Long-Term Follow-up of Patients With Autonomous Thyroid Nodules Treated With Radioiodine.

PURPOSE: This article aims to describe the presentation of Plummer disease and its evolution after radioiodine treatment and determine factors that may influence treatment efficacy. PATIENTS AND METHODS: The sample included retrospective medical records of 165 adult patients with toxic nodular goiter treated with radioiodine between 1997 and 2017, followed up at a single thyroid center. RESULTS: The efficacy of treatment with a single dose of radioiodine was higher than 90%. The mean radioiodine activity was 28.9 ± 3.4 mCi. The mean time between radioiodine performance and hyperthyroidism resolution was 3.6 ± 3.0 months, ranging from 1-12 months. After the first year, 33.9% of the patients were under hypothyroidism, 59.4% under euthyroidism, and 6.7% under hyperthyroidism. Among the nonresponders, the variables that showed statistical difference were the presence of multinodular goiter and the radioiodine activity (mean, 25.5 ± 6.5 mCi; median, 30 [15-30 mCi]). The cumulative rate of hypothyroidism was 48.9% over 20 years of follow-up. CONCLUSIONS: Radioiodine therapy is an effective and safe treatment. In Plummer disease, high rates of euthyroidism are expected after the radioiodine treatment. Therapeutic failure was observed mainly in patients with larger multinodular goiters treated with lower doses of radioiodine. The evolution to hypothyroidism was mostly observed in younger patients with larger and uninodular goiters.

RET 634 germline/gonadal mosaicism generating a second pathogenic amino acid change in multiple endocrine neoplasia type 2A.

Genetic testing for germline RET pathogenic variants, which cause the Multiple Endocrine Neoplasia Type 2 (MEN2) syndrome, has become crucial in managing patients with medullary thyroid carcinoma (MTC). Classically, RET heterozygous missense pathogenic variants are transmitted in a Mendelian autosomal dominant pattern, of which germline/gonadal mosaicism has never been reported. We report the novel occurrence of a MEN2A patient's family in which the siblings inherited three different RET 634 genotypes: wild type (p.Cys634), p.Cys634Gly or p.Cys634Arg heterozygous pathogenic variants. We hypothesized that germline/gonadal mosaicism, derived from an inherited + early somatic mutation in the mother or a double de novo mutation during maternal embryogenesis, led to this rare event in the RET gene. Exome analysis of the proband's deceased mother's paraffin-embedded thyroid tissue confirmed the three nucleotides in the same 634 codon position. For the first time, we describe germline/gonadal mosaicism in RET, generating a second pathogenic amino acid change in the same codon causing MEN2A. Our finding shows that RET parental mosaicism, confirmed by somatic exome sequencing, might explain discrepant genotype cases in siblings with inherited cancers.

Germline genetic variants in pheochromocytoma/paraganglioma: single-center experience.

Pheochromocytoma and paragangliomas (PPGLs) are rare neuroendocrine tumors carrying 25-40% pathogenic germline gene variants (PGVs). We evaluated clinical, laboratory, and germline molecular profile of 115 patients with pathologic (14 patients were relatives from 8 different families recruited for genetic survey) confirmed PPGL followed in our institution. Patients with classic MEN2A/MEN2B phenotypes and at-risk relatives underwent direct analysis of RET proto-oncogene, and the remaining had samples submitted to complete next-generation sequencing aiming 23 PPGL-related genes: ATM, ATR, CDKN2A, EGLN1, FH, HRAS, KIF1B, KMT2D, MAX, MDH2, MERTK, MET, NF1, PIK3CA, RET, SDHA, SDHAF2, SDHB, SDHC, SDHD, TMEM127, TP53, and VHL. We also developed a clinical judgment score (CJS) to determine the probability of patients having a potentially hereditary disease. The resulting genetic landscape showed that 67 patients (58.3%) had variants in at least one gene: 34 (50.7%) had exclusively pathogenic or likely pathogenic variants, 13 (19.4%) had pathogenic or likely pathogenic variants and variant of undetermined significance (VUS), and 20 (29.8%) carried only VUS. PGVs were found in RET (n = 18; 38.3%), VHL (n = 10; 21.3%), SDHB and NF1 (n = 8; 17% each), and MAX, SDHD, TMEM127, and TP53 (n = 1; 2.1% each). Direct genetic testing disclosed 91.3% sensitivity, 81.2% specificity, and 76.4% and 93.3% positive predictive value (PPV) and negative predictive values (NPV), respectively. The CJS to identify patients who would not benefit from genetic testing had 75% sensitivity, 96.4% specificity, and 60% and 98.2% PPV and NPV, respectively. In summary, the landscape of PPGL germline gene variants from 115 Brazilian patients resulted in slightly higher prevalent pathogenic and likely pathogenic variants, especially in the RET gene. We suggest a CJS to identify PPGL patients who would not require initial genetic evaluation, improving test specificity and reducing costs.

