Somatic mutations in the RET proto-oncogene in sporadic medullary thyroid carcinoma

OBJECTIVE We have determined the frequency of specific mutations in the RET proto-oncogene in sporadic medullary thyroid carcinomas (MTCs) and correlated the presence or absence of a codon 918 mutation with the clinical characteristics of these tumours. DESIGN Thirty paraffin-embedded sporadic MTCs and two frozen MTCs were collected for analysis of specific mutations in the RET proto-oncogene in codons 609, 611, 618 and 620 (exon 10); 630 and 634 (exon 11); 768 (exon 13); 883 (exon 15) and 918 (exon 16). A novel primer was designed which introduced a restriction site for RsaI in the presence of the specific codon 918 mutation (ATG → ACG) in these tumour samples. A ‘clinical-genetic’ correlation was performed comparing the presence or absence of the codon 918 mutation with the following clinical characteristics: age at diagnosis, tumour size, presence or absence of metastases, MTC related morbidity, and base line calcitonin levels at diagnosis or most recent follow-up. PATIENTS Patients were classified as having sporadic MTC if there was no family history of C-cell hyperplasia, MTC, phaeochromocytoma or parathyroid disease. Retrospective review of patient records enabled complete clinical data to be obtained in 28 of 32 patients. MEASUREMENTS Base line calcitonin levels were measured by radioimmunoassay or calcitonin enzyme linked immunoassay. Cysteine codons in exons 10 and 11, specifically codons 609, 611, 618, 620, 630 and 634, were screened for the presence of mutations by sequence analysis. Specific mutations occurring at codons 768, 883 and 918 were screened for by restriction endonuclease digestion of PCR products. RESULTS The mutation at codon 918ATG → ACG was found in 21 of 32 (66%) MTCs and the mutation at codon 883GCT → TTT was found in one of 32 MTCs. Where possible, the presence of ‘germline-type’ mutations in codons 609, 611, 618, 620, 630 and 634 were excluded. Ten MTCs did not have a mutation in codons 768, 883 or 918 of the RET proto-oncogene. The presence or absence of the somatic mutation at codon 918 did not correlate with any of the above clinical characteristics. CONCLUSION Somatic mutations in the RET proto-oncogene occur frequently in sporadic MTCs.     

甲状腺髓样癌中RET原癌基因突变

甲状腺髓样癌(MTC)是起源于甲状腺滤泡旁C细胞的恶性肿瘤,分为散发型和遗传型两类,后者是多发性内分泌腺瘤病(MEN)2A、MEN2B和家族性MTC(FMTC)3种遗传性综合征共同的临床特征.RET原癌基因编码跨膜的酪氨酸激酶受体,在细胞的增殖、迁移、分化和生存方面起重要的调节作用.目前,大量研究表明,RET原癌基因不同位点的突变可导致酪氨酸激酶受体不同区域的氨基酸改变,进而引起不同类型的临床表现;同时,其基因多态性也作为一种低外显率突变发挥作用.     

Somatic mutations in the RET proto-oncogene in sporadic medullary thyroid carcinomas

The frequency and prognostic relevance of RET proto-oncogene somatic mutations in sporadic medullary thyroid carcinoma (MTC) remain controversial. In order to study somatic mutations in the RET proto-oncogene in sporadic MTCs found in the Czech population and to correlate these mutations with clinical and pathological characteristics, we investigated 48 truly sporadic MTCs by sequencing classical risk exons 10, 11, 13, 14, 15 and 16. From the 48 tumors studied, 23 (48%) had somatic mutation in the RET proto-oncogene in exons 10, 11, 15 or 16. The classical somatic mutation Met918Thr in exon 16 was only found in 13 tumors (27%). In five cases, multiple somatic mutations and deletions were detected. A statistically significant correlation between the presence of somatic mutation with more advanced pathological TNM stages was observed. Other clinical and pathological characteristics did not show any statistical significant association with the presence or absence of somatic mutation.     

