Mutation of the RET proto-oncogene is correlated with RET immunostaining in subpopulations of cells in sporadic medullary thyroid carcinoma

Mutations in the RET proto-oncogene, which encodes a receptor tyrosine kinase, are associated with the pathogenesis of medullary thyroid carcinoma (MTC). Somatic mutations in RET, predominantly at codon 918, and very rarely at codon 883, have been found in a proportion of sporadic MTC. We have previously shown that approximately 80% of sporadic MTCs had at least one subpopulation with a somatic RET mutation. Uneven distribution of somatic mutation within a single tumor or among metastases from a single individual was notable. In the present study, we sought to correlate RET expression, as demonstrated by RET immunohistochemistry, with mutation status in sporadic MTC for each tumor. Seventy evaluable subpopulations, belonging to 28 unrelated sporadic cases, comprising primary MTC and metastases, were immunostained with two different polyclonal antibodies raised against the C-terminus of RET. The regional presence of codon 918 or 883 seemed to coincide with increased RET immunopositivity in at least 62 of 70 (89%, P < 0.000001) tumor subpopulations. The reasons for this concordance are not entirely clear but could be related to either RNA or protein stability. Preliminary studies have suggested that the presence of somatic codon 918 mutation in MTC has a prognostic significance. If these preliminary results prove true, then given our data, we can further explore the feasibility of RET immunocytochemistry as a rapid assessment for the presence of somatic codon 918 for molecular diagnostic and prognostic purposes.     

Analysis of RET proto-oncogene abnormalities in patients with MEN 2A, MEN 2B, familial or sporadic medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) may occur either as a sporadic or familial (FMTC) disease. Multiple endocrine neoplasia (MEN) type 2, inherited as an autosomal dominant disease, is characterized by coexistence of MTC with other endocrine neoplasia. Activating mutations of the RET proto-oncogene, involving the somatic or the germinal cell lineage, are found in both inherited and acquired forms. In this study, RET mutations were screened in 47 individuals either affected by MTC or belonging to families with hereditary MTC. Exons 10, 11, 13, 14, 15 and 16 of the RET gene were amplified by polymerase chain reaction and examined by DNA sequence and/or restriction enzyme analysis to detect mutations in purified amplicons. Six MEN 2A families with a germline mutation at codon 634, one FMTC family carrying a mutation at codon 618 and two MEN 2B families with a mutation at codon 918 were identified. In affected members of a MEN 2A family no known RET mutations were observed. Besides, we identified a germline mutation in a patient with apparently sporadic MTC and in two out of three sons, indicating the presence of a sporadic misclassified familial disease. In all of the families examined we were able to distinguish the affected vs unaffected (not at risk) members. A somatic mutation of codon 918 was detected in three out of ten patients with apparently sporadic MTC.     

Molecular analysis of the RET proto-oncogene in patients with sporadic medullary thyroid carcinoma: a novel point mutation in the extracellular cysteine-rich domain

A filiform stenosis of the popliteal artery was examined by sonography and the reason for the cystic lesion in the arterial wall was found to be an uncommon type of cystic degeneration of the adventitia. The narrow lumen could be demonstrated by sonography and the lesion was punctured under ultrasound control. A viscous secretion was removed and the patient's claudication was cured without recourse to surgery.     

A new inherited RET proto-oncogene mutation associated with familial medullary thyroid carcinoma and polymorphisms in adjacent regions

A new point mutation, TCG(Ser)-->GCG(Ala) in codon 891, exon 15 of the RET protooncogene was revealed in two patients from a pedigree with familial medullary thyroid carcinoma (FMTC), but not in healthy persons. A linkage analysis with two well-known and two new intragene polymorphisms showed that informative polymorphic markers, the phenotypic expression of the disease, and the mutation are cosegregated in the studied pedigree. Two new polymorphisms, G/A at position-24 of intron 14 and C/T in codon 836 of exon 14, were found in the RET protooncogene. The frequencies of allele 1 of the polymorphic site in codon 836 were the same (0.96) in the Russian and German populations. This was also characteristic of two polymorphisms revealed earlier, namely, the sites in codons 691 (0.80 and 0.81, respectively) and 904 (0.21 and 0.22). However, the frequency of allele 1 of the polymorphisms in intron 14 differed significantly (0.87 and 0.77, respectively).     

