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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 Clin Oncol 223813-3825, 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 clehydrogenase (SDH) complex and mutations of neurofibromatosis type 1 (NF1), BRAF, or KRAS genes (HAS 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 (quadruple(WT) or KITWT /PDGFRA(WT)/SDHWT/RAS-P-WT) remains undefined. The aim of this study is to investigate the genomic profile of KITWT /PDGFRA(WT)/SDHWT/RAS-P-WT GIST, by using a massively parallel sequencing and ruicroarray approach, and compare it with the genoruic profile of other GIST subtypes.

Methods: We performed El whole genome analysis using a massively parallel sequencing approuch on a total of 16 GIST cases (2 KITWT/PDGFRA(WT)/SDFWT and SDHBIHC+/SDHA(IHC+), 2 KITWT/PDGFRA(WT)/SDHA(mut) arid SDHBIHC-/SDHA(IHC-) and 12 cases of KITmut or PDGFRAM(mut) 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/PDGFHA(WT) SDHA(mut) GIST and 19 KITmut or PDGFRA(mut) GIST). The most impressive data were validated by quantitave PCR and Western Blot analysis.

Results: We found that both cases of quadruple(WT) GIST had a genomic profile profoundly different from both either KIT/PDGFRA mutated or SDHA-mutated GIST. In particular, the quadruple(WT) 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 e an integrated genomic picture of KITWT/PDGFRA(WT)/SDHWT/RAS-P-WT GIST, using massively parallel sequencing and gene expression analyses, and found that quadruple(WT) 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 quadruple(WT) GIST represent another unique group within the family of gastrointestintal stromal tumors.