Rearranged During Transfection Mutations Research Articles

In silico profiling and structural insights of missense mutations in RET protein kinase domain by molecular dynamics and docking approach

A major challenge remaining in drug design efforts towards protein kinase is due to the development of drug resistance initiated by the missense mutations in the kinase catalytic domain. Gain or loss of function mutations in the REarranged during Transfection (RET) tyrosine kinase gene have been associated with the development of a wide range of human associated cancers and Hirschsprung's disease. However, to what extent these mutations might affect bio-molecular functions remains unclear. In this article, the functionally significant mutations in RET were screened with the aid of various sequence and structure based in silico prediction methods. We mapped the deleterious mutants, modelled mutant proteins and deciphered the impact of mutations on drug binding mechanisms in the RET crystal structure of PDB ID: with the potential inhibitor vandetanib by docking analysis. Furthermore, molecular dynamics simulations were undertaken to understand the mechanistic action of cancer associated mutations in altering the protein kinase structure, dynamics, and stability. According to our results, the overall effect of V804M, M918T and S922Y were destabilizing and mostly alter the electrostatic component of the binding energy. Specifically, the mutation of gatekeeper residue valine 804 present in the ATP binding pocket affects the protein stability and confers resistance to the drug vandetanib, which was consistent with previously published experimental results. Overall, our findings may provide useful structural insights for in-depth understanding of the molecular mechanism underlying RET mutation and developing effective drugs.

RET mutations in a large indian family with medullary thyroid carcinoma.

Background:Medullary thyroid carcinoma (MTC) is a tumor arising from the para follicular (C) cells of the thyroid gland and can occur either sporadically or as part of an inherited syndrome. A proportion of these cases carry an autosomal dominant mutation in the RET (REarranged during Transfection) proto-oncogene. Screening for these mutations in the affected patients and the carriers “at risk” which includes the first-degree relatives is of utmost importance for early detection and prompt treatment including prophylactic thyroidectomy in cases that harbor these mutations.Results:This report presents details of screening and subsequent follow-up of a large Indian family, where the index case was found to carry p. Cys634Ser mutation involving exon 11 of the RET gene. These data are of value considering the paucity of information within the region in context of screening large families affected by these mutations.

An extended family with familial medullary thyroid carcinoma and Hirschsprung's disease.

Familial medullary thyroid carcinoma (FMTC) is an autosomal dominant inherited disease that has highly characteristic clinical features, including medullary thyroid carcinoma (MTC). Mutation of the RET proto-oncogene is known to be responsible for development of FMTC and for multiple endocrine neoplasia types 2A and 2B. Hirschsprung's disease is the most common form of structural intestinal obstructive disease in human newborns. Hirschsprung's disease is defined by the absence of neural crest-derived enteric ganglia along a variable length of the bowel that invariably involves the rectoanal junction. Co-segregation of FMTC and Hirschsprung's disease is uncommon; nevertheless, in 3 generations of 1 family, we observed 5 patients with FMTC, 2 patients with Hirschsprung's disease, and 1 patient with characteristics of both FMTC and Hirschsprung's disease. Moreover, a Cys620Ser mutation in RET was identified in 4 of the 8 patients. This mutation had both activating and inactivating effects on the RET (REarranged during Transfection) protein. There were individual differences in the penetrance of Hirschsprung's disease due to the RET mutation, but the penetrance of MTC was uniform and high. Genetic testing is important for making decisions about treatment and follow-up in families of this kind.

Central Role of RET in Thyroid Cancer

RET (rearranged during transfection) is a receptor tyrosine kinase involved in the development of neural crest derived cell lineages, kidney, and male germ cells. Different human cancers, including papillary and medullary thyroid carcinomas, lung adenocarcinomas, and myeloproliferative disorders display gain-of-function mutations in RET. Accordingly, RET protein has become a promising molecular target for cancer treatment.

