Maria João Bugalho

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:u2002 Searching for multiple molecular markers in thyroid aspirates appears to be a promising approach for establishing a preoperative diagnosis of papillary thyroid carcinoma (PTC).

Outcomes of adrenal-sparing surgery or total adrenalectomy in phaeochromocytoma associated with multiple endocrine neoplasia type 2: an international retrospective population-based study

BACKGROUNDnThe prevention of medullary thyroid cancer in patients with multiple endocrine neoplasia type 2 syndrome has demonstrated the ability of molecular diagnosis and prophylactic surgery to improve patient outcomes. However, the other major neoplasia associated with multiple endocrine neoplasia type 2, phaeochromocytoma, is not as well characterised in terms of occurrence and treatment outcomes. In this study, we aimed to systematically characterise the outcomes of management of phaeochromocytoma associated with multiple endocrine neoplasia type 2.nnnMETHODSnThis multinational observational retrospective population-based study compiled data on patients with multiple endocrine neoplasia type 2 from 30 academic medical centres across Europe, the Americas, and Asia. Patients were included if they were carriers of germline pathogenic mutations of the RET gene, or were first-degree relatives with histologically proven medullary thyroid cancer and phaeochromocytoma. We gathered clinical information about patientsRET genotype, type of treatment for phaeochromocytoma (ie, unilateral or bilateral operations as adrenalectomy or adrenal-sparing surgery, and as open or endoscopic operations), and postoperative outcomes (adrenal function, malignancy, and death). The type of surgery was decided by each investigator and the timing of surgery was patient driven. The primary aim of our analysis was to compare disease-free survival after either adrenal-sparing surgery or adrenalectomy.nnnFINDINGSn1210 patients with multiple endocrine neoplasia type 2 were included in our database, 563 of whom had phaeochromocytoma. Treatment was adrenalectomy in 438 (79%) of 552 operated patients, and adrenal-sparing surgery in 114 (21%). Phaeochromocytoma recurrence occurred in four (3%) of 153 of the operated glands after adrenal-sparing surgery after 6-13 years, compared with 11 (2%) of 717 glands operated by adrenalectomy (p=0.57). Postoperative adrenal insufficiency or steroid dependency developed in 292 (86%) of 339 patients with bilateral phaeochromocytoma who underwent surgery. However, 47 (57%) of 82 patients with bilateral phaeochromocytoma who underwent adrenal-sparing surgery did not become steroid dependent.nnnINTERPRETATIONnThe treatment of multiple endocrine neoplasia type 2-related phaeochromocytoma continues to rely on adrenalectomies with their associated Addisonian-like complications and consequent lifelong dependency on steroids. Adrenal-sparing surgery, a highly successful treatment option in experienced centres, should be the surgical approach of choice to reduce these complications.

Paragangliomas/Pheochromocytomas: Clinically Oriented Genetic Testing

Paragangliomas are rare neuroendocrine tumors that arise in the sympathetic or parasympathetic nervous system. Sympathetic paragangliomas are mainly found in the adrenal medulla (designated pheochromocytomas) but may also have a thoracic, abdominal, or pelvic localization. Parasympathetic paragangliomas are generally located at the head or neck. Knowledge concerning the familial forms of paragangliomas has greatly improved in recent years. Additionally to the genes involved in the classical syndromic forms: VHL gene (von Hippel-Lindau), RET gene (Multiple Endocrine Neoplasia type 2), and NF1 gene (Neurofibromatosis type 1), 10 novel genes have so far been implicated in the occurrence of paragangliomas/pheochromocytomas: SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, MAX, EGLN1, HIF2A, and KIF1B. It is currently accepted that about 35% of the paragangliomas cases are due to germline mutations in one of these genes. Furthermore, somatic mutations of RET, VHL, NF1, MAX, HIF2A, and H-RAS can also be detected. The identification of the mutation responsible for the paraganglioma/pheochromocytoma phenotype in a patient may be crucial in determining the treatment and allowing specific follow-up guidelines, ultimately leading to a better prognosis. Herein, we summarize the most relevant aspects regarding the genetics and clinical aspects of the syndromic and nonsyndromic forms of pheochromocytoma/paraganglioma aiming to provide an algorithm for genetic testing.

