Somatic and MEN 2A de novo mutations identified in the RET proto-oncogene by screening of sporadic MTC: s

Since germllne mutations in the RET proto-oncogene (RET) predisposingto tumor development In Familial Medullary Thyroid Carcinoma(FMTC), Multiple endocrine neoplasia type 2A (MEN 2A), and Multipleendocrine neoplasia type 2B (MEN 2B) were reported, It has becomepossible to identify gene carriers with a very high degree ofaccuracy. Mutations in FMTC and MEN 2A exclusively affect cystelneresidues in exon 10 and 11 of RET, whereas in MEN 2B codon 918in exon 16 is involved. This latter mutation has also been describedin a subset of apparently sporadic medullary thyroid carcinomas(MTC). Mutations in MEN 2B often occur as de novo germline mutations,whereas de novo mutations have not yet been described In FMTCor MEN 2A. We analyzed ten MTC: s and ten pheochromocytomas,all clinically judged to be sporadically occurring, by directDNA sequencing of exons 10, 11, and 16 of RET. This analysisrevealed a de novo germline mutation of codon 634 in exon 11In a patient with MTC. In addition, somatic mutations of codon918 in exon 16 in six of the remaining MTC: s were found, Interestingly,the presence of this somatic mutation was associated with asignificantly less favorable clinical outcome.     

27-bp Deletion in the ret proto-oncogene as a somatic mutation associated with medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) occurs as a sporadic tumor or in connection with the inherited cancer syndromes of multiple endocrine neoplasia (MEN) types 2A and 2B and familial MTC. Missense RET proto-oncogene mutations of one of cysteine codons in exons 10 and 11 are found in the majority of families with MEN 2A and or familial MTC. In MEN 2B, mutations at codon 918, exon 16, have been identified in most of the affected individuals. In a significant amount of sporadic MTC somatic codon 918 mutations appear. In addition to these, a 6-bp deletion including codon 630 and a 24-bp deletion including codon 634 combined with a 6-bp insertion have been observed. We report on a 27-bp deletion in exon 10 as a somatic mutation associated with a sporadic medullary thyroid carcinoma. Received: 15 September 1997 / Accepted: 19 December 1997     

Mutation of the RET protooncogene in sporadic medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) occurs sporadically or as part of the inherited cancer syndrome multiple endocrine neoplasia (MEN) type 2. In MEN 2A, germline missense mutations are found in one of five cysteine codons within exons 10 and 11 in the extracellular domain of the RET protooncogene. In MEN 2B, germline mutations occur in codon 918 (exon 16) within the catalytic core of the tyrosine kinase domain. To determine if RET mutations similar to those in MEN 2A and 2B play a role in the pathogenesis of sporadic MTC, we analysed 71 sporadic tumours comprising 68 primary tumours and three cell lines, for mutations in RET exons 10, 11, and 16. We found that 23% of sporadic MTC had RET codon 918 mutations, while only 3% had exon 10 mutations, and none had mutations in exon 11. We found no exon 16 mutations in MTC from 14 MEN 2A cases. Thus, exon 10 and 11 mutations, commonly found in familial MTC and MEN 2A, rarely occur in sporadic MTC; somatic mutation of RET codon 918 appears to play a role in the tumourigenesis of a significant minority of sporadic MTC but not MEN 2A tumours. In addition to their biological interest, these findings may have some clinical application in determining whether a patient presenting with isolated MTC is truly sporadic or is part of an inherited cancer syndrome.     

The newly detected mutations in the<Emphasis Type="Italic"> RET</Emphasis> proto-oncogene in exon 16 as a cause of sporadic medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) occurs as a sporadic form or, less frequently, as an autosomal dominant inherited familial disorder. Germline mutations in the RET proto-oncogene in exons 10, 11, 13, 14, 15, and 16 are found in most of the familial cases (nearly 95%). Somatic mutations in sporadic MTC are detected in 23–69% of patients. The most frequent somatic mutation is located in exon 16 at codon 918, and only a small percentage of mutations are found in exons 10, 11, 13, and 15. We have searched for somatic mutations in Czech MTC patients using direct sequencing. We report here two new somatic missense mutations in exon 16 of the RET proto-oncogene associated with the sporadic MTC detected in two Czech men. A homozygous mutation at codon 922 TCC(Ser)CCC(Pro) as a result of loss of heterozygosity was revealed in the first patient. In the second one a heterozygous mutation at codon 930 ACG(Thr)ATG(Met) was found.Abbreviations MEN Multiple endocrine neoplasia - MTC Medullary thyroid carcinoma - PCR Polymerase chain reaction     