Cabozantinib in Progressive Medullary Thyroid Cancer

Cabozantinib, a tyrosine kinase inhibitor (TKI) of hepatocyte growth factor receptor (MET), vascular endothelial growth factor receptor 2, and rearranged during transfection (RET), demonstrated clinical activity in patients with medullary thyroid cancer (MTC) in phase I. We conducted a double-blind, phase III trial comparing cabozantinib with placebo in 330 patients with documented radiographic progression of metastatic MTC. Patients were randomly assigned (2:1) to cabozantinib (140 mg per day) or placebo. The primary end point was progression-free survival (PFS). Additional outcome measures included tumor response rate, overall survival, and safety. The estimated median PFS was 11.2 months for cabozantinib versus 4.0 months for placebo (hazard ratio, 0.28; 95% CI, 0.19 to 0.40; P < .001). Prolonged PFS with cabozantinib was observed across all subgroups including by age, prior TKI treatment, and RET mutation status (hereditary or sporadic). Response rate was 28% for cabozantinib and 0% for placebo; responses were seen regardless of RET mutation status. Kaplan-Meier estimates of patients alive and progression-free at 1 year are 47.3% for cabozantinib and 7.2% for placebo. Common cabozantinib-associated adverse events included diarrhea, palmar-plantar erythrodysesthesia, decreased weight and appetite, nausea, and fatigue and resulted in dose reductions in 79% and holds in 65% of patients. Adverse events led to treatment discontinuation in 16% of cabozantinib-treated patients and in 8% of placebo-treated patients. Cabozantinib (140 mg per day) achieved a statistically significant improvement of PFS in patients with progressive metastatic MTC and represents an important new treatment option for patients with this rare disease. This dose of cabozantinib was associated with significant but manageable toxicity.

Molecular epidemiology of multiple endocrine neoplasia 2: implications for RET screening in the new millenium

Twenty years ago, the groundbreaking discovery that rearranged during transfection (RET) mutations underlie multiple endocrine neoplasia 2 (MEN2) and familial medullary thyroid cancer (FMTC) ushered in the era of personalized medicine. MEN2-associated signs, taking time to manifest, can be subtle. This study sought to clarify to what extent conventional estimates of 1:200 000-500 000 underestimate the incidence of RET mutations in the population. Included in this retrospective investigation were 333 RET carriers born between 1951 and 2000 and operated on at the largest German surgical referral center (286 carriers) or elsewhere (47 carriers). To estimate the incidence of RET mutations, the number of RET carriers born in Germany in five decades (1951-1960, 1961-1970, 1971-1980, 1981-1990, and 1991-2000) was divided by the corresponding number of German live births. Owing to improved diagnosis and capture of FMTC and MEN2 patients, minimum incidence estimates increased over time: overall from 5.0 (1951-1960) to 9.9 (1991-2000) per million live births and year (P=0.008), and by American Thyroid Association/ATA class from 1.7 to 3.7 for ATA class C (P=0.008); from 1.8 to 2.7 for ATA class A (P=0.017); from 1.5 to 2.2 for ATA class B (P=0.20); and from 0 to 1.4 for ATA class D mutations per million live births and year (P=0.008). Based on 1991-2000 incidence estimates the prevalence in Germany is ∼1:80 000 inhabitants. The molecular minimum incidence estimate of ≈1:100 000 was two- to fivefold greater than conventional estimates of 1:200 000-500 000.

Vandetanib

Vandetanib, an orally active, small-molecule, multitargeted tyrosine kinase inhibitor, demonstrates potent inhibitory activity against vascular endothelial growth factor receptor (VEGFR)-2 and -3, epidermal growth factor receptor (EGFR) and the rearranged during transfection (RET) tyrosine kinase receptor. The large (n=331), randomized, double-blind, multinational ZETA trial compared vandetanib at a dosage of 300 mg once daily with placebo in patients with unresectable, locally advanced or metastatic, hereditary or sporadic, medullary thyroid cancer. During a median follow-up period of 2 years, vandetanib demonstrated statistically significant clinical benefits over placebo with respect to the primary endpoint, namely progression-free survival (PFS), and a range of secondary endpoints, which included objective response rate, disease control rate, time to worsening of pain and calcitonin biochemical response rate. The PFS benefit with vandetanib was mostly consistent across patient subgroups based on baseline characteristics and disease status. Although the correlation between RET mutation status and clinical outcome could not be clearly evaluated in this trial, it is notable that, among patients with sporadic disease, vandetanib not only demonstrated a PFS benefit in the subgroup confirmed as having a RET mutation, but also in the subgroup for whom the RET mutation status was unknown. Vandetanib was generally well tolerated in the ZETA trial; the majority of adverse events were manageable according to standard clinical practice alone or in combination with vandetanib dose reductions. The adverse event of most concern is corrected QT interval prolongation, particularly in view of the long terminal elimination half-life of the drug.

