Analysis of mutations in the RET proto-oncogene in patients with medullary thyroid tumor

The spectrum of mutations of the RET protooncogene was analyzed in Russian patients with inherited or sporadic medullary thyroid carcinoma (MTC). Four RET exons (11, 13, 15, and 16) were subjected to molecular analysis, and mutations were revealed and identified in 47.4% (9/19) patients with sporadic MTC. In total, six mutations (including three new ones) were observed. The most common mutation affected codon 918 to cause substitution of methionine with threonine and accounted for 31.6% alleles. Analysis of exons 11 and 16 revealed four mutations in patients with inherited multiple endocrine neoplasia type 2 (MEN 2). Mutations were found in each patient. Thyroidectomy was performed in four asymptomatic carriers of RET mutations from three MET 2A families (in two families, affected relatives had bilateral pheochromocytoma). In two patients, analysis of the surgery material revealed MTC microfoci in both lobes of the thyroid gland. The results provide the ground for constructing a bank of genetic information on Russian MTC patients with the clinically verified diagnosis.     

Germline polymorphisms of RET and GFRA1 genes in patients with medullary thyroid carcinoma

The association of RET and GFRA1 polymorphisms with a predisposition to sporadic medullary thyroid cancer (MTC) and their effects on the clinical features of hereditary and sporadic MTC were studied in 67 MTC patients (22 hereditary and 45 sporadic), 3 asymptomatic carriers of mutant RET, and 178 healthy control residents of Russia. RET exons 8, 10, 11, 13, 14, 15, and 16 and intron 1 along with the GFRA1 5′-UTR were screened by PCR and subsequent direct sequencing or RFLP analysis. Eight polymorphic variants of RET (exons 11, 13, 14, and 15 and introns 1, 8, 13, and 14) and four GFRA1 polymorphisms were detected. Linkage disequilibrium was found between RET variants G691S and S904S, L769L and IVS8, and S836S and IVS13. In sporadic MTC the allele frequency of only one polymorphic RET variant, L769L, was significantly lower than in the control group. In hereditary MTC a significant overrepresentation of the S836S and underrepresentation of the S904S polymorphic variants were observed as compared to groups with sporadic MTC and the controls. Cosegregation was not found between individual polymorphisms and the phenotype of sporadic MTC. In patients with hereditary MTC whose genotype had the polymorphic L769L and the wild-type S836S variants, the disease manifested 20 years later, on average, than in individuals with polymorphic L769L and S836S or with wild-type L769L (P = 0.01). The results suggest a protective role of the L769L polymorphism in sporadic MTC and a modulating effect of the combination polymorphic L769L with wild-type S836S on the clinical outcome of hereditary MTC.     

Adrenal-cortical function in patients with medullary carcinoma of the thyroid and pheochromocytoma.

Medullary carcinoma of the thyroid (MCT) is reported to synthesize ACTH. This ACTH is believed to be responsible for the development of Cushing's syndrome in some patients with MCT. To determine the frequency of occurrence of adrenal cortical overactivity in patients with MCT, we measured plasma cortisol concentration and the urinary excretion of 17-hydroxycorticosteroids, 17-ketosteroids and urinary free cortisol in 22 patients with MCT and 7 patients with MCT plus pheochromocytomas. The patients with MCT and MCT plus pheochromocytoma had similar adrenal cortical function to age and sex matched normal subjects. We conclude that adrenal cortical function is usually normal in patients with MCT.     

RET germline mutations identified by exome sequencing in a Chinese multiple endocrine neoplasia type 2A/familial medullary thyroid carcinoma family

Background

Whole exome sequencing provides a labor-saving and direct means of genetic diagnosis of hereditary disorders in which the pathogenic gene harbors a large cohort of exons. We set out to demonstrate a suitable example of genetic diagnosis of MEN 2A/FMTC (multiple endocrine neoplasia type 2/familial medullary thyroid carcinoma) using this approach.

Methodology/Principal Findings

We sequenced the whole exome of six individuals from a large Chinese MEN2A/FMTC pedigree to identify the variants of the RET (REarranged during Transfection) protooncogene and followed this by validation. Then prophylactic or surgical thyroidectomy with modified or level VI lymph node dissection and adrenalectomy were performed for the carriers. The cases were closely followed up. Massively parallel sequencing revealed four missense mutations of RET. We unexpectedly discovered that the proband''s daughter with MEN 2A-related MTC presented a novel p.C634Y/V292M/R67H/R982C compound mutation, due to the involvement of p.C634Y in the proband with MEN 2A and p.V292M/R67H/R982C in the proband''s husband with FMTC. In the maternal origin, p.C634Y caused bilateral MTC in all 5 cases and bilateral pheochromocytoma in 2 of the 5; the earliest onset age was 28 years. In the paternal origin, one of the six p.V292M/R67H/R982C carriers presented bilateral MTC (70 years old), one only had bilateral C-cell hyperplasia (44 years), two had bilateral multi-nodules (46 and 48 years) and two showed no abnormality (22 and 19 years).