Somatic trinucleotide change encompassing codons 882 and 883 of the RET proto-oncogene in a patient with sporadic medullary thyroid carcinoma

OBJECTIVE: Restriction analysis is a straightforward procedure for mutational analysis. It is commonly used for screening RET mutations. Incomplete digestion is a well-known cause of false results. Herein, we report another limitation of the method. DESIGN AND METHODS: Screening for somatic mutations in RET exons 16, 13 and 15 was performed in a patient with a sporadic medullary thyroid carcinoma. Genetic study was carried out by both restriction analysis and direct sequencing. RESULTS: A somatic trinucleotide change encompassing codons 882 and 883 of the RET proto-oncogene (GTA GCT to GTT TTT) was documented. Particular to this case is the silent mutation (GTA-->GTT) at codon 882. Independently, both the novel silent mutation and the missense mutation at codon 883 may disrupt the same AluI restriction site. Based on the restriction pattern we were able to say that both mutations occurred in the same allele. CONCLUSIONS: Restriction analysis is an easy approach for screening RET mutations; however, it is not enough to assign a final diagnosis.     

New multiple somatic mutations in the RET proto-oncogene associated with a sporadic medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) occurs mostly as a sporadic tumor or in connection with inherited cancer syndromes-multiple endocrine neoplasia (MEN) types 2A and 2B and familial MTC. Germline mutations in the RET proto-oncogene are found in most of the familial cases. Somatic mutations in the RET proto-oncogene are detected in 23%-69% of patients with sporadic MTC. The most frequent somatic mutation is Met918Thr in exon 16 and only a small percentage of mutations in other RET exons have been observed. In a very few cases double mutations were found. Genetic screening for somatic mutations in RET exons 10, 11, 13, 14, 15, and 16 in Czech patients with sporadic MTC was carried out by DNA sequencing. This study presents a new triplesomatic mutation Gly911Asp, Met918Thr, and Glu921Lys in exon 16 of the RET proto-oncogene detected in an 18-year-old Czech male patient. In the second case, a new double-somatic mutation Val591Ile in exon 10 with a concomitant somatic mutation Met918Thr in exon 16 was found in a 77-year-old Czech female patient. These both newly described somatic multiple mutations were revealed in a hemizygous status, the loss of heterozygosity in tumor tissues in comparison with germline DNA was confirmed.     

RET polymorphisms and sporadic medullary thyroid carcinoma in a Portuguese population

The genetic basis of the sporadic form of medullary thyroid carcinoma, derived from "C" cells, is still poorly understood. Somatic mutations of RET proto-oncogene have been reported at a variable frequency ranging from 23% to 69%. The hypothesis that low penetrance factors, such as polymorphisms, might contribute to the phenotype of this neoplasm has been addressed in a few studies conducting to conflicting results. Herein, we studied 100 individuals (50 patients and 50 controls) aiming to compare the frequencies of G691S, L769L, S836S, and S904S RET polymorphisms observed in patients with respect to controls. Furthermore, meta-analysis of published studies including the present results was conducted. To test the contributory role of the above polymorphisms for the development of "C"-cell hyperplasia, we studied a group of 10 individuals selected for having a positive pentagastrin test despite the absence of a RET germline mutation. An over-representation of the G691S polymorphism, particularly in females, was observed in patients with respect to controls, although not reaching the level of significance. Allelic frequencies of the other three polymorphisms were not different in patients and controls. Results obtained in the admittedly small group of individuals with a positive pentagastrin test are unlikely to support a major influence of any polymorphism in the development of "C"-cell hyperplasia. The meta-analysis provided evidence for a significant association of the S691 allele with MTC (odds ratio 1.54, 95% confidence interval 1.12-2.12, p=0.008) and found no significant associations for the other polymorphisms.     

