Erratum to "Gastroenteropancreatic Neuroendocrine Neoplasia Characterization in Portugal: Results from the NETs Study Group of the Portuguese Society of Endocrinology, Diabetes and Metabolism".

[This corrects the article DOI: 10.1155/2019/4518742.].

Gastroenteropancreatic Neuroendocrine Neoplasia Characterization in Portugal: Results from the NETs Study Group of the Portuguese Society of Endocrinology, Diabetes and Metabolism.

BACKGROUND: The incidence of gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) has been increasing in the last five decades, but there is no large-scale data regarding these tumours in Portugal. We conducted a cross-sectional, multicentric study in main Portuguese centers to evaluate the clinical, pathological, and therapeutic profile of GEP-NENs. METHODS: From November, 2012, to July, 2014, data from 293 patients diagnosed with GEP-NENs from 15 centers in Portugal was collected and registered in an online electronic platform. RESULTS: Median age at diagnosis was 56.5 (range: 15-87) years with a preponderance of females (54.6%). The most frequent primary sites were the pancreas (31.1%), jejunum-ileum (24.2%), stomach (13.7%), and rectum (8.5%). Data regarding hormonal status was not available in most patients (82.3%). Stratified by the tumour grade (WHO 2010 classification), we observed 64.0% of NET G1, 24.7% of NET G2, and 11.3% of NEC. Poorly differentiated tumours occurred mainly in older patients (p = 0.017), were larger (p < 0.001), and presented more vascular (p = 0.004) and lymphatic (p = 0.001) invasion. At the time of diagnosis, 44.4% of GEP-NENs presented metastatic disease. Surgery (79.6%) and somatostatin analogues (30.7%) were the most frequently used therapies of GEP-NENs with reported grading. CONCLUSION: In general, Portuguese patients with GEP-NENs presented similar characteristics to other populations described in the literature. This cross-sectional study represents the first step to establish a national database of GEP-NENs that may aid in understanding the clinical and epidemiological features of these tumours in Portugal.

Identification and characterization of two novel germline RET variants associated with medullary thyroid carcinoma.

Activating germline mutations in the RET proto-oncogene are responsible for about 98 % of the familial forms of medullary thyroid carcinoma (MTC), which represent 25 % of all MTC cases. The search for germline mutations in this gene is important for the recognition of hereditary forms of MTC and further identification of at-risk relatives who may benefit from early clinical intervention. Genotype-phenotype correlations are well established for most disease-causing RET mutations, allowing risk stratification. The association of a new RET variant with the MTC phenotype and familial predisposition requires the assessment of its functional and clinical significance. The aim of this study was to evaluate the oncogenic potential of two newly identified RET germline variants associated with late-onset MTC. In vitro functional assays were designed to address the transforming potential of novel RET variants, through their expression in non-transformed cells, and comparing their effect with wild-type RET. The new variants were identified in codons 515 (p.C515W) and 636 (p.T636M) located, respectively, in exons 8 and 11, thus resulting in amino acid substitutions in the extracellular region of the tyrosine kinase receptor RET. Through functional assays, we observed increased cell growth and proliferation, loss of contact inhibition, and a stimulation of cell migration, suggesting that these new RET variants hold some relevant transforming potential. The transforming potential of these novel RET variants was of low-grade, when compared to that of RET MEN2A-causing mutation p.C634R, probably explaining the mild phenotype characterized by late onset and low clinical aggressiveness.

Identification of three new variants of SDHx genes in a cohort of Portuguese patients with extra-adrenal paragangliomas.

BACKGROUND: Extra-adrenal paragangliomas (PGL) are rare neoplasms occurring in sporadic and familial forms, the latter mostly in association with germline mutations of SD- HB, SDHC or SDHD genes. AIM: Characterize frequency and spectrum of germline mutations among a cohort of Portuguese patients with extra-adrenal PGL. DESIGN: Molecular and clinical data were reviewed on 44 patients referred for genetic testing by a single laboratory. RESULTS: Genetic analysis identified 11 patients with head and neck PGL (30.6%) positive for SD- Hx gene mutations (6 SDHD, 4 SDHB, 1 SDHC) and 4 patients with abdominal or pelvic PGL (50%) positive for SDHx gene mutations (4 SDHB). Large deletions made up about 20% of the mutations detected. Mutation carriers were younger and more frequently had multiple or malignant PGL than patients without mutations. Only 11% of the head and neck PGL were secretory. In contrast, 100% of the abdominal or pelvic PGL were secretory. Five patients had a malignant PGL (4 SDHB, 1 apparently sporadic). Three novel mutations were identified: two in the SDHD gene (c.411delT [p.Leu139PhefsX29] and c.371_390del20insGG [p.Ala124_Ala130delinsGly]), one in the SDHB gene (c.49A>G [p.Thr17Ala]). The SDHD variant c.411delT [p.Leu139PhefsX29] was present in 3 apparently unrelated patients. Molecular genetic testing of 22 relatives disclosed 16 mutation carriers. CONCLUSIONS: Genetic analysis identified 15 patients (34.1%) and 16 at-risk individuals (72.7%) positive for SDHx gene mutations. The finding of three novel mutations broadens the mutational profile of the mitochondrial complex II succinate dehydrogenase genes reported in other large European series of patients with paragangliomas. Further studies are needed to clarify whether the high frequency of the SDHD variant c.411delT [p.Leu139PhefsX29] corresponds to a founder mutation.