Conclusions/Significance

The results confirmed the successful clinical utility of whole exome sequencing, and our data suggested that the p.C634Y/V292M/R67H/R982C mutation of RET exhibited a more aggressive clinical phenotype than p.C634Y or p.V292M/R67H/R982C, while p.V292M/R67H/R982C presented a relatively milder pathogenicity of MTC and likely predisposed to FMTC.     

RET and GFRA1 germline polymorphisms in medullary thyroid cancer patients

The role of RET and GFRA1 germline polymorphisms in predisposition to sporadic medullary thyroid cancer (MTC) and polymorphisms' modulation effect on clinical features of inherited and sporadic MTC were investigated. Blood samples from 67 MTC patients (22 hereditary and 45 sporadic), 3 asymptomatic mutant RET gene carriers and 178 ethnically matched healthy control individuals were tested. Screening of RET exons and portion of introns 1, 8, 10, 13, 14, 15, 16 and GFRA1 5'-UTR was performed by means of direct sequencing and PCR-RFLP. 8 polymorphic variants of RET gene (exons 11, 13, 14, 15 and introns 1, 8, 13, 14) and 4 GFRA1 polymorphisms in GFRA1 were detected. Linkage disequilibrium was found between RET variants G691S and S904S, L769L and IVS8, S836S and IVS13. In sporadic MTCs, allelic frequency of only one polymorphic RET variant, L769L, was significantly decreased versus control group. In hereditary MTCs, a significant over-representation of S836S and under-representation of S904S sequence variants were observed as compared to sporadic MTCs and controls. No co-segregation was found between individual polymorphisms and phenotype of sporadic MTC. In patients with inherited MTC whose genotype was presented with polymorphic L769L and wild-type S836S, disease onset occurred 20 years later than in individuals with polymorphic L769L and S836S or wild-type L769L (p = 0.01) suggestive of a possible protective role of L769L in MTC development and modulating effect of a combination of L769L with wild-type S836S on clinical outcome of inherited MTC.     

Melanin-producing medullary thyroid carcinoma with glandular differentiation

A rare case is reported of melanin-producing medullary thyroid carcinoma in a 62-year-old man. Intraoperative imprints of the thyroid tumor revealed numerous detached tumor cells containing large amounts of brown pigment. The Fontana-Masson argentaffin reaction with bleach confirmed that those granules were melanin. Histologically, the tumor was composed of two different components--a medullary area with hyalinized stroma and a follicular area. Melanin was scattered in both areas. The tumor cells in both areas were immunoreactive to carcinoembryonic antigen, calcitonin, gastrin-releasing peptide, somatostatin, met.-enkephalin, neuron-specific enolase, chromogranin and neurofilaments, and negative for thyroglobulin and S-100 protein. The histologic diagnosis was melanin-producing medullary thyroid carcinoma with glandular differentiation. Although various kinds of peptides and amines have been reported to be produced in medullary thyroid carcinoma, melanin production is quite rare; this appears to be only the third reported case.     

Medullary thyroid carcinoma in a child with a new RET mutation and a RET polymorphism

We report a 12 year old boy with an isolated medullary thyroid carcinoma (MTC). A mutation analysis of the RET-proto-oncogene in this boy showed an in frame insertion-deletion mutation (insTTCTdelG) at codon 666 of the RET proto-oncogene. This RET mutation has not been reported previously. The boy's mother and his 82-year-old maternal grandfather showed the same mutation. None of the two ever showed symptoms of MTC. The mother underwent a preventive total thyroidectomy and pathological examination showed C-cell hyperplasia and early MTC. Further genetic analysis showed that the boy inherited a well-known coding polymorphism in exon 11 (G691S) from his father. Therefore the boy is a compound heterozygote for the insertion-deletion mutation at codon 666 and the G691S polymorphism in the RET gene. We hypothesize that the insTTCTdelG mutation at codon 666 is associated with low penetrance for MTC and that the young age of MTC in the reported child results most likely from the additive effects of both mutations (insTTCTdelG and G691S).     