Evolving paradigms for successful molecular imaging of medullary thyroid carcinoma

Medullary thyroid cancer (MTC) is a rare, neuroendocrine tumour (NET) of parafollicular C cells comprising 5–8% of all thyroid cancers with variable biological behaviour and prognosis [1]. Overall mean survival in MTC is 75–85% at 10 years [1–3]. Management of both hereditary (20–30%) and sporadic MTC is challenging due to early cervical lymph node metastases occurring in 50% of patients and distant metastases to the liver, lung and bones being found in 10–20% at diagnosis [1, 3]. MTC secretes several neuroendocrine peptides, of which calcitonin (Ct) and less-specific carcinoembryonic antigen (CEA) are useful tumour markers for diagnosis, surveillance and prognosis. Yet despite the high sensitivity of an elevated serum Ct value as a predictor of disease presence, MTC is an elusive and notoriously problematic tumour to image [1, 3]. Anatomy-based imaging with CT and MRI has exhibited limited sensitivity due to the frequent small size of the metastatic deposits. Although a variety of specific and nonspecific scintigraphic techniques such as Tl, Tc-sestamibi, Tc-tetrofosmin, I/I-metaiodobenzylguanidine, Tc-(V)dimercaptosuccinic acid (DMSA), and Inoctreotide have been used to image MTC, none has emerged as the imaging technique of choice [4, 5]. This raises the question as to why we have been unable to successfully image MTC. The rarity of MTC implies that no single centre can collect a sufficient number of cases, and to date there have been no large, cooperative studies to establish optimal imaging protocols. It has become apparent that MTCs are a heterogeneous group of neoplasms, and the biological behaviour of one MTC may differ significantly from that of another. Imaging approaches that have proven successful, for example using F-fluorodeoxyglucose (FDG) PET/CT in lymphoma and solid cancers and In-octreotide for somatostatin receptor scintigraphy (SRS) in carcinoids, may be too simplistic an imaging approach for MTC, which often appears intermediate in biological behaviour between more aggressive neoplasms characterized by high Ki-67 indices, and hormonally active NETs in which FDG PET has limited value. Genetic testing provides a valuable insight into the phenotypic expression of MTC. Genetic mapping has confirmed the central role of germline mutations of the rearranged during transfection (RET) protooncogene in the pathogenesis of inherited MTC and has shown that somatic RET mutations occur in 50–60% of patients with sporadic MTC, and there have been advances in our understanding of how activated RET tyrosine kinase signalling pathways affect tumour growth and angiogenesis [1, 6–8]. Insights from genetic testing allow informed management decisions. As an example, the dilemma of the timing of prophylactic thyroidectomy in multiple endocrine neoplasia type 2 (MEN2), an autosomal D. Rubello (*) :M. C. Marzola : S. Chondrogiannis Department of Nuclear Medicine, Medical Physics, Radiology, Santa Maria della Misericordia Hospital, Via Tre Martiri 140, 45100 Rovigo, Italy e-mail: domenico.rubello@libero.it

Germline RET sequence variation I852M and occult medullary thyroid cancer: harmless polymorphism or causative mutation?

Rearranged during transfection (RET) gene analysis, widely used to identify carriers at risk of medullary thyroid cancer (MTC), occasionally uncovers novel sequence 'variants of unknown clinical significance' including RET I852M. This study aimed to clarify whether RET I852M represents a harmless polymorphism or a pathogenic mutation. Clinical investigation supported by functional characterization of I852M mutant cells in vitro. Genotype-phenotype correlation including five kindreds from a three-generational Caucasian I852M RET family. A node-negative occult MTC was found in the 64-year-old index patient who had increased basal and stimulated peak calcitonin levels of 190 and 13 307 ng/l, respectively. Her 4-year-old grandson had no histopathological evidence of C-cell disease although his serum calcitonin levels had increased within 5 months from 3·2 to 6·3 ng/l basally and from 17·2 to 24·5 ng/l after pentagastrin stimulation. His mother and two 11- and 1·5-year-old siblings, also carrying the gene, had normal basal and stimulated calcitonin levels and hence did not undergo surgery. Functional characterization of transfected NIH3T3 cells in vitro (cell proliferation rate; cell viability; anchorage-independent cell growth; cell migration; and invasion) indicated that I852M mutant cells have transforming and migratory activities similar to American Thyroid Association (ATA) class A V804M mutants. I852M mutants demonstrated a weaker proliferative potential than fast-proliferating ATA class C C634R mutants and revealed a weaker migratory activity compared with aggressively growing ATA class D A883F mutants. I852M sequence variations represent genuine RET mutations, falling into ATA class A of weakly activating RET germline mutations.