Low frequency of germline mutations in the RET proto-oncogene in patients with apparently sporadic medullary thyroid carcinoma

BACKGROUND AND OBJECTIVES Medullary thyroid carcinoma (MTC) occurs both sporadically and In the autosomal domlnantly inherited multiple endocrine neoplasia (MEN) type 2 syndromes. The distinction between true sporadic MTC and a new mutation familial case is important for future clinical managment of both the patient and family. The susceptibility gene for MEN 2 is theRET proto-oncogene. Systematic analysis for germ-line mutations of theRET proto-oncogene was performed in a series of 67 patients with apparently sporadic MTC to determine whether they were true sporadic cases or unsuspected de novo MEN 2 cases. DESIGN AND PATIENTS Sixty-seven unselected patients with sporadic MTC were randomly ascertained from clinic patients from four centres. The diagnosis of MTC was confirmed by hlstopathology. Germline DNA was extracted from peripheral blood leucocytes or from paraffin-embedded tissue and subsequently used for polymerase chain reaction amplification. MEASUREMENTS Polymerase chain reaction basedRET mutation analysis was performed by direct double-stranded cycle sequencing of axons 10, 11, 13 and 16, within which the majority ofMEN2 mutations have been shown to occur. RESULTS In this series, there was one proven case of germline mutation InRET codon 620, which previously has been shown to be responsible for MEN 2, thus Indicating heritable disease. No germline mutation in codon 918, typical of MEN 2B, was found. CONCLUSIONS A figure of 1.5% germline mutations In 67 apparently sporadic MTC Is lower than the incidence of familial disease reported in previous series Involving clinical and biochemical screening. The presence of a germline mutation in theRET proto-oncogene in a patient with MTC indicates heritable disease. The absence of germlineRET exon 10,11,13 or 16 mutation appears to rule out MEN 2A to a high probability, although the presence of a familial form of MTC other than classical MEN 2A cannot be excluded conclusively.     

甲状腺髓样癌患者RET原癌基因突变的研究

目的研究甲状腺髓样癌(MTC)的RET原癌基因突变情况。方法从12例病理诊断的MTC患者和2例临床上怀疑MTC患者提取外周血基因组DNA,对RET原癌基因第10、11、16外显子进行PCR产物直接测序。结果发现8例患者存在基因突变。1例是第10外显子618密码子TGC/CGC突变;2例是第11外显子634密码子TGC/TAC突变;3例是634密码子TGC/CGC突变;2例是第16外显子918密码子ATG/ACG突变。结论直接基因测序分析能在基因水平诊断MTC,分子遗传学分析使术前诊断该疾病成为可能。     

Long-term outcome in 46 gene carriers of hereditary medullary thyroid carcinoma after prophylactic thyroidectomy: impact of individual RET genotype

OBJECTIVE: In children with RET proto-oncogene mutation, curative treatment of medullary thyroid carcinoma (MTC) is possible by prophylactic thyroidectomy. Recommendations on the timing and extent of thyroidectomy are based upon a model that utilises genotype-phenotype correlations to stratify mutations into three risk groups. DESIGN: We evaluated the long-term outcome (mean follow-up 6.4 years, 15 patients more than 10 years, 26 patients more than 5 years) of operated gene carriers stratified into two risk groups (levels 1 and 2) based on the biological aggressiveness of MTC. RESULTS: In 46 RET gene carriers, prophylactic thyroidectomy was carried out between the ages of 4 and 21 years. Level 1 mutations were harboured by 11 patients (codons 790, 791, 804 and 891). Histology was completely normal in two patients; in seven patients C-cell hyperplasia (CCH) and in two patients T1 tumours were diagnosed. All patients with level 1 mutations were cured. Level 2 mutations were harboured by 35 patients (codons 618, 620, 630 and 634). Histology of these patients showed CCH in 11 patients, T1 tumours in 21, T2 tumour in 1, T3 tumour in 1 and Tx in 1 patient. Histology showed no lymph node involvement. Five patients with level 2 mutations failed to be cured; in two patients, persistence of MTC was diagnosed directly after thyroidectomy and in three during follow-up. In two patients carrying a 634 mutation, other endocrinopathies (hyperparathyroidism and bilateral pheochromocytoma) manifested during follow-up. CONCLUSIONS: If prophylactic thyroidectomy is done at early ages, cure rate is high. Timing and extent of prophylactic thyroidectomy can be modified by individual RET mutation.     