The rare intracellular RET mutation p.S891A in a Chinese Han family with familial medullary thyroid carcinoma

We report intracellular RET mutation in a Han Chinese pedigree with familial medullary thyroid carcinoma (FMTC). Direct sequencing of RET proto-oncogene identified a missense c.2671T>G (p.S891A) mutation in 6 of 14 family members. The single nucleotide polymorphisms c. 135A>G (p.A45A), IVS4+48A>G, c. 1296A>G (p.A432A), c. 2071G>A (p.G691S), c. 2307T>G (p.L769L) and a variant c. 833C>A (p.T278N) were also found in 6 carriers. Among 5 of the 6 carriers presented medullary thyroid carcinoma (MTC) as an isolated clinical phenotype, with elevated basal serum calcitonin (Ct). Two underwent non-normative thyroidectomy either two or four times without physician awareness or diagnosis of this disease at initial treatment, but with elevated Ct. One with elevated pre-Ct accepted total thyroidectomy (TT) with modified bilateral neck dissection (MBiND), and whose seventh posterior rib MTC metastases was confirmed 5 months after surgery. Moreover, results of two affected individuals with elevated Ct were reduced to normal after TT with MBiND or prophylactic VI compartmental dissection. However, only another carrier with the variant p.T278N had slightly elevated Ct rejected surgery and was strictly monitored. Given these case results, we suggest that screening of RET and pre-surgical Ct levels in the management of MTC patients is essential for earlier diagnosis and more normative initial treatment, that FMTC patients with cervical lymph nodes metastases may be cured by TT with MBiND, and that prophylactic VI compartmental dissection should be avoided when Ct levels are low.     

Linkage analysis of hereditary thyroid carcinoma with and without pheochromocytoma

Summary The use of polymorphic DNA segments as markers for the gene for the multiple endocrine neoplasia (MEN) syndrome, type 2a, allows the identification of family members at high risk for developing medullary carcinoma of the thyroid and other tumors, especially pheochromocytoma. Several families have also been identified in which medullary thyroid carcinoma is inherited, but pheochromocytoma is not seen. We have analysed 18 families, 9 with MEN 2A and 9 with medullary carcinoma of the thyroid without pheochromocytoma, with probes specific for the pericentromeric region of chromosome 10 and conclude that the mutations for the two presentations are closely situated. Genetic heterogeneity of the susceptibility locus was not seen among this sample of 18 families. The genetic mutation for medullary carcinoma was in disequilibrium with the marker alleles of the two closely linked probes. IRBPH4 and MCK2. These data suggest that different mutant alleles of the same gene or closely linked mutations account for the variation in penetrance of pheochromocytoma in families with hereditary, medullary thyroid carcinoma.     

A case of extraadrenal pheochromocytoma with papillary thyroid carcinoma.

A 63-year-old housewife with a history of partial thyroidectomy was referred to our hospital because of a neck mass and abdominal tumor. Aspiration biopsy of the neck tumor revealed the recurrence of papillary thyroid carcinoma. Magnetic resonance imaging (MRI) of the abdomen and urinary and plasma catecholamine levels indicated that the tumor beside the abdominal aorta was an extraadrenal pheochromocytoma. Two tumors were excised and histologic studies confirmed the diagnosis. So far two cases of extraadrenal pheochromocytoma with papillary thyroid carcinoma have been reported. The present case indicates that the presence of papillary thyroid carcinoma should be considered in patients with extraadrenal pheochromocytoma.     

Medullary thyroid carcinoma: pitfalls in diagnosis by fine needle aspiration cytology and relationship of cytomorphology to RET proto-oncogene mutations

OBJECTIVE: To elucidate the pitfalls in the diagnosis of medullary thyroid carcinoma (MTC) by fine needle aspiration cytology (FNAC) and the relationship of cytomorphology to RET proto-oncogene mutations. STUDY DESIGN: Cytomorphology was reviewed in the fine needle aspiration slides of 34 patients with MTC proven by surgery and pathology. Germline or somatic RET proto-oncogene mutations were determined using polymerase chain reaction-based sequencing in 15 of 34 patients, and the relationship to cytomorphology was evaluated. RESULTS: Twenty-eight (82.4%) of 34 cases were diagnosed correctly as MTC by FNAC, 3 cases were misdiagnosed as follicular neoplasm and 1 as desmoid, and 2 cases were suspicious for MTC. The main reason for misdiagnosis was overlooking the slight angular shape of the nuclei or atypical changes. In 15 of 34 cases with germline or somatic RET proto-oncogene mutations determined, 10 cases had a germline mutation, and 1 had only a somatic mutation. There were 4 cases that had neither germline nor somatic RET proto-oncogene mutations. Cells with small/round and spindled forms were the predominant findings of codon 918 ATG-->ACG mutation, and cells with small/round and large oval to polygonal forms were the main findings of codon 634 mutations. There were no misdiagnoses in patients with RET proto-oncogene mutations. CONCLUSION: FNAC is a good diagnostic method for MTC. Codon 918 mutation correlates mainly with small/round and spindled cells and codon 634 with small/round, large oval to polygonal forms.     