Correlation of RET somatic mutations with clinicopathological features in sporadic medullary thyroid carcinomas.

Screening of REarranged during Transfection (RET) gene mutations has been carried out in different series of sporadic medullary thyroid carcinomas (MTC). RET-positive tumours seem to be associated to a worse clinical outcome. However, the correlation between the type of RET mutation and the patients' clinicopathological data has not been evaluated yet. We analysed RET exons 5, 8, 10-16 in fifty-one sporadic MTC, and found somatic mutations in thirty-three (64.7%) tumours. Among the RET-positive cases, exon 16 was the most frequently affected (60.6%). Two novel somatic mutations (Cys630Gly, c.1881del18) were identified. MTC patients were divided into three groups: group 1, with mutations in RET exons 15 and 16; group 2, with other RET mutations; group 3, having no RET mutations. Group 1 had higher prevalence (P=0.0051) and number of lymph node metastases (P=0.0017), and presented more often multifocal tumours (P=0.037) and persistent disease at last control (P=0.0242) than group 2. Detectable serum calcitonin levels at last screening (P=0.0119) and stage IV disease (P=0.0145) were more frequent in group 1, than in the other groups. Our results suggest that, among the sporadic MTC, cases with RET mutations in exons 15 and 16 are associated with the worst prognosis. Cases with other RET mutations have the most indolent course, and those with no RET mutations have an intermediate risk.

Searching for RET/PTC rearrangements and BRAF V599E mutation in thyroid aspirates might contribute to establish a preoperative diagnosis of papillary thyroid carcinoma.

OBJECTIVE: Searching for multiple molecular markers in thyroid aspirates appears to be a promising approach for establishing a preoperative diagnosis of papillary thyroid carcinoma (PTC). METHODS: Based on this hypothesis, a total of 63 samples from 55 patients, were collected at random. RNA was extracted from the residue cells inside the needle used for fine needle aspiration cytology (FNAC) and thereafter molecular analysis was carried out both for RETrearrangements (type 1, 2, 3) and BRAF codon 599 mutation molecule. Results were compared with the cytological and histopathological diagnoses in 24 patients submitted to surgery. RESULTS: 58% PTCs presented a genetic alteration either RET/PTC rearrangement, BRAF V599E mutation or both: three cases of PTCs (25%) presented a RET/PTC rearrangement; three cases of PTCs (25%) presented a BRAF V599E mutation and in one case (8%) both alterations were identified. CONCLUSIONS: The present results suggest that searching for multiple molecular markers in thyroid aspirates may enhance the accuracy of FNAC and refine preoperative diagnosis of PTC.

MEN 2A families: from hot spots to hot regions.

The aim of this study was to look for common ancestors among MEN 2A Portuguese families presenting with the same germ-line mutation of the RET proto-oncogene. To address this question from a genetic point of view, we performed haplotype analysis in six out of nine, apparently separate, MEN 2A families using four polymorphic markers. Three families carrying the C634R mutation and presenting the same phenotype shared the same haplotype surrounding the MEN 2A mutation. Moreover, these families were originally from the same geographic region although settled at different places along the country. Altogether, data suggested a common ancestral MEN 2A chromosome for three families. Since MEN 2A is a rare inherited cancer syndrome, identification of common ancestors may draw attention for specific geographic regions from where other affected families may arise at a higher chance and, therefore, termed 'hot regions'.

Detection of thyroglobulin mRNA transcripts in peripheral blood of individuals with and without thyroid glands: evidence for thyroglobulin expression by blood cells.