RET genetic screening of sporadic medullary thyroid cancer (MTC) allows the preclinical diagnosis of unsuspected gene carriers and the identification of a relevant percentage of hidden familial MTC (FMTC)

This study was aimed to demonstrate the clinical benefits of rearranged during transfection (RET) genetic screening in patients with apparently sporadic medullary thyroid cancer (MTC) not only to identify the hereditary nature of the disease in the index case but also to discover family members harbouring the same germline mutations (i.e. gene carriers) who are unaware of their condition. RET genetic screening allowed the identification of germline RET mutations in apparently sporadic MTC resulting in their re-classification as hereditary forms. RET genetic screening was performed in 729 apparently sporadic MTC patients by direct sequencing RET exons 5, 8, 10, 11 and 13-16. Clinical and biochemical evaluation of gene carriers was also performed. We discovered an unsuspected germline RET mutation in 47 of 729 (6·5%) apparently sporadic MTC who were re-classified as hereditary. We found 60 of 146 (41·1%) gene carriers, 35 of whom had biochemical or clinical evidence of MTC. Thirty gene carriers underwent total thyroidectomy and 27 of 30 (90%) were persistently cured after a mean follow-up of 6·0 years. As a further result of RET genetic screening, we observed a significantly higher prevalence of familial medullary thyroid cancer (FMTC) in our series with respect to the largest series of the International RET Consortium (P = 0·0002). RET genetic screening of patients with apparently sporadic MTC represents a major tool for the preclinical diagnosis and early treatment of unsuspected affected family members and allows the identification of a relevant percentage of hidden FMTC.

Abstract 256: Distinct roles for gain and loss of function p53 mutations in medullary thyroid cancer

Abstract Medullary thyroid carcinoma (MTC) accounts for 5-10% of all thyroid cancers and presents as metastatic diseases in ∼50% of patients. Patients with metastatic MTC are typically incurable because of the poor tumor response to current chemotherapeutics and radiation treatment. MTC occurs as sporadic tumors in ∼75% of cases. The remaining cases are familial and associated with either multiple neuroendocrine neoplasia syndromes or familial MTC syndrome. The Rearranged during Transfection (RET) proto-oncogene plays a key role in MTC development: however, the absence of RET mutations in ∼50% of sporadic cases and the variable clinical manifestations in patients with the same RET mutation provide compelling evidence that other genetic alterations are involved in MTC pathogenesis. Recent studies in human tumors and mouse models suggest that the Rb and p53 pathways cooperate to suppress MTC evolution. Although some p53 mutations cause a loss of function, most p53 mutations detected in human cancers result in expression of a mutant p53 protein which may exhibit gain of function properties. The objective of this study was to identify the function of p53 mutant proteins commonly expressed in human cancers in MTC pathogenesis. A conditional mouse model was generated in which MTC is initiated by Rb inactivation. Using this model, the consequences of p53 mutant protein expression on MTC initiation and progression were compared to p53 deletion. Loss of a single copy of p53 led to accelerated medullary thyroid tumor progression resulting in decreased survival. Loss of both p53 alleles resulted in a markedly more severe phenotype leading to a median survival of 4 months. Expression of the structural p53R172H mutant, but not the DNA contact p53R270H mutant, resulted in accelerated death compared with p53 deletion demonstrating that p53R172H has a gain of function property. Interestingly, mice heterozygous for p53R172H/+ and p53R270H/+ showed similar survival as mice with loss of a single p53 allele providing evidence that neither p53 mutant protein functions in a dominant negative fashion to promote MTC initiated by Rb loss. These results clearly demonstrate that p53 suppresses the progression of MTC initiated by Rb loss and indicate that gain of function p53 mutations confer poorer prognosis than p53 loss. Given the critical roles played by the Rb and p53 pathways in tumor response to therapy, identifying key Rb and p53 functions in MTC pathogenesis is important for future design of therapies to combat these currently treatment resistant malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 256.

