Novel Point Mutations and Allele Loss at the RET Locus in Sporadic Medullary Thyroid Carcinomas

Germline mutations in the RET proto-oncogene have been shown to be the underlying cause of multiple endocrine neoplasia type 2 (MEN 2A and 2B) and familial medullary thyroid carcinoma (FMTC). Some cases of sporadic medullary thyroid carcinoma (sporadic MTC) are reported to have specific codon 918, 883 and 768 mutations of the RET gene in tumor tissues. We examined RET gene mutations in 40 Japanese cases who had previously undergone surgery for sporadic MTC. DNA extracted from formalin-fixed tumor tissues and corresponding normal thyroid tissues or peripheral blood leukocytes was analyzed for mutations of exon 10, 11, 13, 14 and 16 of the RET gene by DNA sequencing and by mutation-specific restriction enzyme analysis. Germline RET point mutations were found in six of 40 cases (15%), cysteine residues at codon 618 in two, codon 634 in three and valine residue at codon 804 in one, and were newly identified as heritable MTC. Of the remaining 34 sporadic MTC cases, four (12%) had tumor-specific RET point mutations. Two were found in exon 16; one case showed an ATG to ACG (Met to Thr) mutation at codon 918, and the other showed two point mutations, ATG to ACG (Met to Thr) at codon 918 and GCA to GTA (Ala to Val) at codon 919 with loss of the wild-type allele, suggesting that both alleles at the RET locus were altered. The other two were found in exon 13; one case showed a CCG to TCG (Pro to Ser) mutation at codon 766 and the other showed a silent mutation, GTC to GTT (Val) at codon 778 with loss of the wild-type allele. There was no association of sporadic mutations with recurrence or prognosis in patients with sporadic MTCs. The low rate of somatic RET mutation at codon 918 in our sporadic MTC suggests that as yet unknown factors may be involved. Genetic alterations in both alleles may have an important role in a small fraction of sporadic MTCs.     

Somatic Mutations in RET Exons 12 and 15 in Sporadic Medullary Thyroid Carcinomas: Different Spectrum of Mutations in Sporadic Type from Hereditary Type

Germline mutations in the RET proto-oncogene are responsible for multiple endocrine neoplasia type 2 (MEN 2A and 2B) and familial medullary thyroid carcinoma (FMTC). Point mutations or in-frame deletions of exons 10, 11, 13, 14 and 16 are associated with sporadic medullary thyroid carcinoma (MTC). To understand further the role of the RET gene in sporadic MTC, we examined mutations in exons 12 and 15 of RET in patients with sporadic MTC. DNAs were extracted from 39 formalin-fixed tumor tissues and corresponding normal thyroid tissues or peripheral blood leukocytes. DNA sequencing was used to identify mutations in exons 12 and 15 of RET. In this study, one novel somatic mutation was found in exon 12 and five novel mutations or deletions were found in exon 15. Of the patients with mutations, one had an in-frame 12-bp deletion (nt. 2625-2636), one had point mutations in both codons 884 and 908, and the remaining three had point mutations in codons 748, 876 and 901, respectively. Together with our previous identification of somatic mutations in exons 10, 11, 13, 14 and 16, somatic alterations were found in 10 out of 39 (25.6%) sporadic MTCs. There was no association of RET gene mutations with tumor recurrence or prognosis. These results suggest that mutations occur frequently in the RET coding region in addition to the previously reported mutation hot spots, and there is a different spectrum of mutations between sporadic and hereditary MTC.     

Large-scale Analysis of Mutations in RET Exon 16 in Sporadic Medullary Thyroid Carcinomas in Japan

Germline mutations in the RET proto-oncogene are the cause of multiple endocrine neoplasia type 2 (MEN 2A and 2B) and familial medullary thyroid carcinoma (FMTC). Some cases of sporadic medullary thyroid carcinoma (MTC) have also been reported to have mutations in the RET gene. However, two previous reports have given discrepant results on the frequency of the mutations in RET in sporadic MTCs in Japan. To clarify this problem, we analyzed mutations in RET exon 16 in 72 sporadic MTCs by means of the two methods used in the previous studies, direct sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Mutations in exon 16 were detected in only 2 of 72 cases of sporadic MTC. These results suggest that when a MTC has a mutation in RET exon 16, it is more likely to be a hereditary MTC than a sporadic one in Japan.     