OBJECTIVE: Recent studies have assigned clinical significance and prognostic value to the detection of thyroglobulin (Tg) mRNA in the blood of patients subjected to total thyroidectomy for a papillary or follicular thyroid carcinoma. In this study, we investigated the diagnostic specificity of Tg mRNA detection, analysing blood samples from healthy volunteers and from patients previously subjected to total thyroidectomy for reasons other than a carcinoma of the follicular epithelium. DESIGN AND METHODS: Total RNA was extracted from whole blood, reverse-transcribed and the cDNA amplified for Tg and glyceraldehyde-3-phosphate dehydrogenase with specific primers. Expression levels were analysed by using a semi-quantitative PCR. In a few cases, Lymphoprep gradients were used to separate the mononuclear and polymorphonuclear cells prior to further analysis by reverse transcription/PCR. RESULTS: Our data suggested that all individuals expressed Tg mRNA. Moreover, no differences in the expression levels between subjects with and without thyroid glands were documented. Documentation of Tg expression by the mononuclear and polymorphonuclear layers in patients without thyroid glands support the hypothesis that both lymphocytes and granulocytes express Tg and may justify a background expression in blood, independently of the presence of follicular cells in circulation. CONCLUSIONS: Tg mRNA expression is not limited to follicular cells of the thyroid gland, and its expression by normal blood cells should be considered in tests performed for diagnostic purposes.

Medullary thyroid carcinoma: an accurate pre-operative diagnosis by reverse transcription-PCR.

OBJECTIVE: To study the expression of calcitonin (CT) and thyroglobulin mRNA in samples of leftover cells in needles used for fine-needle aspiration biopsy either from thyroid tumours or cervical lymph nodes. PATIENTS AND METHODS: Specimens were analysed using reverse transcription-polymerase chain reaction; 12 samples from 11 patients were included and molecular diagnosis was compared with cytological or histological diagnosis and serum CT measurements. RESULTS: Transcripts of the CT gene were detected in all six patients with medullary thyroid carcinoma (MTC) but in none of the other patients. CONCLUSIONS: Present data reinforce this technique as a reliable and alternative tool to establish the pre-operative diagnosis of MTC, especially when cytological examination is not conclusive or when cytological information is not in agreement with clinical data. Furthermore, it may be clinically useful to identify those conditions in which increased serum CT in the presence of a thyroid nodule is not due to MTC.

Presence of a Gs alpha mutation in an adrenal tumor expressing LH/hCG receptors and clinically associated with Cushing's syndrome.

We describe the case of a patient with Cushing's syndrome due to a functioning adrenal adenoma. There was a pronounced increase in serum and urinary cortisol after administration of human chorionic gonadotropin. Immunocytochemistry revealed positive immunostaining for LH/hCG receptors. Molecular analysis documented the presence of a gsp mutation at codon 201 (CGT to TGT). The existence of this type of hCG-responsive adrenal tumor may help explain the higher prevalence of cortisol-secreting adrenal tumors versus pituitary-dependent disease in pregnant women with Cushing's syndrome as well as some reported cases of remission following delivery.

Somatic trinucleotide change encompassing codons 882 and 883 of the RET proto-oncogene in a patient with sporadic medullary thyroid carcinoma.

OBJECTIVE: Restriction analysis is a straightforward procedure for mutational analysis. It is commonly used for screening RET mutations. Incomplete digestion is a well-known cause of false results. Herein, we report another limitation of the method. DESIGN AND METHODS: Screening for somatic mutations in RET exons 16, 13 and 15 was performed in a patient with a sporadic medullary thyroid carcinoma. Genetic study was carried out by both restriction analysis and direct sequencing. RESULTS: A somatic trinucleotide change encompassing codons 882 and 883 of the RET proto-oncogene (GTA GCT to GTT TTT) was documented. Particular to this case is the silent mutation (GTA-->GTT) at codon 882. Independently, both the novel silent mutation and the missense mutation at codon 883 may disrupt the same AluI restriction site. Based on the restriction pattern we were able to say that both mutations occurred in the same allele. CONCLUSIONS: Restriction analysis is an easy approach for screening RET mutations; however, it is not enough to assign a final diagnosis.

Multiple endocrine neoplasia type 2A. Study of a family.

INTRODUCTION: Pheochromocytomas (Pheo) can occur sporadically, isolated or in association with other neuroendocrine lesions. In multiple endocrine neoplasia type 2A (MEN-2A), Pheo is associated to medullary thyroid carcinoma (MTC) or its precursor, C-cell hyperplasia (CCH) and parathyroid hyperplasia. Genetic screening provides early diagnosis and preventive treatment. In order to validate DNA analysis as a reliable method of early identification of gene carriers, we compared the results of genetic screening with clinical, biochemical, imaging and pathological findings in the members of an affected family. POPULATION AND METHODS: The diagnosis of a bilateral necrotic Pheo in a female patient led to the study of a family with four generations, aged 3 to 78 years (mean = 30.3 yrs). The study included a clinical examination; basal and pentagastrin stimulated calcitonin values; urinary catecholamines and their metabolites; serum calcium and a genetic study (direct sequence of PCR products from genomic DNA isolated from leucocytes using specific primers in exon 11 of the RET protooncogene of chromosome 10). The radiologic study, gammagraphic study (131I-MIBG) and magnetic resonance study were performed in members with clinical suspicion of Pheo. RESULTS: Seven out of nine patients had a mutation on codon 634 of exon 11 of RET (TGC-CGC), leading to cysteine arginine substitution in the codified protein; all gene carriers had biochemical markers of MTC/CCH and four of Pheo. The Pheo patients underwent adrenalectomy (bilateral in three) and all the gene carriers underwent prophylactic thyroidectomy. The pathologic findings were: MTC in four (metastasized in one); CCH in three and parathyroid hyperplasia in one. CONCLUSIONS: Phenotypic penetration of RET mutation was 100% for MTC/CCH, but only 57% of the gene carriers had Pheo. Genetic screening allowed early prophylactic treatment in four out of seven patients; pathologic findings revealed several evolutionary stages of the disease. Patients not yet showing Pheo are under close clinical and laboratory surveillance.