Somatic mutations of the ret protooncogene in sporadic medullary thyroid carcinoma are not restricted to exon 16 and are associated with tumor recurrence

Germline point mutations in exons 10, 11, and 16 of the ret protooncogene have been identified as causative in multiple endocrine neoplasia type 2 and in familial medullary thyroid carcinoma (MTC). Somatic point mutations of the same gene, exclusively associated with codon 918 of exon 16, have also been reported in few cases of sporadic medullary thyroid carcinoma. We analyzed the blood and tumor DNA of 19 patients with sporadic MTC and 6 patients with primary parathyroid adenoma for point mutations at exons 10, 11, and 16 of the ret protooncogene by restriction analysis of the PCR-amplified product and by sequence analysis of exons 10 and 11. A Cys634-->Tyr mutation was found in both the tumoral and blood DNA of one patient, indicating that he was affected by an hereditary form of MTC, erroneously considered sporadic. In the other 18 patients with MTC, somatic point mutations of ret were found in 8 cases (44.4%). In 5 cases the mutation affected exon 16 (Met918-->Thr), and in 3 cases it affected exon 11 (Cys634-->Arg in 1 and Cys634-->Trp in 2); these 3 mutations were confirmed by sequence analysis. The remaining 10 patients had no mutation in exon 10 by either restriction analysis or sequence analysis. Clinical data showed that 75% of the patients whose tumor carried ret mutation had tumor recurrence and/or increased serum calcitonin concentrations during the postsurgical follow-up period as opposed to 10% of the patients without mutations (P < 0.02, by chi2 analysis). No ret mutation was found in the tumoral DNA from parathyroid adenomas. Our findings indicate that the somatic ret point mutation frequently found in sporadic MTC may affect not only exon 16 but also exon 11 and is associated with less favorable clinical outcome.     

Determinative factors of biochemical cure after primary and reoperative surgery for sporadic medullary thyroid carcinoma

Normalization of calcitonin levels after surgery has been regarded as the most powerful prognostic factor for medullary thyroid carcinoma (MTC). Although the prognosis of patients with persistent hypercalcitoninemia may be acceptable, the biochemical cure rate can be improved by new microdissection techniques. This raises certain questions: Can extension of locoregional lymphadenectomy (LA) further improve biochemical cure and survival after primary or reoperative MTC surgery? Which factors concerning TNM categories are associated with the possibility of postoperative normalization of calcitonin levels? This study included 64 patients with sporadic MTC operated on from 1986 to 1997. Altogether 27 patients underwent primary surgery, and 37 patients were reoperated, performing a microdissection of all four locoregional compartments (four-compartment lymphadenectomy, or 4CLA). For primary MTC the biochemical cure rate was 100% in node-negative patients and 33% in node-positive patients; the latter could be improved to 45% after 4CLA. In contrast to reoperative MTC, the rate of lymph node metastases (LNMs) with primary MTC correlated with the pT category (pT1 33%, pT2 53%, pT3 100%, pT4 100%) but not with age or sex. Again in contrast to reoperative MTC, mediastinal LNMs in primary MTC were present only in patients with a pT4 tumor. At reoperation, 4CLA was able to cure 22% of node-positive patients, 28% without proved distant metastases. No patient with extrathyroidal tumor involvement or distant metastases was biochemically cured after either primary or reoperative surgery. For all node-positive MTC patients, in addition to cervicocentral LA at least a bilateral cervicolateral LA is recommended. Transsternal mediastinal lymph node dissection is indicated in patients with LNMs in the cervicomediastinal transition, facilitating biochemical cure in up to 45% after the first operation and 22% after reoperative surgery of sporadic MTC.     