Risk of malignancy and diagnostic accuracy of fine-needle aspiration biopsy in thyroid nodules with diameters greater than 4 centimeters.

Objective: The risk of malignancy and diagnostic accuracy of fine-needle aspiration biopsy (FNAB) of thyroid nodules (TN) with diameters ≥ 3-4 cm remains controversial. However, some groups have indicated surgical treatment in these patients regardless of the FNAB results. We aimed to evaluate the diagnostic accuracy of the FNAB in systematically resected ≥4 cm TN and if the risk of malignancy is higher in these patients. Subjects and methods: We retrospectively evaluated 138 patients (142 nodules) with TN with diameters ≥4 cm who underwent thyroidectomy. Results: The FNAB results were nondiagnostic/unsatisfactory (ND/UNS) in 2.1% of the cases and benign in 51.4%. They indicated atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) in 23.9% of cases, follicular neoplasia/suspicious for a follicular neoplasm (FN/SFN) in 9.2%, suspicion of malignancy (SUS) in 8.5%, and malignant in 4.9%. The histopathological analysis after thyroidectomy revealed a thyroid cancer rate of 100% in the FNABs classified as malignant, 33.3% in SUS cases, 7.7% in FN/SFN, 17.6% in AUS/FLUS, and 4.1% in benign FNABs. None of the ND/UNS FNABs were malignant. The global malignancy diagnosis was 14.8% (n = 21). However, the rate of false negatives for FNAB was low (4.1%). Conclusion: We showed that the risk of malignancy in nodules with diameters ≥4 cm was higher compared to the risk of thyroid cancer in TN in general. However, we found a low rate of false-negative cytological results; therefore, our data do not justify the orientation of routine resection for these larger nodules.

Risk of malignancy and diagnostic accuracy of fine-needle aspiration biopsy in thyroid nodules with diameters greater than 4 centimeters

ABSTRACT Objective: The risk of malignancy and diagnostic accuracy of fine-needle aspiration biopsy (FNAB) of thyroid nodules (TN) with diameters ≥ 3-4 cm remains controversial. However, some groups have indicated surgical treatment in these patients regardless of the FNAB results. We aimed to evaluate the diagnostic accuracy of the FNAB in systematically resected ≥4 cm TN and if the risk of malignancy is higher in these patients. Subjects and methods: We retrospectively evaluated 138 patients (142 nodules) with TN with diameters ≥4 cm who underwent thyroidectomy. Results: The FNAB results were nondiagnostic/unsatisfactory (ND/UNS) in 2.1% of the cases and benign in 51.4%. They indicated atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) in 23.9% of cases, follicular neoplasia/suspicious for a follicular neoplasm (FN/SFN) in 9.2%, suspicion of malignancy (SUS) in 8.5%, and malignant in 4.9%. The histopathological analysis after thyroidectomy revealed a thyroid cancer rate of 100% in the FNABs classified as malignant, 33.3% in SUS cases, 7.7% in FN/SFN, 17.6% in AUS/FLUS, and 4.1% in benign FNABs. None of the ND/UNS FNABs were malignant. The global malignancy diagnosis was 14.8% (n = 21). However, the rate of false negatives for FNAB was low (4.1%). Conclusion: We showed that the risk of malignancy in nodules with diameters ≥4 cm was higher compared to the risk of thyroid cancer in TN in general. However, we found a low rate of false-negative cytological results; therefore, our data do not justify the orientation of routine resection for these larger nodules.

Comprehensive Assessment of Copy Number Alterations Uncovers Recurrent AIFM3 and DLK1 Copy Gain in Medullary Thyroid Carcinoma.