RET polymorphisms and sporadic medullary thyroid carcinoma in a Portuguese population.

The genetic basis of the sporadic form of medullary thyroid carcinoma, derived from “C” cells, is still poorly understood. Somatic mutations of RET proto-oncogene have been reported at a variable frequency ranging from 23% to 69%. The hypothesis that low penetrance factors, such as polymorphisms, might contribute to the phenotype of this neoplasm has been addressed in a few studies conducting to conflicting results. Herein, we studied 100 individuals (50 patients and 50 controls) aiming to compare the frequencies of G691S, L769L, S836S, and S904S RET polymorphisms observed in patients with respect to controls. Furthermore, metaanalysis of published studies including the present results was conducted. To test the contributory role of the above polymorphisms for the development of “C”-cell hyperplasia, we studied a group of 10 individuals selected for having a positive pentagastrin test despite the absence of a RET germline mutation. An over-representation of the G691S polymorphism, particularly in females, was observed in patients with respect to controls, although not reaching the level of significance. Allelic frequencies of the other three polymorphisms were not different in patients and controls. Results obtained in the admittedly small group of individuals with a positive pentagastrin test are unlikely to support a major influence of any polymorphism in the development of “C”-cell hyperplasia. The meta-analysis provided evidence for a significant association of the S691 allele with MTC (odds ratio 1.54, 95% confidence interval 1.12–2.12, p=0.008) and found no significant associations for the other polymorphisms.

Identification of a novel germline FOXE1 variant in patients with familial non-medullary thyroid carcinoma (FNMTC)

The familial forms of non-medullary thyroid carcinoma (FNMTC) represent approximatelyxa05xa0% of thyroid neoplasms. Nine FNMTC susceptibility loci have been mapped; however, only the DICER1 and SRGAP1 susceptibility genes have been identified. The transcription factors NKX2-1, FOXE1, PAX8, and HHEX are involved in the morphogenesis and differentiation of the thyroid. Recent studies have identified NKX2-1 germline mutations in FNMTC families. However, the role of high-penetrant FOXE1 variants in FNMTC etiology remains unclear. The aim of this study was to investigate the role of FOXE1 germline mutations in the pathogenesis of FNMTC. We searched for molecular changes in the FOXE1 gene in the probands from 60 Portuguese families with FNMTC. In this series, we identified nine polymorphisms and one variant (c.743C>G, p.A248G) which was not previously described. This variant, which involved an amino acid residue conserved in evolution, segregated with disease in one family, and was also detected in an apparently unrelated case of sporadic NMTC. Functional studies were performed using rat normal thyroid cells (PCCL3) clones and human papillary thyroid carcinoma cell line (TPC-1) pools, expressing the wild type and mutant (p.A248G) forms of FOXE1. In these experiments, we observed that the p.A248G variant promoted cell proliferation and migration, suggesting that it may be involved in thyroid tumorigenesis. Additionally, somatic p.V600E BRAF mutations were also detected in the thyroid tumors of two members of the family carrying the p.A248G variant. This study represents the first evidence of involvement of a germline FOXE1 rare variant in FNMTC etiology and suggests that mutations in MAPK pathway-related genes may contribute to tumor development in these familial cases.

The minisequencing method: a simple strategy for genetic screening of MEN 2 families

BackgroundMultiple endocrine neoplasia type 2 is an autosomal dominant disorder. MEN 2A is characterized by medullary thyroid carcinoma, pheochromocytoma and hyperparathyroidism; MEN 2B by medullary thyroid carcinoma, pheochromocytoma and characteristic stigmata. Activating germline mutations of the RET proto oncogene are responsible for this hereditary syndrome. Codon 634 mutations are the most common mutations occurring in MEN 2A families whereas a specific mutation at codon 918 is observed in the great majority of MEN 2B families. Analysis of these codons will provide a final diagnosis in the great majority of affected families making unnecessary further studies. To specifically study the codons 634 and 918 we used a minisequencing method as an alternative method to complete sequencing.ResultsUsing this mutation detection method we were able to reproduce in all cases, representative of 7 families, the information previously obtained by direct sequencing of PCR products. Depending on the number of primers used in the minisequencing reaction, we were able to interrogate either only one nucleotide of the target codon or the three nucleotides simultaneously.ConclusionsThis technique appears as a simple, rapid and efficient method for genetic screening of MEN 2 families. It can be utilized to seek for unknown mutations at specific codons or to screen for previously identified mutations and is therefore of interest to study index cases or individuals at risk. Results suggest that complete sequencing is unnecessary.