Mutations of ret-proto-oncogene in thyroid medullary carcinoma

OBJECTIVE: To determine the importance of the molecular-genetic demonstration of germ-line mutation in the ret protooncogene for therapeutic measures in sporadic and hereditary medullary thyroid carcinoma (MTC). PATIENTS AND METHODS: Several molecular-genetic tests were performed on DNA of 35 families with hereditary and 81 patients with the sporadic form of MTC (isolation of genomic DNA; PCR amplification; DNA sequencing: demonstration of mutation in codon 918 with restriction enzyme FOK 1). RESULTS: A disease risk was demonstrated in 178 individuals among the 35 families, 159 of whom were investigated by molecular-genetic tests: 84 family members were found to be gene carriers. Germ-line mutation had already been suspected on clinical grounds in 76% of the carriers, 24% being discovered in a presymptomatic stage. Six children among the latter were treated prophylactically by thyroidectomy, histological evidence of C-cell hyperplasia being found in all of them, microcarcinomas in three of the older children. There were four patients among the non-carriers on whom thyroidectomy had been performed previously because of a false-positive pentagastrin-test; but germ-line mutation was now excluded. In one family, with familial MTC in two brothers, no mutation in ret-proto-oncogene has been demonstrated. The members of this family must now, as used to be routine, undergo a pentagastrin-test. Three of the 81 patients with "sporadic" MTC had a germ-line mutation, presumably a new one. CONCLUSION: Molecular-genetic tests have further improved the management of families with hereditary MTC and they thus take first place among essential diagnostic procedures. The diagnosis of sporadic MTC can be confirmed by excluding germ-line mutation in the ret-proto-oncogene.     

Differential display in primary and metastatic medullary thyroid carcinoma

Platelet-activating factor (PAF) is a mediator produced in human airways during acute and chronic inflammatory lung diseases. The levels of PAF are regulated by acetylhydrolase (AH), the enzyme that converts PAF to lyso-PAF. To determine whether AH was present in human bronchoalveolar lavage (BAL) fluid, BAL was obtained from normal donors (n = 18) and from adult patients with mild bronchial asthma (n = 15) or with lung fibrosis (n = 15). AH activity was consistently found in the cell-free BAL fluid. BAL-AH is an enzyme different from secretory phospholipase A2 and from plasma AH and erythrocyte AH. Furthermore, BAL-AH is inhibited as much as 95% by exposure to an oxygen radical-generating system (xanthine/xanthine oxidase). BAL-AH is significantly correlated with the number of BAL macrophages (rs = 0.63; p < 0.02). In addition, BAL macrophages release AH both spontaneously and after stimulation with tumor necrosis factor-alpha (TNF-alpha) (100 ng/ml). BAL-AH activity in patients with bronchial asthma (1.32 +/- 0.18 pmol of PAF converted to lyso-PAF/min) is significantly lower than that in normal donors (2.25 +/- 0.26 pmol/min; p < 0.001). In contrast, BAL-AH activity in patients with lung fibrosis (6.13 +/- 0.81 pmol/min) is higher than that found in normal donors (p < 0.01). The variations in BAL-AH activity in patients with bronchial asthma or lung fibrosis are due to a reduction and to an increase, respectively, in the number of active molecules rather than to changes in enzyme affinity. These data demonstrate that human BAL fluid contains an extracellular AH activity that inactivates PAF released in the airways. BAL-AH is secreted by alveolar macrophages and is highly sensitive to oxygen radical-induced damage. The secretion and inactivation of BAL-AH may influence the levels of this enzyme in BAL fluid during acute and chronic inflammatory lung diseases and, ultimately, regulate the proinflammatory activities of PAF in these disorders.     