A Novel Somatic Mutation in the RET Proto-oncogene in Familial Medullary Thyroid Carcinoma with a Germline Codon 768 Mutation

In individuals who carry gcrmline mutations in tumor suppressor genes predisposing them to inherited cancer syndromes, occurrence of somatic mutations in the same genes contributes to tumorigenesis. Germline mutations in the RET proto-oncogene predispose individuals to multiple endocrine neoplasia (MEN) type 2 syndromes. Since these mutations are oncogenic by themselves, somatic mutations in the same gene had been thought unnecessary. Recently, a somatic mutation at codon 918 of RET was reported in medullary thyroid carcinoma (MTC) and C-cell hyperplasia in patients with MEN 2A or familial MTC (FMTC), suggesting its possible contribution to tumorigenesis. We describe here a novel somatic mutation at codon 919 in a patient with FMTC carrying a gcrmline mutation at codon 768 that may also be related to tumor progression.     

RET Proto Oncogene Mutation Detection and Medullary Thyroid Carcinoma Prevention

Thyroid cancer is the most common endocrine neoplasia. The medullary thyroid carcinoma (MTC) is oneof the most aggressive forms of thyroid malignancy,accounting for up to 10% of all types of this disease. Themode of inheritance of MTC is autosomal dominantly and gain of function mutations in the RET proto-oncogeneare well known to contribute to its development. MTC occurs as hereditary (25%) and sporadic (75%) forms.Hereditary MTC has syndromic (multiple endocrine neoplasia type 2A, B; MEN2A, MEN2B) and non-syndromic(Familial MTC, FMTC) types. Over the last two decades, elucidation of the genetic basis of tumorigenesis hasprovided useful screening tools for affected families. Advances in genetic screening of the RET have enabled earlydetection of hereditary MTCs and prophylactic thyroidectomy for relatives who may not show any symptomsof the disease. In this review we emphasize the main RET mutations in syndromic and non syndromic formsof MTC, and focus on the importance of RET genetic screening for early diagnosis and management of MTCpatients, based on American Thyroid Association guidelines and genotype-phenotype correlation.     

Plasma Calcitonin Levels and miRNA323 Expression in Medullary Thyroid Carcinoma Patients with or without RET Mutation

Background: Medullary thyroid cancer (MTC) is an endocrine tumor featuring parafollicular or C-cell differentiation, with calcitonin as a specific biomarker in MTC diagnosis. Germline mutations in the RET proto-oncogene are considered responsible for its familial occurrence and somatic mutations can cause sporadic lesions. MicroRNAs can act as oncogenes or tumor suppressors by inhibiting the expression of target genes.. The aim of this study was to investigate relationships between plasma levels of calcitonin and miRNA323 expression in MTC patients with or without RET mutation. Methods: In this cross-sectional study, MTC lesions (based on pathological confirmation) were investigated. Genomic DNA was extracted and Exons 10 and 11 of RET were genotyped using PCR-sequencing. Division was into two groups of 43 cases each with or without mutation. Plasma levels of calcitonin were determined in both. Results: miRNA323 was measured using real-time-PCR. After performing normality tests, independent T-tests and Mann Whitney tests were used for the statistical comparison of parametric and nonparametric data, respectively. Plasma levels of calcitonin were significantly higher in MTC cases without a RET mutation compared to those with a mutation. Conclusion: There was no significant difference between the two groups regarding the expression of miRNA323 so that this parameter could not be used as a bio-index germ line mutations in MTCs. However, determination of calcitonin levels in plasma might be helpful in this regard.     

