Evidence of a low prevalence of RAS mutations in a large medullary thyroid cancer series.

BACKGROUND: Approximately 60% of sporadic medullary thyroid carcinomas (sMTC) remain orphan of a recognized genetic cause. Recently, a high percentage of RAS point mutations have been described in RET-negative sMTC. The aim of this study was to assess the prevalence of RAS point mutations in a large series of MTC collected in four Italian centers. METHODS: For this purpose, we studied codons 12, 13, and 61 of H-, K-, and N-RAS genes in 188 MTC samples, either hereditary or sporadic, by direct sequencing. Correlations between the RAS mutational status and the clinical-pathological features of MTC patients as well as a meta-analysis of all published data were performed. RESULTS: The prevalence of RAS mutations in the present series of MTC was 10.1%, and 17.6% when considering only RET-negative cases. RAS mutations were found in MTC tumoral tissue, but not in peripheral blood indicating their somatic origin. A novel mutation in codon 72 (M72I) was found, but with a low or null transforming potential. No association was found between the presence of RAS mutations and the clinical-pathological features of the patients. Although not statistically significant, a positive association between the presence of RAS mutations and a better outcome was observed. The meta-analysis of all published studies confirmed a prevalence of 8.8% for RAS mutations in MTC. CONCLUSIONS: The prevalence of RAS mutations in our MTC series was relatively low and consistent with the meta-analysis data. Only somatic RAS mutations were found and only in RET-negative sMTC. Likewise, MTCs that harbor a RAS mutation identify a subgroup of tumors with less aggressive behavior. To our knowledge, this is the largest series of MTCs studied for the presence of mutations in RAS genes and the first meta-analysis on this specific topic.

Low prevalence of the somatic M918T RET mutation in micro-medullary thyroid cancer.

BACKGROUND: The prevalence of RET somatic mutations in sporadic medullary thyroid cancer (MTCs) is ∼40%-50%, and the most frequent somatic mutation is M918T. RET-positive MTCs have been demonstrated to have a more advanced stage at diagnosis and a worse outcome. AIMS: The aim of the present work was to compare the prevalence of RET somatic mutations in sporadic microMTCs (<1 cm) and in larger MTCs. PATIENTS: We analyzed the M918T RET point mutation in 160 sporadic MTC cases. Tumors were classified according to their size: group A, <1 cm; group B, >1 and <2 cm; group C, >2 and <3 cm; and group D, >3 cm. RESULTS: The overall prevalence of the somatic M918T RET mutation was 19.4% (31/160). RET mutations were distributed differently among the four groups. The prevalence was 11.3% (6/53) in group A, 11.8% (8/68) in group B, 31.8% (7/22) in group C, and 58.8% (10/17) in group D, exhibiting an increase with increasing size of the tumor. When comparing the prevalence of mutations in the four groups, we found a lower prevalence in microMTCs (p<0.0001). CONCLUSIONS: The overall prevalence of RET somatic mutations was lower than expected, and the prevalence of the somatic M918T RET mutation was significantly lower in microMTCs than in larger tumors. To explain this finding, we can hypothesize either that other oncogene(s) might be responsible for the majority of microMTC, thus identifying a tumor subset, or that the RET mutation might, or might not, occur later during tumor progression.

Chromosome 10 and RET gene copy number alterations in hereditary and sporadic Medullary Thyroid Carcinoma.

About 30% of hereditary Medullary Thyroid Carcinoma (MTC) have been demonstrated to harbour imbalance between mutant and wild-type RET alleles. We studied the RET copy number alterations (RET CNA) in 65 MTC and their correlation with RET mutation and patients' outcome. Fluorescence in situ Hybridization and Real-time PCR revealed RET CNA in 27.7% MTC but only in a variable percentage of cells. In sporadic MTC, RET CNA were represented by chromosome 10 aneuploidy while in hereditary MTC by RET amplification. A significant higher prevalence of RET CNA was observed in RET mutated MTC (P=0.003). RET CNA was also associated to a poorer outcome (P=0.005). However, the multivariate analysis revealed that only RET mutation and advanced clinical stage correlated with the worst outcome. In conclusion, 30% MTC harbour RET CNA in variable percentage of cells suggesting cell heterogeneity. RET CNA can be considered a poor prognostic factor potentiating the poor prognostic role of RET mutation.

The timing of total thyroidectomy in RET gene mutation carriers could be personalized and safely planned on the basis of serum calcitonin: 18 years experience at one single center.

