Searching for non-RET molecular alterations in medullary thyroid carcinoma: expression analysis by mRNA differential display.

Some 25%-70% of sporadic medullary thyroid carcinomas (MTCs) are associated with somatic mutations within the RET proto-oncogene. In a significant number of MTCs, however, no such genetic variations can be detected, which implies alternative pathogenic molecular alterations. To assess altered RET mutation-specific gene expression, and to identify yet unknown gene transcripts involved in the tumorigenesis of MTC, we performed an expression analysis by mRNA differential display (RT-DD). Snap-frozen tumor tissues and corresponding normal thyroid tissues of 8 patients suffering from MTC (6 sporadic, 2 hereditary tumors) were included in the study; 5/8 MTCs harbored RET point mutations (codons 618, 634, 918). The RT-DD method was refined by use of fluorescence-labeled arbitrary oligonucleotides, electrophoresis on an automated sequencer, and a novel fragment-recovery technique utilizing a high-performance fluorescence scanner. More than 400 differentially expressed mRNA transcripts--representing upregulated or downregulated genes in the compared tissues--were detected. In all, 28 selected fragments were recovered, cloned, sequenced, and identified. Differential expression of gene transcripts with known association to cell proliferation or tumor progression--such as annexin A2, Rab11a, trefoil proteins, superoxide dismutase (SOD1), mitochondrial displacement loop (D-loop), and G protein subunit gamma11--as well as of the neuroendocrine marker chromogranin was observed. Furthermore, several mRNA transcripts of yet unknown genes displayed mutation-specific upregulation or downregulation in MTC. Illumination of the molecular basis especially of C-cell carcinomas without detectable alterations of the RET receptor tyrosine kinase will be required for the development of therapeutic strategies for advanced tumors that cannot be bridled or cured by surgical interventions alone.