罕见RET 原癌基因Y606C 突变所致家族性甲状腺髓样癌家系临床特点分析*

目的：分析讨论RET 原癌基因Y 606C 这一罕见突变所致家族性甲状腺髓样癌（familial medullary thyroid carcinoma ,FMTC）的临床生物学特点。方法：对先期确诊的1 个携带RET 原癌基因Y 606C 种系突变的FMTC 家系成员进行RET 基因种系突变筛查,同时行血清降钙素、甲状旁腺素、颈腹部超声等相关临床检查,对患病成员和携带者给予临床干预,并总结分析上述病例临床生物学特点。结果：包括先证者在内,该家系中10例参与基因筛查,发现6 例携带RET 基因第10外显子Y 606C 突变。其中3 例确诊为FMTC 患者（含先证者）,均为女性,平均发病年龄50.0（47～53）岁。发病成员临床均表现为甲状腺肿物伴降钙素升高,未发现甲状旁腺和肾上腺病变。2 例行全甲状腺切除术,1 例行患侧腺叶切除术。术后病理均确诊为甲状腺髓样癌,其中2 例病理分期均属早期,术后随访期间均达生化治愈。家系中另有3 例为突变携带者,平均年龄33.0（15～55）岁。其中2 例颈部超声均提示甲状腺肿物（考虑良性）,另1 例颈部超声未见异常。除1 例拒绝降钙素检测外,其余2 例血清降钙素均正常范围,建议定期复查。结论：RET 原癌基因Y 606C 突变可以导致FMTC 发病,家系成员发病平均年龄较大,肿瘤分期较早,生化治愈率高,预后较好;家系成员基因检测结合颈部超声和降钙素检查有利于甲状腺髓样癌的早期诊断;对于无症状Y 606C 突变携带者,应根据其降钙素水平及颈部超声检查情况实施个体化临床处置。     

YAP蛋白在甲状腺髓样癌组织中的表达

目的：探讨YAP蛋白在甲状腺髓样癌（ MTC）发生发展中的作用及临床意义。方法采用免疫组织化学方法检测25例MTC、25例正常甲状腺组织中YAP蛋白的表达。结果25例MTC组织中YAP蛋白阳性表达率为88.00%（22/25）,而25例正常甲状腺组织中YAP蛋白表达均为阴性,比较差异有统计学意义（χ2=39.286,P〈0.05）。结论 YAP蛋白在MTC组织中呈明显过表达,在MTC的发生发展中可能起着重要作用,且有望成为具有潜在应用价值的分子标记及治疗靶点。     

甲状腺髓样癌诊治分析

对３８例甲状腺髓样癌的外科治疗及预后因素作初步分析。在手术方式的比较中，一侧腺叶和峡部切除与近全叶切除两种术式比较；功能性颈清术与根治性颈清术比较，两组术后复发率差异均无显著性。本组５年和１０年生存率分别为７８．９％（１５／１９）和６２．５％（５／８）。影响预后的主要因素是原发癌侵犯程度及患者年龄。腺内型及４０岁以下者预后较好。     

Characterization of Wild-Type and Mutated RET Proto-Oncogene Associated with Familial Medullary Thyroid Cancer

Background: We aimed to assess RET proto-oncogene polymorphisms in three different Iranian familieswith medullary thyroid cancer (MTC), and performed molecular dynamics simulations and free energy stabilityanalysis of these mutations. Materials and Methods: This study consisted of 48 patients and their first-degreerelatives with MTC confirmed by pathologic diagnosis and surgery. We performed molecular dynamics simulationsand free energy stability analysis of mutations, and docking evaluation of known RET proto-oncogene inhibitors,including ZD-6474 and ponatinib, with wild-type and mutant forms. Results: The first family consisted of 27people from four generations, in which nine had the C.G2901A (P.C634Y) mutation; the second family consistedof six people, of whom three had the C.G2901T (P.C634F) mutation, and the third family, who included 12individuals from three generations, three having the C.G2251A (P.G691S) mutation. The automated 3D structureof RET protein was predicted using I-TASSER, and validated by various protein model verification programsthat showed more than 96.3% of the residues in favored and allowed regions. The predicted instability indicesof the mutated structures were greater than 40, which reveals that mutated RET protein is less thermo-stablecompared to the wild-type form (35.4). Conclusions: Simultaneous study of the cancer mutations using both insilico and medical genetic procedures, as well as onco-protein inhibitor binding considering mutation-induceddrug resistance, may help in better overcoming chemotherapy resistance and designing innovative drugs.     