RET mutation status in medullary thyroid cancer(MTC) patients and the significance of genetic screening for mutations in their immediate relatives--a preliminary report

Multiple Endocrine Neoplasia (MEN) 2A is an inherited disease characterized by the development of medullary thyroid carcinoma (MTC), pheochromocytoma(PHCH) and hyperparathyroidism(HPT). It has recently been shown to be associated with germline mutations in the RET proto-oncogene. Genetic testing for RET mutations will, therefore allow the identification of people with asymptomatic MEN 2 who can be offered prophylactic thyroidectomy and biochemical screening as preventive measures. No genetic study based on RET mutation detection has been available in India so far. The aim of the present study is to detect the proportion of MTC cases having inherited germline or somatic RET mutations and to identify family members at risk for MEN and, thereby the feasibility of screening for MEN. DNA extracted from the peripheral blood and somatic (tumor) tissues were subjected to PCR using primers for exons 10,11 and 16. A few samples were subjected to direct sequencing. Germline mutations were identified in 3 of 4 MEN 2A patients, 18 of 24 sporadic MTC(SMTC), 2 of 4 children of MEN2A and 8 relatives of SMTC. Common mutation was in exon 10 and 11 (c634). It is recommended that RET mutation analysis and counseling of patients and their immediate relatives be introduced on a regular basis to identify gene carriers.     

Mutation of RET codon 768 is associated with the FMTC phenotype

Multiple endocrine neoplasia type 2A (MEN 2A), type 2B (MEN 2B), and familial medullary thyroid carcinoma (FMTC) are inherited cancer syndromes resulting from mutations in the RET proto-oncogene. Missense mutations of five codons in exons 10 and 11 are found in both MEN 2A and FMTC families, while mutations at codon 768 in exon 13 have been identified in three FMTC families. We report here the results of mutation analysis on a large multi-generation family with multiple cases of medullary thyroid carcinoma (MTC) or C-cell hyperplasia and two individuals with isolated adrenal medullary hyperplasia. A mutation in exon 13, which alters codon 768 from a GAG (Glu) to a GAC (Asp), was found to segregate with the FMTC phenotype in this family but not with the adrenal medullary hyperplasia. These findings suggest that the codon 768 mutation does not predispose to adrenal medullary hyperplasia, but is an accurate predictor of the MTC phenotype in this family.     

Detection of RET proto-oncogene point mutations in paraffin-embedded pheochromocytoma specimens by nonradioactive single-strand conformation polymorphism analysis and direct sequencing.

The suitability of formalin-fixed and paraffin-embedded tumor material was evaluated for molecular analysis of the RET proto-oncogene. We analyzed exons 10, 11, and 16 for point mutations in seven sporadic and six multiple endocrine neoplasia (MEN) 2A-associated pheochromocytomas by a nonradioactive single-strand conformation polymorphism assay followed by nonradioactive direct sequencing of PCR-amplified DNA using an automated DNA sequencer. All MEN 2A-associated pheochromocytomas contained a heterozygous missense germline mutation within cystine codons of the cysteine-rich extracellular domain encoded by exons 10 and 11. Mutations were located in codon 619 (TGC-->TCC; Cys-->Ser) in one, in codon 635 (TGC-->CGC; Cys--Arg) in three, and in codon 635 (TGC-->TAC; Cys-->Tyr) in two pheochromocytomas. No tumor-specific (somatic) mutations were detected in exons 10, 11, and 16 of the sporadic pheochromocytomas. These data support recent findings that germline point mutations that are clustered in distinct cysteine codons of the RET proto-oncogene are involved in the neoplastic phenotype of the MEN 2A syndrome. Our results demonstrate that both nonradioactive single-strand conformation polymorphism and direct sequencing are suitable methods to detect single base substitutions in DNA extracted from archival material.     