TFF1 gene expression in human medullary thyroid carcinoma

This epidemiological study investigated the reasons why children in Northern Ireland who need orthodontic treatment do not receive treatment even when it is provided free by the state. A total of 1584 15- and 16-year-olds were examined in 23 high schools with the Index of Orthodontic Treatment Need. The characteristics of the adolescents who had received orthodontic treatment were compared with those who had a definite need for treatment and yet did not receive treatment or advice. One in 10 of the adolescents examined had an unmet need for orthodontic treatment. Logistic regression analysis was used to assess the influence of 11 variables including socioeconomic status, religion, and standard of dental health on the uptake of orthodontic care. This analysis revealed that the only significant predictors of whether an adolescent received orthodontic treatment was the dental attendance pattern of the adolescent, the adolescent's dental health, and the dental attendance pattern of the adolescent's mother. Those adolescents who had good dental health, who regularly attended a dentist, and whose mother regularly attended a dentist were more likely to receive orthodontic treatment.     

Detection of a homozygous four base pair deletion in the protein X gene in a case of pyruvate dehydrogenase complex deficiency

While the presence of a lipoyl-containing protein (protein X) separate from lipoyl transacetylase in the pyruvate dehydrogenase complex (PDC) has been known for some time, until recently only the cDNA for the yeast enzyme has been cloned. We have cloned, sequenced and characterized the cDNA encoding the human protein X and localized the protein X gene to chromosome 11p13. We also report here a new case of protein X deficiency identified immunologically, with decreased activity of PDC and without mutations in the E1alpha subunit or E1beta subunit. We report that the cDNA and gene of this patient for protein X has a homozygous 4 bp deletion, specifically in the putative mitochondrial targeting signal sequence which results in a premature stop codon. This is the first documented case of a molecular defect in pyruvate dehydrogenase protein X.     

Elevated CEA in breast cancer patients with overlooked medullary thyroid carcinoma

After mastectomy and radiation for breast cancer, two patients were found to have persistent elevated CEA in their serum. This finding was erroneously attributed to occult metastases for the first patient and to local recurrence for the second. Overlooked medullary thyroid carcinoma (MTC) was the causal disease in both patients. A review of the literature stresses the frequency of CEA elevation in serum of MTC patients. A thorough search for any possible cause of elevated levels of CEA is advocated, particularly by thyroid sonogram with a needle aspiration biopsy when a nodule is discovered and by calcitonin assay in the serum.     

The prognostic importance of calcitonin screening in familial medullary thyroid carcinoma

A prospective study of 20 patients with traumatic amputations of the fingers and a thumb was carried out during a six week period at Mpilo Central Hospital, Bulawayo. Most of the adult injuries (17 patients) resulted from industrial accidents (76.5 pc) while two out the three children sustained their injury from domestic accidents. Inadequate safety precaution, ignorance and human error contributed significantly to the injuries. Safety education and preventive measures both at home and in industry should be inoculated into the daily life of individuals.     

Presence of atrial natriuretic peptide in rat thyroid medullary carcinoma cell line

In 1923, Masson described a neoplastic process consisting of papillary hyperplasia of the endothelial cells, with a consequent obliteration of the vascular lumen, followed later by degenerative changes. Masson coined the term vegetant intravascular haemangioendothelioma, however, these days it is more commonly known as papillary endothelial hyperplasia (PEH), or by the pseudonym, Masson's tumour. Although relatively rare, there are numerous accounts of PEH in the literature, describing its predilection for the head and neck region. Our case report describes the finding of a PEH within the paranasal sinuses, a site not previously mentioned even in the largest of series found on literature search. We will then discuss the relevant histological features of the lesion, and its natural history.     

Advances and controversies in the diagnosis and management of medullary thyroid carcinoma

In tasks requiring sustained attention, human alertness varies on a minute time scale. This can have serious consequences in occupations ranging from air traffic control to monitoring of nuclear power plants. Changes in the electroencephalographic (EEG) power spectrum accompany these fluctuations in the level of alertness, as assessed by measuring simultaneous changes in EEG and performance on an auditory monitoring task. By combining power spectrum estimation, principal component analysis and artificial neural networks, we show that continuous, accurate, noninvasive, and near real-time estimation of an operator's global level of alertness is feasible using EEG measures recorded from as few as two central scalp sites. This demonstration could lead to a practical system for noninvasive monitoring of the cognitive state of human operators in attention-critical settings.     