High Affinity Pharmacological Profiling of Dual Inhibitors Targeting RET and VEGFR2 in Inhibition of Kinase and Angiogeneis Events in Medullary Thyroid Carcinoma

Clinical evidence shows that dual inhibition of kinases as well angiogenesis provides ideal therapeutic optionin the treatment of medullary thyroid carcinoma (MTC) than inhibiting either of these with the events separately.Although treatment with dual inhibitors has shown good clinical responses in patients with MTC, it has beenassociated with serious side effects. Some inhibitors are active agents for both angiogenesis or kinase activity.Owing to narrow therapeutic window of established inhibitors, the present study aims to identify high affinitydual inhibitors targeting RET and VEGFR2 respectively for kinase and angiogenesis activity. Establishedinhibitors like Vandetanib, Cabozantinib, Motesanib, PP121, RAF265 and Sunitinib served as query parentcompounds for identification of structurally similar compounds by Tanimoto-based similarity searching with athreshold of 95% against the PubChem database. All the parent inhibitors and respective similar compoundswere docked against RET and VEGFR2 in order to retrieve high affinity compounds with these two proteins.AGN-PC-0CUK9P PubCID: 59320403 a compound related to PPI21 showed almost equal affinity for RET andVEGFR2 and unlike other screened compounds with no apparent bias for either of the receptors. Further, AGNPC-0CUK9P demonstrated appreciable interaction with both RET and VEGFR2 and superior kinase activity inaddition to showed optimal ADMET properties and pharmacophore features. From our in silico investigationwe suggest AGN-PC-0CUK9P as a superior dual inhibitor targeting RET and VEGFR2 with high efficacy whichshould be proposed for pharmacodynamic and pharmacokinetic studies for improved treatment of MTC.     

家族型甲状腺髓样癌家系及临床分析

[目的]探讨家族性甲状腺髓样癌（FMTC）的家系及临床特点。[方法]总结我院自1954年至2000年收治的一组10例家族性甲状腺髓样癌病人。[结果]在147例所收治的甲状腺髓样癌（MTC）中6.8%为FMTC,病人平均年龄为29.8岁,6例（60％）为双侧甲状腺MTC。10例病人来自6个家族,每个亲代不是完全发病甚至晚于子代发病。[结论]FMTC为常染色体显性遗传病,其结果显示FMTC家族中基因携带者不一定发展为MTC。测定血清降钙素水平可作为筛选FMTC家族成员中无临床症状MTC者的一种方法。     

RET mutation p.S891A in a Chinese family with familial medullary thyroid carcinoma and associated cutaneous amyloidosis binding OSMR variant p.G513D

There are no reports on the relationship between familial medullary thyroid carcinoma (FMTC) associated with cutaneous amyloidosis (CA) and RET or OSMR/IL31RA gene mutations. In this study, we investigated a Chinese family with FMTC/CA and found a recurrent RET c.2671T>G (p.S891A) mutation in six of 17 family members. Three of the six p.S891A mutation carriers presented with medullary thyroid carcinoma (MTC). Of them, three (two with and one without MTC) were diagnosed as having combined lichen/macular biphasic CA. We also identified a novel RET variant, c.1573C>T (p.R525W) in five members. Of them, three carriers had no evidence of thyroid/skin or basal serum/stimulated calcitonin abnormalities. In vitro cell proliferation assay indicated that oncogenic activity of RET p.S891A was slightly enhanced by p.R525W, whereas p.R525W alone had no effect on cell proliferation. Meanwhile, we identified a novel OSMR variant, c.1538G>A (p.G513D) in seven members. We noticed that three OSMR p.G513D carriers presenting with CA also had the RET p.S891A mutation. Our investigation indicated that the RET p.S891A mutation combined with OSMR p.G513D may underlie a novel phenotype manifesting as FMTC and CA.     