Combination of RET siRNA and irinotecan inhibited the growth of medullary thyroid carcinoma TT cells and xenografts via apoptosis

Medullary thyroid carcinoma (MTC) is a rare endocrine tumor that frequently metastasizes, and treatment with irinotecan (CPT-11) is limited because of side effects. Mutations in the Rearranged during transfection (RET) proto-oncogene are considered the causative event of MTC. The objective of this study was to examine whether small interfering RNA (siRNA) and its combined treatment with CPT-11 could inhibit MTC cell growth in vitro and in vivo. The transfection of RET siRNA suppressed RET expression, reduced proliferation, and increased caspase-3/7 activity via the down-regulation of Bcl-2 expression. Combined treatments with CPT-11 or SN-38 significantly increased caspase 3/7 activity compared with RET siRNA, CPT-11 or SN-38 treatment alone. Importantly, intratumoral injection of RET siRNA along with intravenous injection of CPT-11 significantly inhibited the tumor growth of MTC xenografts via an increased apoptotic effect. These findings that RET siRNA enhanced sensitivity for CPT-11 will provide a novel strategy for the treatment of MTC with RET mutation.

A novel de novo germ‐line V292M mutation in the extracellular region of RET in a patient with phaeochromocytoma and medullary thyroid carcinoma: functional characterization

In multiple endocrine neoplasia (MEN), rearranged during transfection (RET), gene testing has been extensively exploited to characterize tumour aggressiveness and optimize the diagnostic and clinical management. To report the underlying genetic alterations in an unusual case of MEN type 2 (MEN-2A). Occult medullary thyroid carcinoma (MTC) was diagnosed in a 44-year-old man who had presented with unilateral phaeochromcytoma. DNA extracted from the blood and tumour tissues was analysed for mutations in RET. The transforming potential and mitogenic properties of the identified RET mutation were investigated. The patient carried a novel heterozygous germ-line RET mutation in exon 5 (Val292Met, GTG>ATG) (V292M/RET) with no evidence of additional somatic alterations. The mutation maps to the third cadherin-like domain of RET, which is usually not included in RET screening. Interestingly, MTC with concomitant phaeochromcytoma has never been associated with a RET mutation involving the extracellular cadherin-like domain. V292M/RET was absent in the only two relatives examined. In vitro assays indicate that the mutant has low-grade transforming potential. Complete characterization and classification of all novel RET mutations are essential for extending genetic analysis in clinical practice. Our findings suggest that: (i) in all MEN-2 patients negative for RET hot-spot mutations, testing should be extended to all coding regions of the gene and (ii) the newly identified V292M/RET mutation is characterized by relatively weak in vitro transforming ability.

One hundred and seven family members with the rearranged during transfection V804M proto-oncogene mutation presenting with simultaneous medullary and papillary thyroid carcinomas, rare primary hyperparathyroidism, and no pheochromocytomas: Is this a new syndrome—MEN 2C?

The rearranged during transfection (RET) V804M proto-oncogene mutation is rare and associated with medullary thyroid carcinoma (MTC). We present 40 members from a total cohort of 107 family members with this mutation. Family members were tested for RET mutations, calcitonin levels, and screened for pheochromocytoma and primary hyperparathyroidism (PHPT). Thyroidectomies were performed on 15 members. Surgery and pathology reports were obtained and reviewed. A pedigree was constructed. A high penetrance was found for MTC and simultaneous papillary thyroid carcinoma (PTC; 40%). The incidence of PHPT was low (13%). There were no findings of pheochromocytoma. The course in the first family generation was indolent, with late onset of MTC. The second generation experienced earlier disease development; onset occurred earliest in the third generation. The second generation experienced a higher incidence of PTC than the first. This is the largest family with this mutation reported to date. However, it does not fit the classic familial MTC or MEN 2A cancer syndrome. Considering that PTC is not an incidental finding, but the result of an inherited RET V804 M mutation, we propose to identify this phenotypic expression as a unique syndrome consistent with manifestations of MTC, PHPT, and PTC.