Genetic aberrance of sporadic MEN 2A component tumours: analysis of RET

AIM: The molecular pathogenesis of familial multiple endocrine neoplasia (MEN) type 2 (parathyroid adenoma with medullary thyroid carcinoma and adrenal pheochromocytoma) is associated with a germ-line mutation in the RET proto-oncogene. We undertook this study to clarify the relationship between the tumorigenesis of apparently sporadic MEN type 2 component endocrine tumours and RET mutations. METHODS: Direct sequencing for RET exon 10, 11, 12, 13, 14, 15 and 16 and immunohistochemistry for RET monoclonal antibody were performed on the archival tissues of 84 cases of sporadic endocrine tumours, including 22 medullary thyroid carcinomas (MTCs), 35 adrenal pheochromocytomas (APCs), 18 paragangliomas (PGs), and nine parathyroid adenomas (PTAs). RESULTS: PCR-based direct sequencing revealed somatic point missense mutation within 22.7% of exon 13 of the RET proto-oncogene (four cases of E768D, one case of S7781) in MTCs. No RET genotype and morphological association was observed in MTCs or APCs. APCs revealed significantly lower levels of immunoexpression of RET, even versus PGs. CONCLUSIONS: The genetic mutation in RET is relatively low in incidence, and likely to play an insignificant role in the molecular pathogenesis of sporadic MTC. The molecular bases of PG and APC seem to be different despite their embryological and histological similarities.     

Mutations in the RET proto-oncogene and the von Hippel-Lindau disease tumour suppressor gene in sporadic and syndromic phaeochromocytomas.

Phaeochromocytomas may occur sporadically, or as part of the inherited cancer syndromes multiple endocrine neoplasia (MEN) type 2, von Hippel-Lindau disease (VHL), and, rarely, in type 1 neurofibromatosis. In MEN 2, germline missense mutations have been found in one of eight codons within exons 10, 11, 13, 14, and 16 of the RET proto-oncogene. In VHL, germline mutations within one of the three exons are responsible for the majority of cases. To determine if somatic mutations similar to those seen in the germline in MEN 2 or VHL disease play a role in the pathogenesis of sporadic or familial phaeochromocytomas, we analysed 48 sporadic tumours and tumours from 17 MEN 2 and five VHL patients for mutations in RET exons 9, 10, 11, 13, 14, 15, and 16, and the entire coding sequence of VHL. Five of 48 sporadic phaeochromocytomas had RET mutations within exons 10, 11, and 16. Of these, one was proven to be germline and two were proven to be somatic mutations. Four of 48 had VHL mutations; these included both the bilateral cases in the series (one was proven to be a germline mutation) and two others, of which one was proven somatic.     

Segregation of the V804L mutation and S836S polymorphism of exon 14 of the RET gene in an extended kindred with familial medullary thyroid cancer

In multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid cancer (FMTC), the majority of germline mutations are restricted to specific positions in exons 10 and 11 of the RET gene. However, germline mutations may very occasionally occur in other exons, including exon 14 of the RET gene. Interestingly, an increased frequency of a rare germline sequence variant of the RET exon 14, S836S, has been detected in patients with sporadic medullary thyroid cancer (MTC), and this variant has been proposed to play a role in the genesis of MTC and, perhaps, FMTC. In this study we report the segregation of a germline V804L mutation and a germline sequence variant S836S in exon 14 of the RET gene in an extended Hungarian FMTC kindred comprising 80 individuals of four generations. Molecular analysis of the RET gene was performed by direct DNA sequencing in 23 family members, of whom 12 had the V804L mutation, three had the V804L mutation and S836S polymorphism in separate alleles, and six had the S836S polymorphism, all in heterozygous forms. Two of the family members had neither mutation nor polymorphism of the RET gene. Three of the family members who had the V804L mutation and one member who could not be tested for mutation were operated for non-metastatic MTC, while one member with MTC who had the V804L mutation refused surgery. In all patients affected with MTC, the disease developed relatively late in life and never caused death. None of the other family members carrying the V804L mutation and/or the S836S polymorphism had clinical or biochemical evidence of MTC. These observations suggest that the co-existence of the V804L mutation and S836S polymorphism in separate alleles does not seem to aggravate the relatively low-risk disease phenotype characteristic in most patients with codon 804 mutations of the RET exon 14.     