甲状腺髓样癌降钙素及其基因相关肽检测的临床意义——附88例报告(英文)

目的研究甲状腺髓样癌患者降钙素及其基因相关肽的变化规律,指导临床治疗方案的选择和患者预后的判断。方法回顾性研究包括对58例甲状腺髓样癌病例血清中降钙素的分析及应用免疫组织化学方法观察相应标本中降钙素及其基因相关肽的表达情况;对新入组30例病例进行放射免疫学测定。结果 (1)术后1个月降钙素水平正常与升高的患者之间,肿瘤复发存在显著性差异(P<0.01)。(2)约98%的患者肿瘤标本降钙素染色呈阳性,而降钙素基因相关肽的阳性率为87.8%。(3)部分术前降钙素水平正常的患者降钙素基因相关肽水平升高。(4)术后1周左右降钙素下降至一稳定水平。结论降钙素可以作为指导甲状腺髓样癌诊断和治疗的重要指标,检测降钙素基因相关肽有助于部分降钙素阴性的甲状腺髓样癌患者术前诊断。     

Frequent Somatic Mutations of the APC and p53 Genes in Sporadic Ampullary Carcinomas

Although a close relation of somatic mutations of the adenomatous polyposis coli gene with ampullary carcinomas in familial adenomatous polyposis patients has been reported, the possible association with sporadic ampullary neoplasms has not been fully examined. We have therefore investigated loss of heterozygosity at the adenomatous polyposis coli locus and the mutational status of a portion of the adenomatous polyposis coli gene, including the mutation cluster region, in 17 ampullary carcinomas of non–familial adenomatous polyposis patients. Alteration of the adenomatous polyposis coli gene was found in 8 of 17 (47.1%) cases, as missense or insertion mutations, with or without loss of heterozygosity. Additional investigation of ⁁53 (cxons 5–8) and K–ras (codons 12 and 13) gene mutations revealed a striking mutational pattern of thep53 gene. Nine of the 17 cases demonstrated a total of 12 mutations, 6 clustered at codon 189 and 3 at codon 166. Furthermore, 5 of the 12 mutations were nonsense mutations. Regarding the K–ras gene, 4 of the 17 (23.5%) cases had mutations in codon 12, 3 of the 4 cases being derived from the intraduodenal bile duct. The findings indicate that alterations of the adenomatous polyposis coli and the p53 genes are relatively frequent in sporadic ampullary carcinomas. In particular, the clustering at specific p53 codons might offer an etiological clue to clarify ampullary carcinogenesis. Mutations of the K–ras gene, on the other hand, might be characteristic of intraduodenal bile duct origin.     

甲状腺髓样癌降钙素及其基因相关肽检测的临床意义--附88例报告

Objective： To investigate the changes of calcitonin （CT） and calcitonin gene-related peptide （CGRP） in patients with medullary thyroid carcinoma （MTC）. Methods： Fifty-eight cases of MTC were selected and the relationship between the CT levels and metastasis was investigated. The immunohistochemical method was used to detect the expression of CT and CGRP in the 58 samples of MTC tissues. The CT and CGRP in 30 newly diagnosed MTC inpatients were measured before operation and in the first few days after operation using a radioimmunoassy. Results： （1） The rate of residual tumor had a significant difference between the normal serum CT group one month after operation and the elevated group at the same period （P〈0.01）. （2） Immunohistochemical study revealed the positive rate of CT was about 98%, and that of the CGRP was 87.8%. （3） Part of the patients had an elevated CGRP levels while CT levels was normal. （4） The serum CT levels were decreased to a stable range one week after operation. Conclusion： CT is a useful index to evaluate the efficacy of surgical treatment. The measurement of serum CGRP is helpful in the diagnosis of MTC, especially for those whose preoperative CT levels are normal.     