Multiple endocrine neoplasia-type medullary thyroid carcinoma in three generations of a family

The familial accumulation of a multiple endocrine neoplasia (MEN) type 2a medullary thyroid carcinoma, is described based on the retrospective analysis of a family history. The proband was characterized by medullary carcinoma (MC) combined with phaeochromocytoma, her child had been shown to suffer from MC. In the third generation of the family C-cell hyperplasia and bilateral adrenal hyperplasia occurred. It is shown that in order to arrive at a correct decision as regards the therapy to be used, modern laboratory tests (serum calcitonin, CEA-analysis) and diagnostic imaging methods (ultrasound, computer tomography, magnetic resonance imaging, positron emission tomography, metaiodobenzylguanidine scintigraphy) should be used. It is emphasized that the available therapeutic means (surgery, radiotherapy, nuclear medicine) have to be carefully selected and, if necessary, combined. In medullary thyroid carcinoma associated tumours in other endocrine organs should be expected to occur. Family screening using blood chemical and genetic tests are recommended in asymptomatic cases, since their surgical treatment can in this way lead to complete recovery.     

Use of somatostatin analogue scintigraphy in the localization of recurrent medullary thyroid carcinoma

Detection of recurrence of medullary thyroid carcinoma (MTC) remains a diagnostic problem. Increased serum tumour marker levels frequently indicate recurrence while conventional imaging techniques (CIT) are non-diagnostic. In this study, we performed indium-111 octreotide scintigraphy and CIT in a series of 20 patients with MTC presenting with elevated serum tumour markers after surgery. 111In-octreotide whole-body studies detected 15 pathological uptake foci in 11 of the 20 patients studied and CIT detected 17 lesions in 11 of the 20 patients. Ten patients underwent reoperation, five of them with positive 111In-octreotide scintigraphy and CIT and two with positive isotopic exploration and negative CIT. Surgical findings demonstrated that the results of isotopic study and CIT had been false-positive for MTC in one case (sarcoidosis). The six patients with true-positive 111In-octreotide studies had significantly higher basal calcitonin (CT) and carcinoembryonic antigen (CEA) levels than the patients with negative isotopic studies. The expression of somatostatin receptor (SSTR) subtypes by PC-PCR could be investigated in four cases with a positive isotopic study. Among the three cases with a true-positive study, SSTR2, the SSTR subtype that preferentially binds to the somatostatin analogue octreotide, was detected in two, SSTR5 was demonstrated in the three, and SSTR3 was detected in one. No subtype of SSTR was detected in the case with a final diagnosis of sarcoidosis. We conclude that 111In-octreotide has limited sensitivity in detecting recurrence in patients with MTC, although its sensitivity may improve with high serum CT levels. This radionuclide imaging technique should be employed when conventional imaging techniques are negative or inconclusive or when the presence of somatostatin receptors may provide the basis for treatment with somatostatin analogues.     

C-cell hyperplasia and medullary thyroid carcinoma in patients routinely screened for serum calcitonin