Risk profiles and penetrance estimations in multiple endocrine neoplasia type 2A caused by germline RET mutations located in exon 10

Multiple endocrine neoplasia type 2 is characterized by germline mutations in RET. For exon 10, comprehensive molecular and corresponding phenotypic data are scarce. The International RET Exon 10 Consortium, comprising 27 centers from 15 countries, analyzed patients with RET exon 10 mutations for clinical-risk profiles. Presentation, age-dependent penetrance, and stage at presentation of medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism were studied. A total of 340 subjects from 103 families, age 4-86, were registered. There were 21 distinct single nucleotide germline mutations located in codons 609 (45 subjects), 611 (50), 618 (94), and 620 (151). MTC was present in 263 registrants, pheochromocytoma in 54, and hyperparathyroidism in 8 subjects. Of the patients with MTC, 53% were detected when asymptomatic, and among those with pheochromocytoma, 54%. Penetrance for MTC was 4% by age 10, 25% by 25, and 80% by 50. Codon-associated penetrance by age 50 ranged from 60% (codon 611) to 86% (620). More advanced stage and increasing risk of metastases correlated with mutation in codon position (609→620) near the juxtamembrane domain. Our data provide rigorous bases for timing of premorbid diagnosis and personalized treatment/prophylactic procedure decisions depending on specific RET exon 10 codons affected.     

散发性嗜铬细胞瘤患者RET原癌基因突变检测

目的在嗜铬细胞瘤散发患者中进行RET原癌基因突变筛查。方法收集42例病理诊断确诊为嗜铬细胞瘤的患者基因组DNA，其中12例为外周血基因组DNA，30例为嗜铬细胞瘤病理切片组织中提取的基因组DNA。对RET原癌基因第10和第11外显子，采用DNA测序技术进行基因突变筛查。结果在42例中，2例在RET基因的第11外显子存在基因突变。1例634位密码子由TGC突变为TAC，另1例632位密码子由GAG突变为AAG。结论在嗜铬细胞瘤患者中存在RET原癌基因突变携带者，有必要对散发的嗜铬细胞瘤患者进行RET原癌基因的常规突变筛查。     

Novel germline RET mutation segregating with papillary thyroid carcinomas.

The RET proto-oncogene is responsible for inherited medullary thyroid cancer syndromes. RET is also found mutated in sporadic medullary thyroid cancer (MTC) and rearranged in sporadic papillary thyroid carcinomas. Here, we describe a previously unreported germline RET mutation at codon 603 in exon 10 associated with both MTC and nonmedullary thyroid cancer (NMTC) in a kindred. RET may thus not be excluded as a potential candidate for predisposition to some forms of NMTC.     

Molecular Diagnosis of Multiple Endocrine Neoplasia (MEN) in Paraffin-Embedded Specimens

In this article, we summarize our recent findings on rearranged during transfection (RET) mutations in a series of 46 sporadic as well as multiple endocrine neoplasia (MEN) type 2-associated tumors and present results of our family screening efforts to identify MEN 2 and MEN 1 gene carriers. A nonisotopic polymerase chain reaction-based single-strand conformation polymorphism (PCR-SSCP) analysis and heteroduplex gel electrophoresis method was used to screen DNA extracted from archival specimens of 22 patients with MEN 2-associated and 24 patients with sporadic tumors for mutations inRET exons 10, 11, 13, and 16. Point mutations were identified by nonisotopic cycle sequencing of PCR products using an automated DNA sequencer. We found six different missense germline mutations at cysteine residues encoded by exons 10 and 11 in all patients with MEN 2A or familial medullary thyroid carcinoma (FMTC). The frequency of mutations at codon 634 was higher in patients with MEN 2A than with FMTC and a₆₃₄ Cys→Arg mutation was associated with parathyroid disease. A germline Met→Thr point mutation at codon 918 of theRET tyrosine kinase domain encoded by exon 16 was identified in all MEN 2B patients. Nonpredicted inheritable medullary thyroid carcinomas (MTCs) were detected in two patients and a mosaic postzygotic mutation was found in one additional patient. Tumor-specific (somatic) Met→Thr point mutations at codon 918 were identified in 5 of 13 sporadic MTCs and 2 of 8 sporadic pheochromocytomas (PCCs). The remaining sporadic tumors lacked mutations in all fourRET exons tested. In exon 13, a nucleic acid polymorphism (CTT/CTG; Leu) at codon 769 was identified, which is present in approx 40% of the examined population. Our study demonstrates that the molecular methods used are not only suitable to identify asymptomatic individuals at risk for MEN 2A, FMTC, and MEN 2B, but also to distinguish sporadic from inherited tumors using archival tissue specimens; and that more tumors than clinically expected are inheritable, indicating the need for genetic analysis of all MTC and PCC patients.     