Medullary thyroid carcinoma (MTC) is a malignant tumor originating from thyroid C-cells that can occur either in sporadic (70-80%) or hereditary (20-30%) form. In this study we aimed to identify recurrent copy number alterations (CNA) that might be related to the pathogenesis or progression of MTC. We used Affymetrix SNP array 6.0 on MTC and paired-blood samples to identify CNA using PennCNV and Genotyping Console software. The algorithms identified recurrent copy number gains in chromosomes 15q, 10q, 14q and 22q in MTC, whereas 4q cumulated losses. Coding genes were identified within CNA regions. The quantitative PCR analysis performed in an independent series of MTCs (n = 51) confirmed focal recurrent copy number gains encompassing the DLK1 (14q32.2) and AIFM3 (22q11.21) genes. Immunohistochemistry confirmed AIFM3 and DLK1 expression in MTC cases, while no expression was found in normal thyroid tissues and few MTC samples were found with normal copy numbers. The functional relevance of CNA was also assessed by in silico analysis. CNA status correlated with protein expression (DLK1, p = 0.01), tumor size (DLK1, p = 0.04) and AJCC staging (AIFM3p = 0.01 and DLK1p = 0.05). These data provide a novel insight into MTC biology, and suggest a common CNA landscape, regardless of if it is sporadic or hereditary MTC.

Revised criteria for diagnosis of NIFTP reveals a better correlation with tumor biological behavior.

The recent reclassification of a follicular variant of papillary thyroid carcinoma (FVPTC), subset as noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), aims to avoid overtreatment of patients with an indolent lesion. The diagnosis of NIFTP has recently been revisited using more rigid criteria. This study presents histological and molecular findings and a long clinical follow-up of 94 FVPTC, 40 cases of follicular adenoma (FTA) and 22 cases of follicular carcinoma (FTC) that were classified before the advent of the NIFTP reclassification. All slides were reviewed using these rigid criteria and analysis of numerous sections of paraffin blocks and reclassified as 7 NIFTPs, 2 EFVPTCs, 29 infiltrative FVPTC (IFVPTCs), 57 invasive EFVPTC (I-EFVPTCs), 39 FTAs and 22 FTCs. Remarkably, EFVPTC and NIFTP patients were all free of disease at the end of follow-up and showed no BRAF mutation. Only one NIFTP sample harbored mutations, an NRAS Q61R. PAX8/PPARG fusion was found in I-EFVPTCs and FTC. Although additional studies are needed to identify a specific molecular profile to aid in the diagnosis of lesions with borderline morphological characteristics, we confirmed that the BRAF V600E mutation is an important tool to exclude the diagnosis of NIFTP. We also show that rigorous histopathological criteria should be strongly followed to avoid missing lesions in which more aggressive behavior is present, mainly via the analysis of capsule or vascular invasion and the presence of papillary structures.

Searching for mutations in the HNF1B gene in a Brazilian cohort with renal cysts and hyperglycemia

ABSTRACT Objective To verify the presence of variants in HNF1B in a sample of the Brazilian population selected according to the presence of renal cysts associated with hyperglycemia. Subjects and methods We evaluated 28 unrelated patients with clinical suspicion of HNF1B mutation because of the concomitant presence of diabetes mellitus (DM) or prediabetes and renal cysts. Genotyping was accomplished using Sanger sequencing or multiplex ligation-dependent probe amplification (MLPA). In positive cases, available relatives were recruited. Results We found two patients with HNF1B mutations. The first presented the variant p.Pro328Leufs*48(c.983delC) and had DM, renal cysts, and hypomagnesemia. The second presented a heterozygous whole gene deletion in HNF1B, DM, renal cysts, body and tail pancreatic agenesis, and hypomagnesemia; this alteration was also found in his two siblings and his father. Conclusion The recruitment of suspected cases of HNF1B gene mutations in Brazilians due to hyperglycemia and renal cysts presents two positive cases. Our cases contribute to the annotation of clinical and biochemical phenotypes of this rare form of maturity-onset diabetes of the young (MODY).

Searching for mutations in the HNF1B gene in a Brazilian cohort with renal cysts and hyperglycemia.

OBJECTIVE: To verify the presence of variants in HNF1B in a sample of the Brazilian population selected according to the presence of renal cysts associated with hyperglycemia. SUBJECTS AND METHODS: We evaluated 28 unrelated patients with clinical suspicion of HNF1B mutation because of the concomitant presence of diabetes mellitus (DM) or prediabetes and renal cysts. Genotyping was accomplished using Sanger sequencing or multiplex ligation-dependent probe amplification (MLPA). In positive cases, available relatives were recruited. RESULTS: We found two patients with HNF1B mutations. The first presented the variant p.Pro328Leufs*48(c.983delC) and had DM, renal cysts, and hypomagnesemia. The second presented a heterozygous whole gene deletion in HNF1B, DM, renal cysts, body and tail pancreatic agenesis, and hypomagnesemia; this alteration was also found in his two siblings and his father. CONCLUSION: The recruitment of suspected cases of HNF1B gene mutations in Brazilians due to hyperglycemia and renal cysts presents two positive cases. Our cases contribute to the annotation of clinical and biochemical phenotypes of this rare form of maturity-onset diabetes of the young (MODY).