Targeting the RET Pathway in Thyroid Cancer

The RET (rearranged during transfection) protooncogene encodes a single pass transmembrane receptor that is expressed in cells derived from the neural crest and the urogenital tract. As part of a cell-surface complex, RET binds glial derived neurotrophic factor (GDNF) ligands in conjunction with GDNF-family alpha co-receptors (GFRalpha). Ligand-induced activation induces dimerization and tyrosine phosphorylation of the RET receptor with downstream activation of several signal transduction pathways. Activating germline RET mutations play a central role in the development of the multiple endocrine neoplasia (MEN) syndromes MEN2A, MEN2B, and familial medullary thyroid carcinoma (FMTC) and also in the development of the congenital abnormality Hirschsprung's disease. Approximately 50% of patients with sporadic MTC have somatic RET mutations, and a significant portion of papillary thyroid carcinomas result from chromosomal inversions or translocations, which activate RET (RET/PTC oncogenes). The RET protooncogene has a significant place in cancer prevention and treatment. Timely thyroidectomy in kindred members who have inherited a mutated RET allele, characteristic of MEN2A, MEN2B, or FMTC, can prevent MTC, the most common cause of death in these syndromes. Also, recently developed molecular therapeutics that target the RET pathway have shown activity in clinical trials of patients with advanced MTC, a disease for which there has been no effective therapy.

Mutational Spectrum of Multiple Endocrine Neoplasia Type 2 and Sporadic Medullary Thyroid Carcinoma in Taiwan

Multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominant cancer predisposition syndrome, and > 95% of MEN 2 patients carry rearranged during transfection (RET) protooncogene mutants. We aimed to elucidate the genotype and phenotype relationship of RET proto-oncogene mutations in Taiwanese subjects with medullary thyroid cancer (MTC). We genotyped the MEN-2-associated germ-line mutations by PCR-based sequencing of the RET gene. DNA was extracted from a total of 69 members from eight unrelated families with individuals affected by MTC, and from seven sporadic cases of MTC. RET mutations were found in four MEN 2A families, all at codon 634 (one with C>R, two with C>F, and one with C>W). One MEN 2A patient carried a de novo mutation at codon 634 (C>R). In two families of MEN 2B, all carried the mutation at codon 918 (M>T). These two cases of MEN 2B were all de novo mutations. One family of familial MTC or unclassified MEN 2 carried the codon 620 (C>F) mutation. Among the seven sporadic cases of MTC, none was found to carry any mutation in hotspot exons. Only two non-synonymous variants (T278N/exon 4 and D489N/exon 7) were found in two cases. However, these two variants were not uncommon in our elderly population. We found that all eight MTC patients with a family history or with the other phenotypes of MEN 2 had RET mutations, whereas no significant RET mutation was found in seven patients with isolated MTC without family history and other endocrine diseases. Molecular scanning of the RET gene in MEN 2 and MTC in Taiwanese patients probably should be limited to exons 10, 11 and 16, initially to be cost-effective.

Correlation of RET somatic mutations with clinicopathological features in sporadic medullary thyroid carcinomas

Screening of REarranged during Transfection (RET) gene mutations has been carried out in different series of sporadic medullary thyroid carcinomas (MTC). RET-positive tumours seem to be associated to a worse clinical outcome. However, the correlation between the type of RET mutation and the patients' clinicopathological data has not been evaluated yet.We analysed RET exons 5, 8, 10–16 in fifty-one sporadic MTC, and found somatic mutations in thirty-three (64.7%) tumours. Among the RET-positive cases, exon 16 was the most frequently affected (60.6%). Two novel somatic mutations (Cys630Gly, c.1881del18) were identified. MTC patients were divided into three groups: group 1, with mutations in RET exons 15 and 16; group 2, with other RET mutations; group 3, having no RET mutations. Group 1 had higher prevalence (P=0.0051) and number of lymph node metastases (P=0.0017), and presented more often multifocal tumours (P=0.037) and persistent disease at last control (P=0.0242) than group 2. Detectable serum calcitonin levels at last screening (P=0.0119) and stage IV disease (P=0.0145) were more frequent in group 1, than in the other groups.Our results suggest that, among the sporadic MTC, cases with RET mutations in exons 15 and 16 are associated with the worst prognosis. Cases with other RET mutations have the most indolent course, and those with no RET mutations have an intermediate risk.