A Korean Family of Familial Medullary Thyroid Cancer with Cys618Ser RET Germline Mutation

Familial medullary thyroid carcinoma (FMTC) is caused by autosomal dominant gain-of-function mutations in the RET proto-oncogene. An identifiable RET mutation can be detected in about 85% of FMTC families. The majority of germline mutations in FMTC have been found in exons 10 and 11 of the RET proto-oncogene, specifically within the cysteine codons 609, 611, 618, 620, and 634. We screened members of a large Korean family that had a history of FMTC by genetic analyses, and propose a therapeutic approach for managing the disorder. We report a RET mutation in exon10, codon 618 that causes substitution of a cysteine by a serine in the cysteine-rich domain of the RET receptor in a three-generation FMTC family composed of 30 members with 11 carriers. Nine of the gene carriers were clinically affected. The FMTC with cysteine RET mutations found in the Korean population is consistent with the clinical pattern reported worldwide; to date there have been no ethnic differences identified for FMTC. Our results suggest that this genetic profile might be associated with usually aggressive clinical course with regional lymph node metastasis but late onset of MTC.     

Absence of RET proto-oncogene point mutations in sporadic hyperplastic and neoplastic lesions of the parathyroid gland.

We investigated the possible role of RET proto-oncogene mutations in the development of sporadic hyperplastic, benign, and malignant parathyroid lesions. DNA extracted from paraffin-embedded specimens of forty parathyroid lesions was screened for RET proto-oncogene point mutations in exons 10, 11, and 16 by nonisotopic polymerase chain reaction-based single-strand conformation polymorphism and heteroduplex gel electrophoresis. The nucleotide sequence of samples with aberrant band patterns was identified by nonisotopic direct sequencing of polymerase chain reaction-amplified DNA. Parathyroids of seven patients with multiple endocrine neoplasia type 2A (MEN 2A) and MEN 2B served as positive controls. None of the eight hyperplastic lesions, three cases of parathyromatosis, ten parathyroid adenomas, eleven carcinomas or one normal parathyroid gland contained mutations in each of the three RET exons tested. Six MEN-2A-associated hyperplastic glands exhibited identical band shifts in the polymerase chain reaction single-strand conformation polymorphism analysis of exon 11, which corresponded to a Cys 634-->Arg substitution in the nucleotide sequence analysis (TGC-->CGC), whereas in the MEN 2B parathyroid specimen a point mutation was found at codon 918 of exon 16 (ATG-->ACG), causing a Met 918-->Thr substitution. Our data indicate that RET mutations of the MEN 2 loci in exons 10, 11, and 16 are not involved in the development of sporadically occurring benign or malignant parathyroid lesions. Furthermore, our results are in accordance with the observation that MEN 2A patients with Cys 634-->Arg (germline) mutations have a higher risk of developing parathyroid disease than those with other mutations at codon 634.     

Mutational analysis of the RET proto-oncogene in 71 Japanese patients with medullary thyroid carcinoma

