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).     

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.     

Immune response against medullary thyroid carcinoma (MTC) induced by parental and/or interleukin-2-secreting MTC cells in a rat model of human familial medullary thyroid carcinoma

 The existence of inherited aggressive forms of medullary thyroid carcinoma (MTC), and their resistance to all classical therapies, make it a prime candidate for adoptive immunotherapy. As a prelude to a vaccine for the protection of family members at risk of developing the disease, we investigated the immunological antitumour response provoked by the 6/23 rMTC cell line, compared to that of the same cells engineered to secrete interleukin-2 (rMTC-IL2), in an animal model of familial human MTC, the inbred strain of Wag/Rij rats. The rMTC cells developed a tumour that invaded the whole neck 15 days after orthotopic injection (into the thyroid), while the rMTC-IL2 cells were progressively rejected. Co-injection of rMTC-IL2 with the parental cells induced the rejection of the rMTC transplants. When injected, both tumoral cell types showed a similar positive immunoreaction with anti-MHC class I (major histocompatibility complex class I) antibodies. They both recruited natural killer cells and eosinophils at the site of injection. In addition, CD8⁺ T lymphocytes infiltrated the rMTC-IL2 cells, and eosinophil recruitment was amplified. Neutrophils, macrophages and CD4⁺ T lymphocytes were scarce. Our results suggest that the CD8⁺ T lymphocytes are implicated in the antitumour reaction elicited by the Il-2-transfected cells. As these effectors are known to induce a specific immunological response, including memory, such a protocol should be tested as a vaccine on the young population genetically at risk of developing a MTC. Received: 18 December 1995 / Accepted: 21 August 1996     

Activation of the Erk8 mitogen-activated protein (MAP) kinase by RET/PTC3, a constitutively active form of the RET proto-oncogene

Mitogen-activated protein (MAP) kinases have a central role in several biological functions, including cell adhesion and spreading, chemotaxis, cell cycle progression, differentiation, and apoptosis. Extracellular signal-regulated kinase 8 (Erk8) is a large MAP kinase whose activity is controlled by serum and the c-Src non-receptor tyrosine kinase. Here, we show that RET/PTC3, an activated form of the RET proto-oncogene, was able to activate Erk8, and we demonstrate that such MAP kinase participated in RET/PTC3-dependent stimulation of the c-jun promoter. By using RET/PTC3 molecules mutated in specific tyrosine autophosphorylation sites, we characterized Tyr(981), a known binding site for c-Src, as a major determinant of RET/PTC3-induced Erk8 activation, although, surprisingly, the underlying mechanism did not strictly depend on the activity of Src. In contrast, we present evidence that RET/PTC3 acts on Erk8 through Tyr(981)-mediated activation of c-Abl. Furthermore, we localized the region responsible for the modulation of Erk8 activity by the RET/PTC3 and Abl oncogenes in the Erk8 C-terminal domain. Altogether, these results support a role for Erk8 as a novel effector of RET/PTC3 and, therefore, RET biological functions.     

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.     

Mutational spectrum in the neurofibromatosis type 2 gene in sporadic and familial schwannomas

Using a heteroduplex approach and direct sequencing, we have completed the screening of approximately 88% of the neurofibromatosis type 2 (NF2)-coding sequence of DNA extracted from 33 schwannomas from NF2 patients and from 29 patients with sporadic schwannomas. The extensive screening has resulted in the identification of 33 unique mutations. Similarly to other human genes, we have shown that the CpG sites are more highly mutable in the NF2 gene. The frequency, distribution, and types of mutations were shown to differ between the sporadic and familial tumors. The majority of the mutations resulted in protein truncation and were consistent with more severe phenotype, however three missense mutations were identified during this study and were all associated with milder manifestations of the disease. Received: 25 September 1995 / Revised: 19 December 1995     

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.     

Spinocerebellar ataxias in Spanish patients: genetic analysis of familial and sporadic cases

Autosomal dominant cerebellar ataxias (ADCA) are a clinically heterogeneous group of neurodegenerative disorders caused by unstable CAG repeat expansions encoding polyglutamine tracts. Five spinocerebellar ataxia genes (SCA1, SCA2, SCA3, SCA6 and SCA7) and another related dominant ataxia gene (DRPLA) have been cloned, allowing the genetic classification of these disorders. We present here the molecular analysis of 87 unrelated familial and 60 sporadic Spanish cases of spinocerebellar ataxia. For ADCA cases 15% were SCA2, 15% SCA3, 6% SCA1, 3% SCA7, 1% SCA6 and 1% DRPLA, an extremely rare mutation in Caucasoid populations. About 58% of ADCA cases remained genetically unclassified. All the SCA1 cases belong to the same geographical area and share a common haplotype for the SCA1 mutation. The expanded alleles ranged from 41 to 59 repeats for SCA1, 17 to 29 for SCA2, 67 to 77 for SCA3, and 38 to 113 for SCA7. One SCA6 case had 25 repeats and one DRPLA case had 63 repeats. The highest CAG repeat variation in meiotic transmission of expanded alleles was detected in SCA7, this being of +67 units in one paternal transmission and giving rise to a 113 CAG repeat allele in a patient who died at 3 years of age. Meiotic transmissions have also shown a tendency to more frequent paternal transmission of expanded alleles in SCA1 and maternal in SCA7. All SCA1 and SCA2 expanded alleles analyzed consisted of pure CAG repeats, whereas normal alleles were interrupted by 1–2 CAT trinucleotides in SCA1, except for three alleles of 6, 14 and 21 CAG repeats, and by 1–3 CAA trinucleotides in SCA2. No SCA or DRPLA mutations were detected in the 60 sporadic cases of spinocerebellar ataxia, but one late onset patient was identified as a recessive form due to GAA-repeat expansions in the Friedreich’s ataxia gene. Received: 6 January 1999 / Accepted: 18 March 1999     

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.     