MiR-129-5p is down-regulated and involved in the growth,apoptosis and migration of medullary thyroid carcinoma cells through targeting RET

Dysregulation of the REarranged during Transfection proto-oncogene (RET) pathway and microRNA (miRNAs) are crucial for the development of medullary thyroid carcinomas (MTC). Here we demonstrate that miR-129-5p is down-regulated in MTC tissues and cell lines and inhibits RET expression by directly binding its 3′ untranslated regions. Ectopic expression of miR-129-5p significantly decreases cell growth, induces apoptosis and suppresses migration ability in MTC cells through decreasing the phosphorylated AKT, thus functioning as a tumor suppressor. These findings give new clues for understanding MTC carcinogenesis and may help in developing a therapeutic approach for the treatment of RET-activated MTC.     

Imaging and uptake mechanism of 131I-meta-iodobenzylguanidine in medullary thyroid carcinoma

131I-meta-iodobenzylguanidine (131I-MIBG) was also taken up by medullary thyroid carcinoma (MTC) as well as by pheochromocytoma in two patients with Sipple's syndrome. However, the mechanism of 131I-MIBG uptake by MTC has not been clarified yet. We measured tissue catecholamine levels in three MTC, since MTC can produce several active substances. Catecholamines were detected in various amounts in all MTC, but not in normal thyroid tissues. These findings suggest that MTC can produce catecholamines and therefore, 131I-MIBG is taken up and stored in catecholamine vesicles of MTC, like pheochromocytoma and neuroblastoma. We conclude that 131I-MIBG may be applied not only to diagnosis but also for the treatment of patients with MTC.     

Chemotherapy with doxorubicin in progressive medullary and thyroid carcinoma of the follicular epithelium.

Twenty-two patients (mean age 61) with metastasizing, progressive, nonradioiodine-accumulating thyroid carcinoma of the follicular epithelium were treated with doxorubicin between 2000 and 2005. Tumors were histologically classified as follicular in 15 patients (68%) and papillary in 7 patients (32%). In addition, nine patients (mean age 51 years) with medullary thyroid carcinoma were treated with doxorubicin between 1997 and 2005. Treatment consisted of doxorubicin: either 8 cycles of 15 mg/m2 weekly or 3 cycles of 60 mg/m2 every 3 weeks, repeated once, depending on response and side effects. The effect of therapy was evaluated by radiographic imaging, [18F] FDG-PET, and bone scans. In patients with papillary or follicular thyroid carcinoma, 5% had a partial regression over 6 months, 42% had stable disease for a median of 7 months (range: 1-22), and 53% had continuous progression established over 5 months (range: 1-11). Three patients died before completing chemotherapy. In patients with medullary thyroid carcinoma, 11% had a partial regression over 6 months followed by stable disease for 3 months, 11% had stable disease over 7 months, and 79% demonstrated progressive disease established over 5 months (range: 2-12). Doxorubicin can be a valid chemotherapy option, especially for advanced or metastatic thyroid carcinoma of the follicular epithelium.     

Occult medullary carcinoma of thyroid with lymph node metastases: a case report

BACKGROUND: Occult thyroid malignancies presenting with secondary neck masses as the first clinical manifestation is well known. Although rare, medullary carcinoma serves a potential source for lymph node metastases. The characteristic cytomorphology of medullary thyroid carcinoma (MTC) should clinch the diagnosis. Further, fine needle aspiration cytology (FNAC) of the ultrasonography-detected occult nodules in thyroid serves as a useful preoperative diagnostic tool. CASE: A 22-year-old man presented with left-sided neck masses of 1 year duration. FNAC smears of the neck masses revealed cytomorphology characteristic of MTC. Ultrasonography of the thyroid led to ruling out the presence of an occult nodule and detected an 8-mm nodule in the left thyroid lobe. Ultrasound-guided FNAC of the nodule showed features similar to those with FNAC of the neck masses. Surgical resection of thyroid and neck masses further confirmed the diagnosis of a primary occult MTC with lymph node metastases. CONCLUSION: FNAC smears of lymph node masses showing the distinct cytomorphology of MTC should prompt suspicion for occult primary in thyroid. Ultrasound-guided FNAC of these occult nodules, if detected, further serves a diagnostic tool for accurate preoperative diagnosis when metastasis presents as the first clinical manifestation of an occult primary.