Evaluation of a DHPLC-based assay for rapid detection of RET germline mutations in Italian patients with medullary thyroid carcinoma

Causative gain-of-function mutations of the RET tyrosine-kinase receptor gene have been reported in more than 95% of inherited cases of medullary thyroid carcinoma (MTC; OMIM# 155240). Most RET activating mutations are clustered in mutational "hot spots" in exons 10, 11, 13, 14, 15 and 16 and are usually detected by single-strand conformation polymorphism (SSCP) followed by direct sequencing. To improve sensitivity, time and costs of mutational screening we have developed a denaturing high performance chromatography (DHPLC) protocol, based on the detection of heteroduplex molecules by ion-pair reverse-phase liquid chromatography under partially denaturing conditions. The mutational screening of RET exons 10, 11, 13-16 was performed in a total of 111 subjects, including 45 MTC patients and 49 relatives with known RET mutations and 17 individuals, being at risk of hereditary MTC and carrying unknown RET alleles. Heteroduplex peaks with a distinct and reproducible DHPLC elution profile allowed the detection of both rare and common RET mutations. Overall, the DHPLC-based methodology showed a high level of sensitivity and accuracy, nearing 100%. Furthermore, our protocol showed the ability to identify: 1) all the mutated codons of RET located in the "hot spots" domain; 2) the different point mutations occurring in the same codon of RET gene; 3) less frequent or rare mutations; 4) polymorphisms. As such, it can be proposed as a relatively simple and highly accurate method for a rapid genetic testing for members of MTC families.     

Significance of the RET proto-oncogene polymorphisms in Turkish sporadic medullary thyroid carcinoma patients

Several single nucleotide polymorphisms (SNP) of the RET gene have been identified in medullary thyroid carcinoma (MTC) patients as well as in the general population. However, the relevance of SNP for MTC patients is still controversial, whether these allelic variants play other interacting, predisposing or modifying roles in clinical behavior of MTC. The aim of this work is to elaborate allelic frequencies of the RET proto-oncogene polymorphisms in Turkish sporadic MTC patients and to demonstrate if there is an association between SNP and the clinical disease features, specifically the age at onset of MTC and lymph node involvement at diagnosis. We analyzed the allelic frequencies of SNP of the exon 11, 13, 14 and 15 of the RET proto-oncogene in blood samples from 50 sporadic MTC patients, using the polymerase chain reaction methodology followed by DNA sequencing. The observed allelic frequencies were 24% for G691S polymorphism in exon 11, 29% for L769L polymorphism in exon 13, 5% for S836S polymorphism in exon 14, and 26% for S904S polymorphism in exon 15. These frequencies are similar to those reported in other countries. We did not observe any significant association of all four SNP with the age at onset of MTC. Our results indicate a possible association between the presence of lymph node involvement at the time of diagnosis (extent of disease) and L769L or S836S polymorphism. However, it is not possible to draw definitive conclusions that these two polymorphisms play a significant role in clinical behavior of MTC. Further studies are needed to evaluate the role of this polymorphism in the clinical behavior of MTC.     

