A Novel Germline Deletion of p.C630 in RET Causes MTC and Promotes Cell Proliferation and Sensitivity to Pralsetinib.

Context: Medullary thyroid cancer (MTC) is usually caused by gain-of-function mutations in the proto-oncogene RET. Objective: This study aimed to determine the underlying mechanism in a male patient diagnosed with MTC at age 51 years. Methods: Genomic DNA extracted from leukocytes or tumor tissues of patients was used for next-generation sequencing (NGS)-panel sequencing and Sanger sequencing. Wild-type (WT) and p.C630 deletion RET were expressed in HEK 293T cells. Activation of phosphorylation of the crucial tyrosine-905 of RET and MAPK/ERK was analyzed by Western blotting. The effect of RET mutants on cell viability and colony formation ability was determined by CCK8 assay and a colony forming assay. Results: NGS-Panel sequencing revealed a 3-nucleotide/1-amino acid C630 in-frame deletion in exon 11 of RET (c.1887_1889delGTG p.C630del). In vitro expression showed that phosphorylation of the crucial tyrosine 905 was much stronger in the p.C630del RET mutant than in WT RET, indicating ligand-independent activation of the Ret protein tyrosine kinase. Furthermore, p.C630del RET mutant induced strong activation of the MAPK/ERK pathway. In addition, p.C630del RET mutant cells exhibited increased HEK 293T cell viability and colony formation compared with WT RET cells. Pralsetinib (BLU-667), a highly selective RET inhibitor, inhibited the viability of WT RET and p.C630del RET mutant-transfected HEK 293T cells (IC50s: 18.54 and 16.49 μM after treatment for 24 hours), followed by inhibition of the RET-induced MAPK/ERK pathway. Conclusion: The finding in our patient with MTC was a 3-base-pair deletion in exon 11 of RET, a p.C630 deletion not previously reported. The p.C630del RET stimulates cell proliferation by increasing ligand-independent phosphorylation and activation of MAPK/ERK pathway, demonstrating the pathogenic nature of the mutation. We therefore recommend screening panel sequence of RET in MTC patients with indications of a genetic cause. [ABSTRACT FROM AUTHOR]