Abstract
Background:
Multiple endocrine neoplasia type 2 (MEN2) is an autosomal dominant disorder caused by mutations in the RET proto-oncogene. MEN2 is classified into two subtypes, MEN 2A and 2B. MEN2B is characterized by early-onset and aggressive medullary thyroid carcinoma (MTC), pheochromocytoma, and characteristic physical features.
Patient Findings:
We present a 39-year-old male with early-onset metastatic MTC diagnosed at the age of 13 years and physical features typical for MEN2B such as marfanoid habitus, mucosal neuromas, and thickened eyelids. The patient has two first-degree relatives (mother and maternal uncle) with MTC and pheochromocytoma. The mother has similar facial features. RET sequencing revealed a novel tandem RET E768D/L790F germline mutation in exon 13. The patient's mother has the same RET variant. For functional in vitro characterization, wild-type RET, RET E768D, RET L790F, the double RET E768D/L790F mutant, and RET M918T were expressed in HEK293 cells. The novel double RET E768D/L790F mutant increased ligand-independent RET phosphorylation, activation of the mitogen-activated protein kinase (MAPK)-pathway, and colony formation similar to the classical MEN2B RET M918T mutation.
Summary:
In this male patient with a MEN2B-like phenotype, we identified a novel double RET germline mutation, E768D/L790F. Functional characterization of the double mutant shows similar transforming capacity as RET M918T.
Introduction
The hallmark of multiple endocrine neoplasia type 2 (MEN2) is medullary thyroid carcinoma (MTC). MEN2A is characterized by MTC, typically diagnosed in adult life, pheochromocytoma and primary hyperparathyroidism. MEN2B comprises early-onset aggressive MTC, pheochromocytoma, skeletal abnormalities, ganglioneuromatosis, and typical ophthalmologic features, among others (1). MEN2 is caused by germline RET mutations leading to ligand-independent RET autophosphorylation with subsequent downstream mitogen-activated protein kinase (MAPK)- and PI3K-pathway activation (2).
Case Description
We present a 39-year old Caucasian patient diagnosed with MTC in 1994 at the age of 13 years (III.1; Fig. 1). He underwent calcitonin screening after his mother (II.2) and maternal uncle (II.1) were diagnosed with MTC and bilateral pheochromocytoma in 1993, raising the suspicion of MEN2.
The uncle (II.1; index patient) has been diagnosed with MTC and pheochromocytoma at the age of 36 years and died 2 years later. No written records are available. The patient's mother was diagnosed at age 34 in 1993. She underwent total thyroidectomy (TNM pT2pN0) and her calcitonin levels have remained undetectable postoperatively. The patient's uncle and mother did not develop pHPT. The mother suffers from chronic constipation and has facial features characteristic of MEN2B, including neuromas of the eyes and lips. The patient's grandfather (I.1) died at the age of 45 and apparently had similar facial features and a thyroid tumor of unknown histology.
The reported patient was referred to our center in January 2016 with disseminated metastases to mediastinal lymph nodes, bone, liver, and newly diagnosed lung metastases. The patient showed a typical MEN2B phenotype with marfanoid habitus, mucosal neuromas, thickened eyelids, and enlarged nodular lips but no evidence of pheochromocytoma.
We carefully reviewed the patient's medical history, although written medical records from 1994 to 2001 were unfortunately missing. According to the patient, thyroidectomy was performed in 1994 followed by neck dissection in 1995 because of persistently high calcitonin levels. Liver metastases were diagnosed in 2001 and treated with peptide receptor radionuclide therapy (PRRT, 131I-MIBG and DOTATATE) in 2010 and embolization therapy in 2011 and 2012. Bone metastases were diagnosed between 2006 and 2009. The patient's calcitonin and carcinoembryonic antigen (CEA) levels are summarized in Figure 1b. The calcitonin doubling time remained longer than 2 years, while the CEA doubling time was 1.7 years (04/2015–12/2018). Screening for pheochromocytoma with measurement of plasma metanephrines and normetanephrines remained negative, and imaging studies did not reveal adrenal lesions.
Therapeutic options were discussed in an interdisciplinary setting, considering the patient's general condition (ECOG 1-2) and symptoms such as chronic diarrhea, weight loss (body mass index 17 kg/m2), and lower back pain. In 2016, the patient underwent radiotherapy (40 Gy) for symptomatic spinal bone metastases and bisphosphonate therapy was initiated. Tyrosine kinase inhibitor therapy was recommended multiple times but refused by the patient. The patient died in February 2020.
Molecular Analyses
As MEN2B disease was clinically evident, Sanger Sequencing of RET exons 5, 8, 10, 11, 13, 14, 15, and 16 was performed of DNA isolated from peripheral blood to determine the patient's genotype. Two missense mutations were identified in the same allele of exon 13 that cause amino changes from glutamic acid 768 to aspartic acid (c.2304G>C (p.E768D; GAG>GAC), and leucine 790 to phenylalanine (c.2370G>T (p.L790F; TTG>TTT)). The patient's mother also has the germline RET E768D/L790F mutation.
Functional Characterization of the RET Mutations
Functional characterization of the novel tandem RET mutation was performed as previously described (3) and the results are summarized in Figure 1c–e. Expression vectors (template pBabe-RET9 WT; provided by Massimo Santoro, Pisa, Italy) encoding RET9 E768D, RET9 L790F, RET9 M918T, and RET9 E768D/L790F were used for all experiments.
We observed a significantly higher activation of the MAPK/extracellular-signal-regulated kinase (ERK) signaling pathway in RET9 E768D/E790F expressing cells compared with RET9 WT and the single mutations RET9 E768D and RET9 L790F in double-luciferase reporter assays (Fig. 1c). Ligand-independent autophosphorylation of the double mutant RET was markedly increased compared with RET WT and similar to the M918T mutation (Fig. 1d). A colony formation assay showed that HEK293 cells transfected with RET9 E768D/L790F form significantly more colonies than cells transfected with RET9 WT, RET9 E768D, or RET9 L790F vectors (Fig. 1e).
Discussion
This is the first description of a patient with a double RET E768D/L790F mutation associated with a MEN2B phenotype. Single RET mutations in codon 768 and 790 are described in MEN2A patients and are considered moderate risk variations according to the American Thyroid Association (ATA) classification (4). The data presented provide in vitro evidence of cooperative transforming activity of the double mutant that is similar to the classical MEN2B M918T mutation.
Footnotes
Author Disclosure Statement
No competing financial interests exist.
Funding Information
Deutsche Forschungsgemeinschaft DFG Grants FU356/7-2 (to D.F.), MO1018/2-2 (to L.C.M) Interne Forschungsförderung Essen (IFORES) program, Faculty of Medicine, Duisburg-Essen.