Genotype and phenotype landscape of MEN2 in 554 medullary thyroid cancer patients: the BrasMEN study.

Multiple endocrine neoplasia type 2 (MEN2) is an autosomal dominant genetic disease caused by RET gene germline mutations that is characterized by medullary thyroid carcinoma (MTC) associated with other endocrine tumors. Several reports have demonstrated that the RET mutation profile may vary according to the geographical area. In this study, we collected clinical and molecular data from 554 patients with surgically confirmed MTC from 176 families with MEN2 in 18 different Brazilian centers to compare the type and prevalence of RET mutations with those from other countries. The most frequent mutations, classified by the number of families affected, occur in codon 634, exon 11 (76 families), followed by codon 918, exon 16 (34 families: 26 with M918T and 8 with M918V) and codon 804, exon 14 (22 families: 15 with V804M and 7 with V804L). When compared with other major published series from Europe, there are several similarities and some differences. While the mutations in codons C618, C620, C630, E768 and S891 present a similar prevalence, some mutations have a lower prevalence in Brazil, and others are found mainly in Brazil (G533C and M918V). These results reflect the singular proportion of European, Amerindian and African ancestries in the Brazilian mosaic genome.

Integration of a postoperative calcitonin measurement into an anatomical staging system improves initial risk stratification in medullary thyroid cancer.

OBJECTIVE: Staging systems applied to medullary thyroid cancer (MTC) rely on initial clinical and pathological features and do not consider the response to treatment. To determine whether MTC staging can be improved by incorporating the first postoperative calcitonin measurement. PATIENTS AND MEASUREMENTS: Eighty-five patients being monitored for MTC (median follow-up 5 years) were retrospectively classified according to both the American Joint Committee on Cancer (AJCC) and the proposed combined risk stratification system (low, intermediate and high risk), which incorporates the first postoperative calcitonin measurement, using the outcomes no evidence of disease (NED), biochemical evidence of disease, structurally identifiable disease and death. RESULTS: Ninety per cent of AJCC I patients were classified as NED at final follow-up. When we added a postoperative calcitonin measurement, 95% low-risk patients were classified as NED at final follow-up. AJCC stages I and IV were associated, respectively, with no occurrence and a high rate (63%) of structurally identifiable disease. Stages II and III yielded similar predictions of structurally identifiable disease, 13% and 14%, respectively. When we included the postoperative calcitonin level, the patients with structural evidence of disease included none from the low-risk group, 10% from the intermediate group and 63% from the high-risk group. The proportion of variance explained analysis (PVE) was better for the combined risk stratification system (54%) than for the AJCC system alone (32%). CONCLUSION: Including the first postoperative calcitonin measurement with the anatomical staging system can better predict the clinical outcome of patients with MTC and refine the follow-up of these patients.

Measurement of calcitonin and calcitonin gene-related peptide mRNA refines the management of patients with medullary thyroid cancer and may replace calcitonin-stimulation tests.

BACKGROUND: Serum calcitonin (sCT) is the main tumor marker for medullary thyroid cancer (MTC), but it has certain limitations. Various sCT assays may have important intra-assay or interassay variation and may yield different and sometimes conflicting results. A pentagastrin- or calcium-stimulation calcitonin (CT) test may be desirable in some situations. Alternatively, or in the absence of the stimulation test, mRNA detection offers the advantages of being more comfortable and less invasive; it only requires blood collection and has no side effects. The objective of this study was to investigate the applicability of measuring calcitonin-related polypeptide alpha (CALCA) gene transcripts (CT-CALCA and calcitonin gene-related peptide [CGRP]-CALCA) in patients with MTC and in relatives diagnosed with a RET mutation and to test mRNA as an alternative diagnostic tool for the calcitonin-stimulation test. METHODS: Twenty-three healthy controls and 26 individuals evaluated for MTC were selected, including patients with sporadic or hereditary MTC and RET mutation-carrying relatives. For molecular analysis, RNA was extracted from peripheral blood, followed by cDNA synthesis using 3.5 µg of total RNA. Quantitative real-time polymerase chain reaction (RT-qPCR) was performed with SYBR Green and 200 nM of each primer for the two specific mRNA targets (CT-CALCA or CGRP-CALCA) and normalized with the ribosomal protein S8 as the reference gene. RESULTS: We detected CALCA transcripts in the blood samples and observed a positive correlation between them (r=0.946, p<0.0001). Both mRNAs also correlated with sCT (CT-CALCA, r=0.713, p<0.0001; CGRP-CALCA, r=0.714, p<0.0001). The relative expression of CT-CALCA and CGRP-CALCA presented higher clinical sensitivity (86.67 and 100, respectively), specificity (97.06 and 97.06), positive predictive value (92.86 and 93.75), and negative predictive value (94.29 and 100), than did sCT (73.33, 82.35, 64.71, and 87.50, respectively). In addition, the CALCA transcript measurement mirrored the response to the pentagastrin test. CONCLUSION: We demonstrate that the measurement of CALCA gene transcripts in the bloodstream is feasible and may refine the management of patients with MTC and RET mutation-carrying relatives. We propose considering the application of this diagnostic tool as an alternative to the calcitonin-stimulation test.

Extended RET gene analysis in patients with apparently sporadic medullary thyroid cancer: clinical benefits and cost.

RET sequencing has become an important tool in medullary thyroid cancer (MTC) evaluation and should be performed even in the absence of family history of MTC. The most commonly studied exons in index cases are 8, 10, 11, and 13-16. To address the ATA guidelines regarding the sequencing of the entire coding region of RET, we selected 50 patients with sporadic MTC (sMTC) without mutations in the hot spot regions of RET for extended investigation of exons 1-7, 9, 12, 17, 18, and 19. Twenty-seven of 50 patients presented with one or more features suggesting familial disease. We found only a new RET variant (p.Gly550Glu) in one patient with MTC. Several polymorphisms were observed, and their frequency was histogram scaled by exons and introns. Eight patients were also included for somatic mutation search. We estimated the sequencing cost by stratifying into four investigation approaches: (1) hot spot exons in a new patient, (2) the remaining exons if the hot spots are negative in a patient with suspected familial disease, (3) a relative of a carrier for a known RET mutation, and (4) tumor sequencing. In spite of the increasing number of variants being described in MTC, it appears that there is no direct clinical benefit in extending RET germ line analysis beyond the hot spot regions in sMTC. The cost evaluation in apparent sMTC using a tiered approach may help clinicians make more suitable decisions regarding the benefits of investigating only the hot spots against the entire coding region of RET.