Study of Calcitonin and Calcitonin Gene-related Peptide in Patients with Medullary Thyroid Carcinoma

OBJECTIVE To provide a basis for treatment and prognosis for MTC patients by investigating CT and CGRP.METHODS Fifty-eight cases of MTC were selected and the relationship between the CT levels and metastasis was investigated. An immunohistochemical method was used to detect the expression of CT and CGRP in the 58 samples of MTC tissues. The CT and CGRP of new MTC inpatients were measured before operation and in the first few days after operation using a radioimmunoassy.RESULTS 1 ) The metastatic rate of cervical lymph nodes was a significantly different between the normal serum CT group and the elevated group (P＜0.01). 2) The rate of residual tumor was significantly different between the normal serum CT group one month after operation and the elevated group at the same period (P＜0.01). 3) In the immunohistochemical study we found the positive rate of CT was about 98%, and the CGRP was 87.8%. 4) Part of the patients had an elevated CGRP level while CT levels were normal. 5) The serum CT levels dropped to a stable range one week after operation.CONCLUSION The CT levels are a useful index to evaluate the probability of cervical lymph node metastasis and the efficacy of surgical treatment.Measurement of the serum CGRP is an aid for the diagnosis of MTC,especially for those patients whose preoperative CT levels are normal.     

Detection of Gastrin-Releasing Peptide mRNA in Small Cell Lung Carcinomas and Medullary Thyroid Carcinomas Using Synthetic Oligodeoxyribonucleotide Probes

Human gastrin-releasing peptide (GRP) mRNA was detected in thetumor tissues of medullary thyroid carcinomas and small celllung carcinomas using synthetic oligodeoxyribonucleotldes ashybridization probes. The amount of GRP mRNA was estimated byradiodensitometric hybridization assay. A good correlation wasfound between the amount of GRP mRNA and the concentration ofimmuno-reactive GRP in the tumor tissues.     

Development of High-grade Renal Cell Carcinomas in Rats Independently of Somatic Mutations in the Tsc2 and VHL Tumor Suppressor Genes

Ferric nitrilotriacetate (Fe-NTA) induces renal proximal tubular damage that ultimately leads to a high incidence of renal cell carcinoma (RCC) in rats. The RCCs are characterized by 1) high incidence of pulmonary metastasis and peritoneal invasion, 2) high incidence of tumor-associated mortality and 3) possible involvement of reactive oxygen species in carcinogenesis. The present study investigated the possible role of Tsc2 and VHL tumor suppressor genes in this model. Thirty-four Fe-NTA-induced primary RCCs and 20 other primary or metastatic tumors of rats were searched for genetic alteration in all the coding exons of both genes by polymerase chain reaction-single-strand-conformation polymorphism analysis and sequencing in conjunction with morphological evaluation. In the Fe-NTA-induced RCCs, frequency of metastasis or invasion was proportionally associated with the nuclear grade of the tumor (grades 1–3). Only one Fe-NTA-induced RCC of grade 1 revealed missense mutations with loss of heterozygosity in exon 10 of the Tsc2 gene (codons 334, GTG (Val) to GCG (Ala), and 336, TAT (Tyr) to CAT (His)). No mutation was found in the VHL gene. The results suggest that 1) high-grade RCCs can develop in the absence of mutations in the Tsc2 and VHL genes in rats, and that 2) Tsc2 gene somatic mutation can nonetheless be one of the causes of non-Eker rat RCCs.     

A Novel Somatic Point Mutation of the RET Proto-oncogene in Tumor Tissues of Small Cell Lung Cancer Patients

We examined whether the novel point mutation from GCC (Ala) to GAC (Asp) at codon 664 in exon 11 of RET proto-oncogene, which we had found in two small cell lung carcinoma (SCLC) cell lines, existed in genomic DNA of tumor tissues of the two SCLC patients from whom these SCLC cell lines were derived. Sequence analysis revealed that point mutation identical to that of the SCLC cell lines was present in amplified alleles of single-strand conformational variants in genomic DNA of the tumor tissues, whereas it was not detected in genomic DNA of non-tumor tissues of the patients. These results indicate that this mutation had initially occurred in the SCLC patients and was of somatic origin.