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.

A pioneering RET genetic screening study in the State of Ceará, Brazil, evaluating patients with medullary thyroid cancer and at-risk relatives: experience with 247 individuals

ABSTRACT Objective: Initial diagnosis of medullary thyroid carcinoma (MTC) is frequently associated with advanced stages and a poor prognosis. Thus, the need for earlier diagnoses and detection in relatives at risk for the disease has led to increased use of RET genetic screening. Subjects and methods: We performed RET screening in 247 subjects who were referred to the Brazilian Research Consortium for Multiple Endocrine Neoplasia (BRASMEN) Center in the State of Ceará. Direct genetic sequencing was used to analyze exons 8, 10, 11, and 13-16 in MTC index cases and specific exons in at risk relatives. Afterward, clinical follow-up was offered to all the patients with MTC and their affected relatives. Results: RET screening was performed in 60 MTC index patients and 187 at-risk family members. At the initial clinical assessment of the index patients, 54 (90%) were diagnosed with apparently sporadic disease and 6 (10%) diagnosed with hereditary disease. After RET screening, we found that 31 (52%) index patients had sporadic disease, and 29 (48%) had hereditary disease. Regarding at-risk relatives, 73/187 were mutation carriers. Mutations in RET codon 804 and the rare p.M918V mutation were the most prevalent. Conclusions: Performing RET screening in Ceará allowed us to identify a different mutation profile in this region compared with other areas. RET screening also enabled the diagnosis of a significant number of hereditary MTC patients who were initially classified as sporadic disease patients and benefited their relatives, who were unaware of the risks and the consequences of bearing a RET mutation.

LIMD2 Is Overexpressed in BRAF V600E-Positive Papillary Thyroid Carcinomas and Matched Lymph Node Metastases.

We previously described that LIM domain containing 2 (LIMD2) overexpression was closely correlated with metastatic process in papillary thyroid carcinoma (PTC). We here evaluated the expression of LIMD2 in a series of non-metastatic and metastatic PTC and their matched lymph node metastases via immunohistochemistry. LIMD2 was expressed in 74 (81%) of primary PTC and 35 (95%) of lymph node metastases. Sub-analysis performed in 37 matched samples demonstrated that in four cases, LIMD2 is expressed in lymph node metastases, while it is not expressed in primary tumors. Moreover, in eight cases, the staining intensity of LIMD2 was stronger in the patient-matched lymph node metastases than in the primary tumors. Next, the expression of LIMD2 was correlated with clinical pathological parameters and BRAF V600E and RET/PTC mutational status. The expression of LIMD2 in primary tumors was correlated with the presence of BRAF V600E mutation (P = 0.0338). Western blot analysis in thyroid cell lines demonstrated that LIMD2 is expressed in two PTC cell lines, while it is not expressed in normal thyroid and follicular thyroid carcinoma cell lines. Importantly, its expression was higher in a PTC cell line that harbors BRAF V600E mutation than in a PTC cell line that harbors RET/PTC1. The available genomic profiling data generated by The Cancer Genome Atlas Research Network confirmed that LIMD2 expression is higher in BRAF-like PTC samples. Our data suggest that LIMD2 may play an important role in the metastatic process of PTC, predominantly in BRAF V600E-positive tumors.

A pioneering RET genetic screening study in the State of Ceará, Brazil, evaluating patients with medullary thyroid cancer and at-risk relatives: experience with 247 individuals.

OBJECTIVE: Initial diagnosis of medullary thyroid carcinoma (MTC) is frequently associated with advanced stages and a poor prognosis. Thus, the need for earlier diagnoses and detection in relatives at risk for the disease has led to increased use of RET genetic screening. SUBJECTS AND METHODS: We performed RET screening in 247 subjects who were referred to the Brazilian Research Consortium for Multiple Endocrine Neoplasia (BRASMEN) Center in the State of Ceará. Direct genetic sequencing was used to analyze exons 8, 10, 11, and 13-16 in MTC index cases and specific exons in at risk relatives. Afterward, clinical follow-up was offered to all the patients with MTC and their affected relatives. RESULTS: RET screening was performed in 60 MTC index patients and 187 at-risk family members. At the initial clinical assessment of the index patients, 54 (90%) were diagnosed with apparently sporadic disease and 6 (10%) diagnosed with hereditary disease. After RET screening, we found that 31 (52%) index patients had sporadic disease, and 29 (48%) had hereditary disease. Regarding at-risk relatives, 73/187 were mutation carriers. Mutations in RET codon 804 and the rare p.M918V mutation were the most prevalent. CONCLUSIONS: Performing RET screening in Ceará allowed us to identify a different mutation profile in this region compared with other areas. RET screening also enabled the diagnosis of a significant number of hereditary MTC patients who were initially classified as sporadic disease patients and benefited their relatives, who were unaware of the risks and the consequences of bearing a RET mutation.