Molecular genetics of medullary thyroid carcinoma: the quest for novel therapeutic targets

Medullary thyroid carcinoma (MTC) is a rare tumour arising from neural crest-derived parafollicular C-cells. Metastatic MTC patients are incurable because the cancer does not respond to radiotherapy or chemotherapy. The REarranged during Transfection (RET) proto-oncogene plays a key role in the development of MTC. However, one-half of the sporadic MTC do not carry RET mutations. Mice models and early evidence obtained in human samples suggest that other genes, including those encoding components of the RB1 (retinoblastoma) and TP53 tumour-suppressor pathways, may be involved in MTC formation. Here, we review the data on the involvement of genes acting in the RET and RB1/TP53 pathways in MTC. Understanding genetic lesions that occur in MTC is a prerequisite to identifying molecular therapeutic targets in MTC and in improving the efficacy of RET-targeted therapies.

RET gene mutations are not a common cause of congenital solitary functioning kidney in adults.

Sir, RET (rearranged during transfection) is a transmembrane receptor tyrosine kinase. Loss-of-function RET mutations occur in Hirschsprung disease, while gain-of-function mutations cause multiple endocrine neoplasia type 2 (MEN2) and familial medullary thyroid carcinoma (FMTC) [1]. In mice, Ret initiates mouse kidney development by enhancing ureteric bud outgrowth [2]. Renal agenesis occurs in mice with homozygous null mutation of either Ret, Gdnf or Gfra1, the latter two genes, respectively, coding for a key ligand that activates Ret and a Ret co-receptor [2]. In humans, unilateral renal agenesis occurs in around 0.01% births and results in a solitary functioning kidney, whereas bilateral renal agenesis and/or severe dysplasia occurs in a similar frequency and usually leads to neonatal death [3]. A role for RET in human kidney development was suggested by a report of unilateral renal agenesis in two members of a FMTC family with RET mutation [4]. More recently, RET mutations were reported in 7 of 19 fetuses with bilateral renal agenesis, and in 2 of 10 with unilateral agenesis and contralateral dysplasia [5]. However, the prevalence of RET mutations in adults with congenital solitary functioning kidney is unknown. A cohort of 14 subjects were selected for our current study. They were living adults born with a solitary functioning kidney, one of whom had two failed pregnancies with fetuses affected by malformed kidneys. We screened exons 1–20 and the conserved splice-sites of RET using direct sequencing. None of the cohort were known to have Hirschsprung disease or MEN2/FMTC or other recognized genetic syndrome. We did not identify any mutations in the 14 adults, and thus it is unlikely that RET mutations will be a common cause of solitary functioning kidney in adults. Because mutations of hepatocyte nuclear factor 1B have been reported in the context of solitary functioning kidney [6], we also screened this gene by sequencing and by dosage analysis but failed to find mutations. Other genes such as GDNF and GFRA1 have yet to be elucidated in our cohort. Our study was in adults while Skinner et al. [5] studied fetuses. We therefore suggest that human RET mutations causing renal disease lead to severe renal failure incompatible with postnatal survival. Mutations of nephrogenesis genes other than RET should be sought to explain human congenital solitary functioning kidney. Conflict of interest statement. None declared.

Routine screening for germline RET mutations is recommended for all patients with medullary thyroid cancer

All patients with medullary thyroid cancer (MTC) are routinely screened for germline mutations in the rearranged during transfection (RET) proto-oncogene. In this Practice Point commentary, we discuss a cross-sectional study by Machens and Dralle, who performed germline RET proto-oncogene mutation analysis in patients with MTC at a tertiary referral centre in Germany to reveal patterns of familial disease presentation over the generations. The study identified a typical distribution of germline RET mutations (subclassified into three accepted risk categories) in the study population, consistent with the reported literature. The investigators also attempted to obtain data on the time of appearance of RET germline mutations in previous generations of the families. The natural history of MTC and penetrance of specific RET mutations could not be clearly identified in each family because of the cross-sectional nature of the study. Nevertheless, the study re-establishes the importance of routine screening for germline RET mutations in all new cases of MTC.