Serum vascular endothelial growth factor levels in patients with medullary thyroid carcinoma

BACKGROUNDnHigh levels of vascular endothelial growth factor (VEGF) have been reported in patients with cancers of different origins. There are no data comparing serum VEGF levels of medullary thyroid carcinoma (MTC) patients with that of the healthy subjects.nnnOBJECTIVEnWe tried to assess whether serum VEGF concentration in MTC patients is correlated with tumour extension and whether this marker might be used to further refine the selection of candidates for future therapies with receptor tyrosine kinase inhibitors.nnnMETHODSnSera from 57 individuals divided into five groups: group I, healthy individuals (n=14); group II, MTC patients in remission (n=10); group III, MTC patients with residual disease (n=12); group IV, MTC patients with loco-regional disease (n=11) and group V, MTC patients with distant metastases (n=10) were analysed for serum VEGF and calcitonin (CT) levels.nnnRESULTSnAnalysis of serum VEGF did not disclose significant differences among the five groups. Mean serum VEGF level of patients with distant metastases was not significantly different from that observed in healthy individuals (319.4+/-49.78 vs 313.7+/-43.13 ng/l). Serum VEGF levels correlated positively with serum CT (r=0.4891; P=0.0394) for CT values below 2500 ng/l whereas there was no correlation for CT values above this threshold.nnnCONCLUSIONSnSerum VEGF levels in MTC patients are not significantly different from those found in healthy individuals and did not correlate with the extension of disease.

In vitro assays to test the interference of anti-thyroglobulin antibodies on thyroglobulin measurement

Objective To assess the interference of anti-thyroglobulin antibodies (TgAb) on serum thyroglobulin (Tg) measurement by inxa0vitro experiments. Design Re-evaluation of Tg concentration after dilution with different TgAb-positive sera. On a first step, dilutions of the same Tg with different TgAb sera were performed and on a second step, different Tgs were diluted with the same TgAb serum. Methods Tg measurements were performed using an immunometric (IMA) chemiluminescence assay. TgAb measurements were performed using two methods: immunoflurimetric assay (UNICAP 100) and IMA chemiluminescent assay (IMMULITE 2000). Results Dilution of a known concentration of Tg with different TgAb-positive sera resulted in a variation of the final concentration of Tg ranging from −24 to −79%. A weak correlation was observed between the TgAb concentration and the percentage of the Tg deviation. Dilution of different Tgs with the same TgAb-positive serum illustrated how the same TgAb positive serum may determine a high interference or a neutral effect. Conclusions Present results suggest that the interference on Tg measurement observed in the presence of TgAb may result not only from the anti-thyroglobulin antibodies, but also from the thyroglobulin itself.

Uncommon association of cerebral meningioma, parathyroid adenoma and papillary thyroid carcinoma in a patient harbouring a rare germline variant in the CDKN1B gene

Multiple endocrine neoplasia type 4 (MEN 4) is a novel form of multiple endocrine neoplasia caused by mutations in the CDKN1B gene. Its clinical presentation includes MEN 1-related tumours such as parathyroid and anterior pituitary tumours in possible association with gonadal, adrenal, renal and thyroid tumours as well as facial angiofibromas, colagenomas and meningiomas. We describe the case of a patient with meningioma, papillary thyroid carcinoma, parathyroid adenoma and, additionally, Hürthle cell adenoma, cholesteatoma and uterine leiomyomas. Considering that this association could represent a MEN 4-like phenotype, we looked for germline mutations in the CDKN1B gene. A rare heterozygous single nucleotide substitution c.397C>A was identified. Its role as a susceptibility factor remains to be established.