Multiple endocrine neoplasia types 2A and 2B (MEN2A and MEN2B) and familial medullary thyroid carcinomas (FMTC) are caused by germline mutations in the RET proto-oncogene. To investigate the spectrum of RET mutations among Japanese patients, we screened the RET gene in 71 patients with thyroid carcinomas. The panel included representatives of 44 families carrying FMTC or MEN2, 22 sporadic medullary thyroid carcinomas (MTCs), and five MTCs without familial information. Mutations in nucleotide sequences encoding one of three specific cysteine residues in the extracellular domain of the RET protein were found in 33 of the 34 MEN2A patients and in five of the six FMTC patients examined. A mutation at codon 918, causing the substitution of threonine for methionine in the tyrosine kinase domain of the protein, was found in germline DNAs of all four patients with MEN2B and in two of the 22 patients with sporadic MTCs; codon 918 was mutated somatically in tumor DNAs from three other sporadic cases. Germline mutations of codon 768, GAG to GAC (Glu to Asp), were detected in one FMTC, in one patient with sporadic MTC, and in one of the patients without familial information. Two somatic mutations, an Asp to Gly substitution at codon 631 and a Cys to Arg substitution at codon 634, had not been reported previously. Of five germline mutations found among the 22 sporadic cases, four were confirmed as de novo mutations since in each case neither parent carried the mutation. As nearly one-fourth of the patients with sporadic MTCs carried germline mutations and 50% of their children are expected to develop MTC and other endocrine tumors, these results indicated the importance of careful clinical surveillance of family members of any patient with MTC. Received: August 22, 1997 / Accepted: October 22, 1997     

The distribution of constitutional and somatic mutations in the neurofibromatosis 2 gene

Constitutional heterozygous inactivating mutations in the neurofibromatosis 2 (NF2) tumor suppressor gene cause the autosomal dominant disease NF2, and biallelic inactivating somatic NF2 mutations are found in a high proportion of unilateral sporadic vestibular schwannoma (USVS) and sporadic meningioma. We surveyed the distributions of constitutional NF2 mutations in 823 NF2 families, 278 somatic NF2 mutations in USVS, and 208 somatic NF2 mutations in sporadic meningioma. Based on the available NF2 mutation data, the most dominant influence on the spectra of mutations in exons 1-15 are C>T transitions that change arginine codons (CGA) to stop codons (TGA) due to spontaneous deamination of methylcytosine to thymine in CpG dinucleotides. The paucity of reported mutations in exon 9 and the absence of reported mutations in exons 16 and 17 may be related to structure-function relationships in the NF2 protein.     

Detection of Molecular Alterations in Medullary Thyroid Carcinoma Using Next-Generation Sequencing: an Institutional Experience

Medullary thyroid carcinoma (MTC) harbors rearranged during transfection (RET) gene and rarely RAS gene mutations. The knowledge of the type of gene mutation in MTC is important to determine the treatment of the patients and the management of their family members. Targeted next-generation sequencing with a panel of 47 genes was performed in a total of 12 cases of sporadic (9/12) and hereditary MTC (3/12). Two of three hereditary MTCs had RET/C634R mutation, while the other one harbored two RET mutations (L790F and S649L). All the sporadic MTC had RET/M918T mutation except one case with HRAS mutation. Next-generation sequencing (NGS) can provide comprehensive analysis of molecular alterations in MTC in a routine clinical setting, which facilitate the management of the patient and the family members.     

Gastrointestinal stromal tumors with exon 8 c-kit gene mutation might occur at extragastric sites and have metastasis-prone nature

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the human gut. Most sporadic GISTs have somatic gain-of-function mutations of the c-kit gene. The mutations are frequently found at exon 11, sometimes at exon 9 and rarely at exon 13 or 17. Recently, exon 8 c-kit gene mutations were reported in very minor proportion of sporadic GISTs. We also found 3 GISTs with exon 8 c-kit gene mutations in approximately 1,000 sporadic GISTs examined. In the present report, we showed the clinicopathological data of those GISTs. One case had a deletion of codon 419 of aspartate, and 2 cases had a substitution of 3 amino acids of codon 417 to codon 419 to tyrosine. The former was the same mutation recently reported in 2 GIST cases, but the latter has not been reported in any GISTs. All three cases occurred at extragastric sites and two of three showed distant metastasis. Since the remaining case was regarded as high risk for recurrence, imatinib adjuvant treatment has been done without evidence of metastasis. Our results confirmed the idea that exon 8 mutations are minor but actually existing abnormalities in sporadic GISTs, and suggested that such GISTs have a feature of extragastric development and a metastasis-prone nature. Since the exon 8 mutations appeared to be really sensitive to imatinib as shown in the present case study, accurate genotyping including exon 8 of the c-kit gene is necessary in GISTs to predict response to imatinib in both the unresectable/metastatic and adjuvant settings.