In vitro transforming potential, intracellular signaling properties, and sensitivity to a kinase inhibitor (sorafenib) of RET proto-oncogene variants Glu511Lys, Ser649Leu, and Arg886Trp

Abstract

Multiple endocrine neoplasia type 2 and a subset of apparently sporadic medullary thyroid carcinoma (AS-MTC) are caused by germ line activating point mutations of the rearranged during transfection (RET ) proto-oncogene. RET encodes a receptor with tyrosine kinase activity that targets several intracellular signaling cascades, such as RAS-RAF-ERK1/2, PIK3-AKT, and STAT transcription factors. The objective of this study was to assess the function of three germ line RET variants Arg886Trp, Ser649Leu, and Glu511Lys of undetermined pathogenic significance, which were found in three kindreds of isolated AS-MTC. For this purpose, we employed vectors expressing each of the RET variants and measured the number of NIH3T3 transformation foci and soft agar colonies, the degree of activation of known RET intracellular signaling targets (ERK1/2, STAT1, STAT3, and TCF4), and the extent of ERK1/2 inhibition on sorafenib treatment. We found that RET variants Arg886Trp and Glu511Lys have shown increased in vitro transforming potential in a glial-derived neurotrophic factor-dependent manner. In contrast, the Ser649Leu variant did not significantly increased the number of foci and agar colonies relative to wild-type RET (RET-WT). The variants Glu511Lys and Arg886Trp showed 10-and 12.5-fold ERK1/2 activation respectively, that was significantly higher than that observed for RET-WT (fivefold). Increased levels of STAT1 and TCF4 activation were only observed for RET Arg886Trp (2.5-and 3-fold versus 1.2-and 2-fold in RET-WT respectively). The three RET variants analyzed here were sensitive to treatment with sorafenib. In conclusion, our results allow to classify previously uncharacterized RET genotypes, which may be of use to define follow-up and therapeutic regimens.

Mutational load in carotid body tumor

“…It was deposited to dbSNP from the gnomAD database as a germline variant. In ClinVar, another allele was reported with conflicting interpretations of pathogenicity found in hereditary cancer-predisposing syndrome (uncertain significance) and multiple endocrine neoplasia (MEN) type 2 (uncertain significance/likely benign) characterized by medullary thyroid carcinoma, pheochromocytomas, and hyperparathyroidism [70][71][72]. In this patient, we also identified pathogenic and likely pathogenic somatic variants in TP53 gene (NM_000546.5: c.842A > T, p.Asp281Val (chr17: 7,577,096, rs587781525) and NM_000546.5: c.A170A > G, p. Asp57Gly (chr17: 7,579,517) that are the same in Pat104.…”

Next-Generation Sequencing in the Diagnosis of Rare Pediatric Sinonasal Tumors

“…27 Furthermore, activating germline mutations in the RET gene are associated with multiple endocrine neoplasia type 2 (MEN2), medullary thyroid carcinoma (MTC), pheochromocytoma, and rarely in other carcinomas. 31,32 As only the tumor was profiled by NGS, and since the possibility existed that the RET variant was a germ line change and not a true somatic mutation, we pursued targeted Sanger sequencing for the RET variant, using DNA isolated from a sample of the patient's blood. This variant, c.1531G>A (p.Glu511Lys), was detected in the blood at an allele frequency of *50%, confirming its germline status.…”

“…The first patient was referred at age 65 years. 31,32 She passed the variant to 3 of her 4 children (ages 49, 51, and 56), none of whom showed signs of MEN2, such as MTC, pheochromocytoma, or hyperparathyroidism. The second patient underwent thyroidectomy at age 66 and showed no features of MEN2 during 10 years of follow-up.…”

RET Proto-Oncogene Variants in Patients with Medullary Thyroid Carcinoma from the Mediterranean Basin: A Brief Report

“…This is not the case though based on the identified RET -proro-oncogene pathogenic variants reported so far. In the recent comprehensive review by Maciel et al [ 10 ], only 3 out of 20 (p.Cys634Arg, p.Cys634Tyr, and p.Met918Thr) reported RET proto-oncogene pathogenic variants are common between Spanish and Portuguese patients [ 27 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 ] ( Table 1 ). In Portugal, a total of fourteen RET pathogenic variants have so far been reported.…”