To elucidate the differential reactivity of pulmonary microvessels in the acini to hypoxia, excessive CO2, and increased H+, we investigated changes in the diameter of precapillary arterioles, postcapillary venules, and capillaries in isolated rat lungs on exposure to normocapnic hypoxia (2% O2), normoxic hypercapnia (15% CO2), and isocapnic acidosis (0.01 mol/L HCl). Microvascular diameters were precisely examined using a real-time confocal laser scanning luminescence microscope coupled to a high-sensitivity camera with an image intensifier. Measurements were made under conditions with and without indomethacin or N(omega)-nitro-L-arginine methyl ester to assess the importance of vasoactive substances produced by cyclooxygenase (COX) or NO synthase (NOS) as it relates to the reactivity of pulmonary microvessels to physiological stimuli. We found that acute hypoxia contracted precapillary arterioles that had diameters of 20 to 30 microm but did not constrict postcapillary venules of similar size. COX- and NOS-related vasoactive substances did not modulate hypoxia-elicited arteriolar constriction. Hypercapnia induced a distinct venular dilatation closely associated with vasodilators produced by COX but not by NOS. Arterioles were appreciably constricted in isocapnic acidosis when NOS, but not COX, was suppressed, whereas venules showed no constrictive response even when both enzymes were inhibited. Capillaries were neither constricted nor dilated under any experimental conditions. These findings suggest that reactivity to hypoxia, CO2, and H+ is not qualitatively similar among intra-acinar microvessels, in which COX- and NOS-associated vasoactive substances function differently.     

Prophylactic thyroidectomy for medullary thyroid carcinoma in gene carriers of MEN2 syndrome

BACKGROUND/PURPOSE: Although medullary thyroid carcinoma (MTC) can occur sporadically, in the pediatric population it is most often associated with the multiple endocrine neoplasia syndrome (MEN type 2). Traditional screening was based on evaluation of basal and stimulated serum calcitonin levels. The recent cloning of the MEN2 gene on the RET proto-oncogene of chromosome 10 now allows for testing of gene carrier status in individuals at risk who could benefit from prophylactic treatment. The current study was undertaken to determine the appropriate age for safe total prophylactic thyroidectomy. METHODS: Over a 16-year period, 12 patients with a family history of MEN2A and one with a MEN2B underwent total thyroidectomy and central neck dissection without parathyroid autotransplantation. Four patients (31%) were treated previously for Hirschsprung's disease. RESULTS: In seven patients (mean age, 11.8 years) undergoing biochemical screening for diagnosis, multifocal MTC and C cell hyperplasia (CCH) were found in all the resected specimens. Of six patients identified with genetic screening (mean age, 9.1 years), two had elevated stimulated calcitonin levels, one (age 14) had evidence of MTC, and one (age 6) had CCH. Four patients with normal calcitonin levels had no evidence of MTC (ages 6, 8, 10) but there was one occurrence of CCH (age 11). No permanent postoperative hypoparathyroidism or recurrent laryngeal nerve damage occurred in this series. With a mean follow-up of 4 years (range, 1 to 14 years), the overall disease-free survival is 100%. CONCLUSIONS: From this study the authors conclude that total thyroidectomy can be performed safely in children and should be the treatment of choice in patients with a family history of MEN2A carrying a germinal RET mutation even if the serum basal or stimulated serum calcitonin level is normal. Total thyroidectomy should be performed as early as 5 years of age before the occurrence of CCH or MTC.     

Usefulness of the genetic study in the diagnosis of medullary carcinoma of the thyroid

Germ-line mutations in the RET proto-oncogene are associated with multiple endocrine neoplasia type 2A (MEN 2A) and with familial medullary thyroid carcinoma (FMTC). Detection of these mutations allows the identification of the affected kindred members, who will develop medullary thyroid carcinoma (MTC) in 100% of cases. We studied 24 patients of two kindreds (MEN 2A and FMTC). Basal calcitonin levels and pentagastrin-stimulated calcitonin were measured in all patients. The RET mutations were detected by DNA analysis. The RET mutations were identified in 14 patients. Two of them had been operated in the past, 2 refused operation and 4 were living abroad. In the 6 remaining, only one showed a thyroid mass, basal calcitonin was normal in all patients except one, and pentagastrin-stimulated calcitonin was negative in 2 patients. Total thyroidectomy was performed in all cases. Histology showed C-cell hyperplasia in all patients and MTC in 5 of them. In MEN 2A and FMTC DNA analysis allows the identification of RET mutation carriers, in which presymptomatic thyroidectomy allows and improvement in survival.