Beyond RET: potential therapeutic approaches for advanced and metastatic medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) is a rare calcitonin‐producing neuroendocrine tumour that originates from the parafollicular C‐cells of the thyroid gland. The RET proto‐oncogene encodes the RET receptor tyrosine kinase, which has essential roles in cell survival, differentiation and proliferation. Activating mutations of RET are associated with the pathogenesis of MTC and have been demonstrated in nearly all hereditary and in 30–50% of sporadic MTC cases, making this receptor an excellent target for small‐molecule inhibitors for this tumour. Clinical trials of small organic inhibitors of tyrosine kinase receptors (TKIs) targeting the RET receptor have shown efficacy for treatment of metastatic MTC with 30–50% of patients responding to these agents. Despite the importance of the RET receptor in MTC, it is clear that other signal transduction pathways, tyrosine kinase receptors, and tumour suppressor genes are involved in MTC tumourigenesis and progression. A better understanding of molecular cross‐talk between these signal pathways and the RET receptor may lead to combinatorial therapy that will improve outcomes beyond what is currently possible with RET‐directed TKIs. Finally, there is evidence that immunological‐based therapy using dendritic cell vaccination strategies have been effective for reducing tumour mass in a small number of patients. The identification of additional MTC‐specific tumour antigens and a better understanding of specific epitopes in these tumour antigens may lead to improvement of response rates.     

Analysis of RET polymorphisms and haplotypes in the context of sporadic medullary thyroid carcinoma.

CONTEXT: Little is known about the etiology of sporadic medullary thyroid carcinoma (sMTC). While germline gain-of-function mutations in the RET proto-oncogene cause hereditary MTC, the molecular mechanisms leading to the sporadic forms remain obscure. Our group had evidence about the existence of a low-penetrance susceptibility locus for sMTC in linkage disequilibrium with RET variants S836S/IVS1-126G>T, and probably in 5' with respect to both variants. In this study we sought to identify such locus. On the other hand, because an overrepresentation of G691S/S904S variants in patients with sMTC had been previously reported, we sought to determine if such association was present in our series. DESIGN: We performed a case-control study analysing a wide spectrum of RET variants in the 5' region of the gene, as well as the variants G691S/S904S. Haplotype distribution were also analyzed. A total of 58 patients with sMTC were included in the study. In addition, 100 unselected, unrelated race-, age-, and gender-matched normal controls were also evaluated. MAIN OUTCOME: Although the overrepresentation of IVS1-126G>T remains present in our current sMTC series, thus supporting our previous hypothesis, no differences were obtained among cases and controls in the distribution of the variants tested upstream this position. On the other hand, the frequency and distribution of G691S/S904S variants were similar in both groups of study, leading to exclude their role in sMTC in our series. CONCLUSIONS: These findings would suggest that the major genetic events contributing to the appearance of sMTC may reside in several different RET loci. In this way, we could hypothesize about the existence of at least two sMTC loci, linked to S836S-IVS1-126G>T, or to G691S-S904S, respectively.     

Polymorphisms in the RET proto-oncogene and the phenotypic presentation of familial medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) occurs in a sporadic or as an autosomal dominant hereditary form. Inherited forms of MTC are related to mutations in the RET proto-oncogene. We screened genomic DNA from 11 patients with MTC for mutations in exons 10, 11, 13, 14, 15, and 16 of the RET proto-oncogene. Subsequently, we also evaluated a family of 1 patient with presumed diagnosis of sporadic MTC. Three patients with MEN2A from two unrelated families presented mutations in exon 11 (C634Y and C634R). A heterozygous mutation in exon 14 (V804M) was identified in the patient with the presumed sporadic MTC. We also observed two different polymorphisms: S904S in exon 15 (2 patients) and L769L in exon 13 (4 patients). The L769L polymorphism has been associated with earlier onset of sporadic MTC. On the other hand, mutations in exon 14 are associated with MTC of later onset and lower aggressiveness. Indeed, the carrier of the V804M mutation associated with L769L polymorphism presented MTC at 32 years of age, in contrast to her asymptomatic mother, who had only the V804M mutation and had MTC diagnosed by fine-needle aspiration biopsy at 60 years of age. In conclusion, the present study confirms the need for genetic screening to differentiate sporadic and hereditary forms of MTC. In addition, the genetic study allows the identification of asymptomatic carriers of hereditary forms of MTC. Finally, we speculated that the L769L polymorphism of the RET proto-oncogene may be related to earlier age of onset in the patient with the V804M mutation.