ACTH-producing carcinoma of the pituitary with haematogenic metastases.

The case report is presented of a 47-year-old white woman with Cushing's disease treated by bilateral adrenalectomy in June 1981. A first computed tomography (CT) scan in September 1984 showed a voluminous pituitary adenoma with invasion of the sphenoid sinus and left parasellar extension. The tumour increased progressively in size, a suprasellar extension developed and the optic chiasm was eventually affected. In March 1988 and June 1989 the patient underwent two surgeries for the pituitary tumour, the second followed by radiotherapy. During this period, the ACTH values varied between 100 pmol/l and 403 pmol/l (normal: < 13 pmol/l). After radiotherapy, a progressive shrinking of the tumour was observed and the ACTH concentrations decreased to a lowest value of 27.5 pmol/l. The patient was clinically well until September 1993 when, suddenly, the plasma ACTH concentration increased to very high levels (greater than 965 pmol/l). There was no evidence of tumour growth on the sellar CT scan. In January 1995, an ACTH-producing pituitary carcinoma was diagnosed, based on the presence of bone metastases. The patient died in May 1995.

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

Germline point mutations in the RET proto-oncogene are associated with multiple endocrine neoplasia type 2 (2A and 2B) and familial medullary thyroid carcinoma. On the other hand, somatic point mutations of RET have been described in a subset of sporadic medullary thyroid carcinomas (MTCs). We examined tumor and blood DNA of thirteen apparently sporadic MTC patients for mutations in RET exons 10, 11, 13, 15 and 16 to determine whether they had true sporadic tumors or either de novo or occult germline mutations. Three different somatic missense mutations were documented in seven patients. In five patients a mutation in exon 16, codon 918, (ATG-->ACG) causing a Met-->Thr substitution was found. In the remaining two patients the mutation affected exon 11: codon 630 in one case and codon 634 in the other. In both cases a T-->C transversion was identified causing a Cys-->Arg substitution. In conclusion, absence of a germline mutation in RET exons 10, 11, 13 or 16 is evidence against an inherited form in all cases. In seven patients, identification of a somatic mutation supported the previous clinical diagnosis of sporadic medullary thyroid carcinoma; in one of them we identified a hitherto undescribed somatic point mutation at codon 630.

Clinical use of molecular information in the management of multiple endocrine neoplasia type 2A.

One hundred and ninety-seven members of 28 kindreds with multiple endocrine neoplasia type 2A (MEN 2A) were screened for RET proto-oncogene exon 10 and 11 mutations. Seventy-one known affected individuals had mutations of codons 609, 618, 620 or 634, whereas 53 unaffected individuals had no abnormalities. Nineteen out of 54 individuals of unknown status, mostly children, had RET mutations. Four of these children had thyroidectomy based on this analysis and were found to have C-cell abnormalities. We identified one false negative mutation analysis because of a codon 691 polymorphism. We conclude that RET mutational analysis is a cost-effective and accurate method for determination of gene carrier status in MEN 2A.

[Ret proto-oncogene mutations associated with type 2 multiple endocrine neoplasms (MEN 2). Clinical implications]. / Mutações no Ret Proto-oncogene em associação com neoplasias endócrinas múltiplas tipo 2 (MEN 2). Implicações clínicas.

Multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominant cancer syndrome. Clinical features and diagnostic aspects of this syndrome are reviewed with a focus on progress biochemical screening to genetic analysis of those at risk. The clinical implications resulting from the application of the new methods of molecular biology are discussed.

Calcitonin-producing insulinoma: clinical, immunocytochemical and cytogenetical study.

The case of a patient with a large goitre associated with hypercalcitoninaemia and fasting hypoglycaemia is reported. Pentagastrin (PG) test was negative. Repeated measurements of fasting glycaemia, insulin and C peptide established the diagnosis of insulinoma. After localization by endoscopic ultrasonography, a distal pancreatectomy was performed and a small insulinoma was recovered. Glycaemia and calcitonin (CT) became normal. The tumour cells displayed a strong immunoreactivity for insulin and CT. Cytogenetical evaluation of the tumour revealed a translocation t(1;9) (p13;p22).