An Investigation of miRNAs in the Pathogenesis of Pediatric/Wild-Type Gastrointestinal Stromal Tumor
Lorna Kelly, Su-Young Kim, Kenneth Bryan, Maria Debiec-Rychter, Maureen J O'Sullivan. Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland
Background: Gastrointestinal stromal tumour [GIST] is characterized by activating mutations of tyrosine kinase III receptors KIT/PDGFRA, or rarely BRAF, yet a wild-type [WT] form, commonest in childhood, shows similar downstream KIT signalling but without activating TK mutation. The wild-type form is poorly responsive to targeted therapy with tyrosine kinase inhibitors such as imatinib. Genomic losses at 14q, 22q, 1p and 9p occur in adult but not pediatric GIST, which shows no consistent copy number alterations. These findings prompted our investigation of epigenetic mechanisms including miRNAs to explain cell biological dysregulation in wild-type/pediatric GIST.
Design: DNA and RNA were extracted from 45 adult and 29 pediatric GISTs. KIT exons 9, 11, 13, 17 and PDGFRA exons 12, 14, 18 were amplified from DNA, the products examined by high resolution melt analysis and those with aberrant curves sequenced. RNA applied to TaqMan Low Density Arrays (TLDAs) for miRNA profiling, examined 667 miRNAs per sample. Validation was by individual TaqMan assays. Hierarchical clustering was performed using Spearman's rank correlation and Ward's linkage. The methylation status of the 14q32 region was analyzed by allele-specific, methylation-sensitive PCR of bisulfite-converted DNA.
Results: Unsupervised hierarchical clustering clearly separated adult and pediatric cases. The adult group could be further split by differential expression of 47 miRNAs located on 14q32, due to differential allelic methylation.
Differential miRNA expression between wild-type/pediatric and mutant GISTs revealed numerous miRNAs >5-fold differentially expressed and of statistical significance (p<0.05). Potential interactions between diametrically expressed miRNA and mRNA revealed significantly more interactions than expected, particularly for IGF1R.
Conclusions: This work has produced unprecedented distinct miRNA profiles for adult and pediatric GISTs. Adult GISTs are further split by high expression of a miRNA cluster on 14q32, not explained by 14q loss, but by differential allelic methylation. miRNA/mRNA interaction analysis reveals several significant interactions between miRNAs and genes important in WT GIST, which we are currently investigating.
Monday, March 19, 2012 1:00 PM
Poster Session II # 235, Monday Afternoon