyclin- dependent kinases (NTRK2 and CDK6) and one member of the ETS-transcription factor family (ERG). CONCLUSION: We report for the first time an integrated genomic picture of KITWT/PDGFRAWT/SDHWT/RAS-PWT GIST, using massively parallel sequencing and gene expression analyses, and found that quadrupleWT GIST have an expression signature that is distinct from SDH-mutant GIST as well as GIST harbouring mutations in KIT or PDGFRA. Our findings suggest that quadrupleWT GIST represent another unique group within the family of gastrointestintal stromal tumors

Design, synthesis and biochemical evaluation of inhibitors of aromatase

The importance of inhibiting the cytochrome P-450 enzyme, aromatase, in the treatment of hormone-dependent breast cancer is discussed, and the current inhibitors reviewed. A number of novel inhibitors of aromatase have been synthesised based on Evans' chiral auxiliary, namely 4-benzyl-2-oxazolidinone. The derivatisation of the 4-benzyl group is described involving nitration and reduction reactions to give the 4-(4'-aminobenzyl)-2-oxazolidinone - the 4-amino group being required to ligate the cytochrome P-450 haem group of aromatase. Alkylation of the oxazolidinone ring is also considered in an attempt to investigate the affect of hydrophobicity on the inhibitory activity of these compounds. The reactions were, in general, found to proceed without major problems and in good yield to give a range of N-alkylated 4-(4'-aminobenzyl)-2-oxazolidinone compounds, the alkylation involved the methyl to the decyl derivatives of both the R and S enantiomers. These potential inhibitors were then subjected to biochemical evaluation using the standard literature procedure (as previously described by Thompson and Siiteri) involving the radiometric analysis of the aromatase catalysed reaction. A number of problems were encountered, however, during the course of the initial screening and only limited results were obtained, for example compounds 61 and 63 gave 39% inhibition at 100¡..tM. After these initial results, the assay failed to provide any more useful data. Extensive analysis of the assay procedure showed that the enzyme had denatured over time - it was discovered that other non-cytochrome P-450 enzymes within the sample (such as oestrone sulphatase) were found to retain their activity. Due to a lack of time, however, new placental preparations could not be prepared and thorough evaluation of the inhibitory activity of the compounds was not achieved