Analysis of sister-chromatid exchanges (SCEs) in familial and sporadic multiple sclerosis

The levels of sister-chromatid exchanges (SCEs) were calculated in 17 multiple sclerosis (MS) patients (14 familial and 3 sporadic cases) and 16 healthy controls matched for sex and age. The SCEs were significantly increased for MS patients (p = 0.0002), while there was no statistically significant difference between men and women and between younger and older subjects in both groups. Such factors as familial occurrence and severity of MS, smoking habits, and distribution of lymphocyte subpopulations were discussed. Although there was a significant difference between the MS patients taking medication and the patients taking none (p = 0.038), the latter were still significantly different from the controls (p = 0.035), supporting the fact that the disease itself increases SCEs. Our study, done with 2 doses of BrdU, also shows that the increased SCEs in MS patients are not due to a hypersensitivity to this substance known to be an inducer of SCEs. Thus we suggest that the increased SCEs found are probably disease-related.     

A rare haplotype of the RET proto-oncogene is a risk-modifying allele in hirschsprung disease

Hirschsprung disease (HSCR) is a common genetic disorder characterized by intestinal obstruction secondary to enteric aganglionosis. HSCR demonstrates a complex pattern of inheritance, with the RET proto-oncogene acting as a major gene and with several additional susceptibility loci related to the Ret-signaling pathway or to other developmental programs of neural crest cells. To test how the HSCR phenotype may be affected by the presence of genetic variants, we investigated the role of a single-nucleotide polymorphism (SNP), 2508C-->T (S836S), in exon 14 of the RET gene, characterized by low frequency among patients with HSCR and overrepresentation in individuals affected by sporadic medullary thyroid carcinoma. Typing of several different markers across the RET gene demonstrated that a whole conserved haplotype displayed anomalous distribution and nonrandom segregation in families with HSCR. We provide genetic evidence about a protective role of this low-penetrant haplotype in the pathogenesis of HSCR and demonstrate a possible functional effect linked to RET messenger RNA expression.     

Tumor-associated macrophages (TAMs) form an interconnected cellular supportive network in anaplastic thyroid carcinoma

Background

A relationship between the increased density of tumor-associated macrophages (TAMs) and decreased survival was recently reported in thyroid cancer patients. Among these tumors, anaplastic thyroid cancer (ATC) is one of the most aggressive solid tumors in humans. TAMs (type M2) have been recognized as promoting tumor growth. The purpose of our study was to analyze with immunohistochemistry the presence of TAMs in a series of 27 ATC.

Methodology/Principal Findings

Several macrophages markers such as NADPH oxidase complex NOX2-p22phox, CD163 and CD 68 were used. Immunostainings showed that TAMs represent more than 50% of nucleated cells in all ATCs. Moreover, these markers allowed the identification of elongated thin ramified cytoplasmic extensions, bestowing a “microglia-like” appearance on these cells which we termed “Ramified TAMs” (RTAMs). In contrast, cancer cells were totally negative. Cellular stroma was highly simplified since apart from cancer cells and blood vessels, RTAMs were the only other cellular component. RTAMs were evenly distributed and intermingled with cancer cells, and were in direct contact with other RTAMs via their ramifications. Moreover, RTAMs displayed strong immunostaining for connexin Cx43. Long chains of interconnected RTAMs arose from perivascular clusters and were dispersed within the tumor parenchyma. When expressed, the glucose transporter Glut1 was found in RTAMs and blood vessels, but rarely in cancer cells.

Conclusion

ATCs display a very dense network of interconnected RTAMs in direct contact with intermingled cancer cells. To our knowledge this is the first time that such a network is described in a malignant tumor. This network was found in all our studied cases and appeared specific to ATC, since it was not found in differentiated thyroid cancers specimens. Taken together, these results suggest that RTAMs network is directly related to the aggressiveness of the disease via metabolic and trophic functions which remain to be determined.     

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.     

Hereditary multiple exostoses (EXT): mutational studies of familial EXT1 cases and EXT-associated malignancies.

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by the formation of cartilage-capped prominences that develop from the growth centers of the long bones. EXT is genetically heterogeneous, with three loci, currently identified on chromosomes 8q24.1, 11p13, and 19q. The EXT1 gene, located on chromosome 8q24.1, has been cloned and is encoded by a 3.4-kb cDNA. Five mutations in the EXT1 gene have been identified--four germ-line mutations, including two unrelated families with the same mutation, and one somatic mutation in a patient with chondrosarcoma. Four of the mutations identified resulted in frameshifts and premature termination codons, while the fifth mutation resulted in a substitution of leucine for arginine. Loss of heterozygosity (LOH) analysis of chondrosarcomas and chondroblastomas revealed multiple LOH events at loci on chromosomes 3q, 8q, 10q, and 19q. One sporadic chondrosarcoma demonstrated LOH for EXT1 and EXT3, while a second underwent LOH for EXT2 and chromosome 10. A third chondrosarcoma underwent LOH for EXT1 and chromosome 3q. These results agree with previous findings that mutations at EXT1 and multiple genetic events that include LOH at other loci may be required for the development of chondrosarcoma.