BACKGROUND: Medullary thyroid carcinoma (MTC) is a calcitonin (CT)-producing C-cell tumor. In hereditary cases, a germline RET mutation is found in 98% of families. Because MTC is cured only if intrathyroidal, prophylactic thyroidectomy is recommended in the gene carrier (GC). AIMS: The aim was to determine whether thyroidectomy performed when stimulated CT becomes detectable is as safe as prophylactic thyroidectomy and to identify the serum CT cutoff able to distinguish intrathyroidal from extrathyroidal MTC. PATIENTS: Eighty-four GC were prospectively enrolled; 53 of the 84 underwent total thyroidectomy, one refused surgery, and 30 with normal basal and stimulated CT were under surveillance. The follow-up ranged from 2 to 18 yr. RESULTS: GC operated on for elevated stimulated CT included 27 GC with a positive peak CT at the screening and four cases who became positive after 4 yr. All of them had intrathyroidal MTC and no node metastases; all were cured after a mean follow-up of 7.5 yr. Among those operated on for detectable basal CT, intrathyroidal tumors were found when CT was below 60 pg/ml, whereas either node metastases or larger tumors were observed when CT was above 60 pg/ml. No correlation among serum CT, age, and type of RET mutation was observed. Thirty GC were still biochemically negative at the annual control. CONCLUSIONS: The time of thyroidectomy in GC with negative CT could be personalized and safely planned when stimulated CT becomes positive, independent of the type of RET mutation and patient's age. In this series, a basal CT below 60 pg/ml was always associated to an intrathyroidal localization of MTC.

Multiple endocrine neoplasia type 2 syndromes (MEN 2): results from the ItaMEN network analysis on the prevalence of different genotypes and phenotypes.

OBJECTIVE: Multiple endocrine neoplasia type 2 (MEN 2) is a genetic disease characterized by medullary thyroid carcinoma (MTC) associated (MEN 2A and 2B) or not familial MTC (FMTC) with other endocrine neoplasia due to germline RET gene mutations. The prevalence of these rare genetic diseases and their corresponding RET mutations are unknown due to the small size of the study population. METHODS: We collected data on germline RET mutations of 250 families with hereditary MTC followed in 20 different Italian centres. RESULTS AND CONCLUSIONS: The most frequent RET amino acid substitution was Val804Met (19.6%) followed by Cys634Arg (13.6%). A total of 40 different germline RET mutations were present. Six families (2.4%) were negative for germline RET mutations. The comparison of the prevalence of RET germline mutations in the present study with those published by other European studies showed a higher prevalence of Val804Met and Ser891Ala mutations and a lower prevalence of Leu790Phe and Tyr791Phe (P<0.0001). A statistically significant higher prevalence of mutations affecting non-cysteine codons was also found (P<0.0001). Furthermore, the phenotype data collection showed an unexpected higher prevalence of FMTC (57.6%) with respect to other MEN 2 syndromes (34% MEN 2A and 6.8% of MEN 2B). In conclusion, we observed a statistically significant different pattern of RET mutations in Italian MEN 2 families with respect to other European studies and a higher prevalence of FMTC phenotype. The different ethnic origins of the patients and the particular attention given to analysing apparently sporadic MTC for RET germline mutations may explain these findings.

Expression analysis of facilitative glucose transporters (GLUTs) in human thyroid carcinoma cell lines and primary tumors.

Fluorine-18-fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET) is based on cell capability to take-up glucose. While a significantly higher expression of the glucose transporter GLUT1 has been reported in thyroid tumors only few data are available on the expression of other GLUT isoforms. We studied several GLUT isoforms expression in thyroid tumor cell lines deriving from anaplastic (ARO, FRO), papillary (NPA), follicular (WRO) and medullary (TT) human thyroid carcinoma. GLUT1 and GLUT3 were also studied in 157 human thyroid malignant and benign tissues. Quantitative Real-time RT-PCR analysis revealed that GLUT1 mRNA levels were higher in less-differentiated cells (ARO, FRO) while GLUT3 mRNA levels were prevalent in well-differentiated cells (NPA, WRO). Accordingly, Western blot showed high expression and correct membrane targeting of GLUT1 protein in ARO and FRO and of GLUT3 protein in NPA and WRO. All cell lines were able to take-up different rates of (3)H-deoxy-glucose. The analysis of GLUT1 and GLUT3 mRNA expression in human thyroid tissues showed the prevalence of GLUT1, but not of GLUT3, in malignant with respect to normal tissues. Finally, both GLUT1 and GLUT3 showed a slightly higher expression in anaplastic than in well-differentiated tumors. In conclusion, we showed that GLUT1 and GLUT3 were the most important glucose transporters in the thyroid tumoral cells. In particular GLUT1 was the most prevalent in less-differentiated cells (ARO and FRO) while GLUT3 was the most prevalent in well-differentiated cells (NPA and WRO). A similar pattern of expression was found for GLUT1 but not for GLUT3 in human thyroid tumors.

Prognostic significance of somatic RET oncogene mutations in sporadic medullary thyroid cancer: a 10-year follow-up study.

BACKGROUND: Medullary thyroid carcinoma (MTC) is a well-differentiated thyroid tumor that maintains the typical features of C cells. An advanced stage and the presence of lymph node metastases at diagnosis have been demonstrated to be the most important bad prognostic factors. Somatic RET mutations have been found in 40-50% of MTCs. Although a relationship between somatic mutations and bad prognosis has been described, data are controversial and have been performed in small series with short-term follow ups. The aim of this study was to verify the prognostic value of somatic RET mutations in a large series of MTCs with a long follow up. METHODS: We studied 100 sporadic MTC patients with a 10.2 yr mean follow-up. RET gene exons 10-11 and 13-16 were analyzed. The correlation between the presence/absence of a somatic RET mutation, clinical/pathological features, and outcome of MTC patients was evaluated. RESULTS: A somatic RET mutation was found in 43 of 100 (43%) sporadic MTCs. The most frequent mutation (34 of 43, 79%) was M918T. RET mutation occurrence was more frequent in larger tumors (P=0.03), and in MTC with node and distant metastases (P<0.0001 and P=0.02, respectively), thus, a significant correlation was found with a more advanced stage at diagnosis (P=0.004). A worse outcome was also significantly correlated with the presence of a somatic RET mutation (P=0.002). Among all prognostic factors found to be correlated with a worse outcome, at multivariate analysis only the advanced stage at diagnosis and the presence of a RET mutation showed an independent correlation (P<0.0001 and P=0.01, respectively). Finally, the survival curves of MTC patients showed a significantly lower percentage of surviving patients in the group with RET mutations (P=0.006). CONCLUSIONS: We demonstrated that the presence of a somatic RET mutation correlates with a worse outcome of MTC patients, not only for the highest probability to have persistence of the disease, but also for a lower survival rate in a long-term follow up. More interestingly, the presence of a somatic RET mutation correlates with the presence of lymph node metastases at diagnosis, which is a known bad prognostic factor for the definitive cure of MTC patients.

RET genetic screening in patients with medullary thyroid cancer and their relatives: experience with 807 individuals at one center.

BACKGROUND: Germline RET gene mutations are causative of multiple endocrine neoplasia (MEN) 2 and may be identified by genetic screening. Three different syndromes are distinguished: MEN 2A, when medullary thyroid carcinoma (MTC) is associated with pheochromocytoma and/or parathyroid adenomas; MEN 2B, when accompanied by a marfanoid habitus and/or pheochromocytoma; and familial medullary thyroid carcinoma (FMTC), when only MTC is present. PATIENTS AND METHODS: During the last 13 yr, we performed RET genetic screening in 807 subjects: 481 with apparently sporadic MTC, 37 with clinical evidence of MEN 2, and 289 relatives. Genomic DNA was extracted from the blood of all subjects, and exons 10, 11, 13, 14, 15, and 16 were analyzed by direct sequencing after PCR. RESULTS: We unexpectedly discovered a germline RET mutation in 35 of 481 (7.3%) apparently sporadic MTC patients. A germline RET mutation was also found in 36 of 37 patients with clinical evidence of hereditary MTC. The distribution of RET mutations in cysteine and noncysteine encoding codons was significantly different in the two groups of patients, with the prevalence of RET mutations in noncysteine codons being higher in MTC that presented as apparently sporadic (P < 0.0001). A total of 34 FMTCs (75.5% of all FMTC) arrived with apparent sporadic MTC, with no familial history of other MTC cases. According to genetic screening and clinical data, our 72 families were classified as follows: 45 FMTC (62.5%), 22 MEN 2A (30.5%), and five MEN 2B (7%). CONCLUSIONS: In this large series of MTC, hereditary forms, mainly FMTC, were clinically unsuspected in 7.3% of apparently sporadic cases. As a consequence, the prevalence of FMTC in our series is higher than that previously reported (60 vs. 10%). In these cases, RET mutations were more prevalently located in noncysteine codons. Data derived from our series helped elucidate the role of RET genetic screening for the identification of all forms of MEN 2, and especially for FMTC, which are frequently clinically misdiagnosed as nonheritable, sporadic cases.

Identification of a novel point mutation in the RET gene (Ala883Thr), which is associated with medullary thyroid carcinoma phenotype only in homozygous condition.

The RET protooncogene mutations responsible for multiple endocrine neoplasia type 2 are inherited as autosomic dominant traits. We describe here a novel germline homozygous mutation in exon 15 of the RET gene that determines an amino acid substitution (Ala->Thr) at codon 883. The index case was a 51-yr-old patient with an apparently sporadic form of medullary thyroid cancer (MTC). RET gene mutations screening was performed in exons 10, 11, 13, 14, 15, and 16 by automatic sequence analysis. An unexpected homozygous GCT->ACT point mutation was found at codon 883 in exon 15 and confirmed by restriction analysis (Alu I). The presence of the two chromosomes 10 was confirmed by fluorescence in situ hybridization analysis on lymphocytes. As expected on the basis of the homozygosity of the index case, the parents were consanguineous (second-degree cousins). Eight relatives were further investigated: the mother, two sisters, and the son were positive for heterozygous RET mutation. The mother (82 yr old) showed a nodular goiter but was negative both for basal and pentagastrin stimulated calcitonin. The young son (15 yr old) and the two sisters (63 and 58 yr old) did not show any clinical and/or biochemical sign of MTC. One brother (59 yr old) was negative both for RET mutation and clinical/biochemical examination. The other brother, 56 yr of age, was positive for both homozygous RET mutation and serum calcitonin. When operated, the histological examination of the thyroid showed the presence of MTC and C cell hyperplasia. In conclusion, we identified a new germline RET gene mutation during a routine RET gene screening of an apparently sporadic MTC case. This mutation showed a very low transforming activity as demonstrated by the absence of MTC phenotype in heterozygous subjects. The possibility that the homozygous gene carriers were indeed carrying a germline loss of heterozygosity was excluded by fluorescence in situ hybridization analysis for RET gene performed on lymphocytes derived from one homozygous patient. The analysis of several RET polymorphisms also confirmed the presence of two mutated alleles in MTC affected patients and both mutated and wild-type allele in heterozygous subjects.

RET exon 11 (G691S) polymorphism is significantly more frequent in sporadic medullary thyroid carcinoma than in the general population.

The RET protooncogene is constitutively activated by point mutations in hereditary medullary thyroid carcinomas (MTCs). RET somatic point mutations have also been reported in 40-50% of sporadic MTCs. Several single nucleotide polymorphisms of the RET gene have been described in the general population as well as in patients with MTC. These allelic variants do not seem to confer any transforming activity to the tyrosine kinase domain of the RET gene. Because the exon 11 RET polymorphism determines an important aminoacidic variation (G691S), we studied its frequency in 212 subjects, 106 sporadic MTC patients and 106 normal age-, sex-, race-, and geographic origin-matched controls. In 46 cases of sporadic MTCs, we also studied the cosegregation of somatic RET gene mutation and G691S polymorphism as well as the linkage of the polymorphism with RET germline mutation in 60 members of eight multiple endocrine neoplasia type 2 families. The influence of this polymorphism on the RET gene transcription has also been studied. In parallel we analyzed the frequencies of another three neutral polymorphisms (L769L, S836S, S904S). We found a statistically significant (P = 0.029) higher allelic frequency of G691S polymorphism in MTCs (27.83%) than that found in normal controls (18.86%), at variance with the three neutral polymorphisms whose frequencies were not different in patients and controls. With this study we excluded the influence of the G691S polymorphism on RET mRNA expression, the development of the somatic RET mutation, the linkage with the germline RET mutation, the younger onset of the MTCs, and the clinical outcome of the disease. A putative role of the G691S polymorphism as genetic modifier in the normal subjects remains to be established.

Medullary and papillary tumors are frequently associated in the same thyroid gland without evidence of reciprocal influence in their biologic behavior.

Papillary thyroid microcarcinoma (mPTC), is a very frequent incidental finding with a frequency varying from a few percent to 35% at postmortem histopathologic examinations. However, the presence of mPTC in patients undergoing thyroidectomy for multinodular goiter (MNG) and for Graves' disease (GD) has been found to be lower. Patients with medullary thyroid carcinoma (MTC) and papillary thyroid carcinoma (PTC) association have been published as anecdotal case reports, as well as kindred with familial MTC or multiple endocrine neoplasia (MEN) 2A with some members simultaneously affected by MTC and PTC. We studied the prevalence and the biological behavior of MTC associated with PTC, with particular attention to those cases in which a mPTC was incidentally found. Twenty-seven of 196 (13.8%) MTC cases showed an association with PTC and in particular 21 of 190 (11.05%) with an incidental mPTC. This percentage is higher than that reported in the literature on the association of mPTC with GD (2.8%-4.5%) and MNG (3%). Also the percentage of the more general association of MTC/PTC, not restricted to mPTC, found in our series (13.8%) is higher than that reported in studies that analyzed the prevalence of PTC (any size) in patients treated for MNG (7.5%). A similarly high percentage of MTC/PTC had not been reported before and in particular there are no reports on large series of MTC/PTC. We also analyzed the epidemiologic, clinical, and pathologic features of MTC associated and not associated with PTC without finding any difference. In particular the outcome of the MTC did not appear to be influenced by the presence of the PTC and the specific radioiodine treatments. Moreover, although we cannot completely exclude a shared pathogenic event as the cause of both MTC and PTC, the molecular analysis of RET gene alterations did not show any common mutation.