THE COMBINED USE OF CALCITONIN DOUBLING TIME AND 18F-FDG PET/CT IMPROVES PROGNOSTIC VALUES IN MEDULLARY THYROID CARCINOMA: THE CLINICAL UTILITY OF 18F-FDG PET/CT.

OBJECTIVE: Calcitonin and carcinoembryonic antigen (CEA) doubling times are established prognostic markers in medullary thyroid cancer (MTC). On the other hand, 18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) shows an increased rate of detection with high blood tumor marker levels in several cancers. This study aimed to analyze the ability of 18F-FDG PET/CT to determine prognosis in the follow-up of patients with MTC. METHODS: Medical records of 17 patients with MTC who underwent 18F-FDG PET/CT were analyzed retrospectively. All patients were classified into two groups: stable disease or progressive disease. RESULTS: Eight patients presented with progressive disease, and all of them showed 18F-FDG uptake (100%), compared to only 3 of 9 patients who presented in stable condition (33%). 18F-FDG PET/CT results were able to distinguish progressive from stable disease (P = .009). Calcitonin levels >4,020 pg/mL (P = .0004), CEA levels >26.8 ng/mL (P = .04), and a calcitonin doubling time <24.1 months (P = .015) were associated with progressive disease in our cohort. The proportion of variance explained that predicted progressive disease was 32% for 18F-FDG uptake, 27.1% for a calcitonin doubling time of 24.1 months, and 41.2% for doubling time plus 18F-FDG PET/CT. CONCLUSION: 18F-FDG uptake was able to distinguish progressive from stable disease. However, this tool should not replace the validated calcitonin doubling time, but rather the combination of information could improve the clinical re-assessment and better identify high-risk patients who require more careful surveillance. ABBREVIATIONS: CEA = carcinoembryonic antigen CT = computed tomography 18F-FDG = 18F-fluorodeoxyglucose MTC = medullary thyroid cancer PET = positron emission tomography PVE = proportion of variance explained sCT = serum calcitonin SUV = standard uptake value US = ultrasound.

Evidence for the founder effect of RET533 as the common Greek and Brazilian ancestor spreading multiple endocrine neoplasia 2A.

OBJECTIVES: About one-quarter of patients with medullary thyroid cancer (MTC) have inherited disease due to mutations in the RET gene. A rare mutation in exon 8 (G533C) of RET, previously described in a large Brazilian family with MEN2A, also appeared to be clustering in Greece, whereas it was rarely reported in other ethnic groups. The aim of this study was to identify a possible common ancestry between these carriers. PATIENTS AND METHODS: Twelve RET G533C mutation carriers, four randomly selected from the Brazilian cohort and eight from apparently unrelated Greek families, were studied for a possible common ancestral origin. RET flanking microsatellite markers at chromosome 10q (D10S197, D10S196, D10S1652 and D10S537) were used. RESULTS: Genomic DNA analysis using these markers showed that many of these apparently unrelated individuals shared a common haplotype indicating a common ancestral origin. CONCLUSION: Our data suggest that Brazilian and Greek patients with MTC carrying the G533C mutation in exon 8 of RET gene originate from a common ancestor. Due to historical reasons, we speculate that the more plausible explanation for the origin of this mutation is in Greece.

Down-regulation of Kir2.6 channel by c-termini mutation D252N and its association with the susceptibility to Thyrotoxic Periodic Paralysis.

Inward rectifying potassium - Kir - channels drive the resting potential to potassium reversal potential and, when disrupted, might be related to muscular diseases. Recently, Thyrotoxic Periodic Paralysis (TPP) has emerged as a channelopathy related to mutations in KCNJ18 gene, which encodes Kir2.6 channel. TPP is a neuromuscular disorder characterized by a triad of muscle weakness, hypokalemia, and thyrotoxicosis, the latter being essential for the crisis. Direct sequencing revealed two heterozygous mutations - D252N and R386C - in two TPP patients. KCNJ18 cDNAs were cloned into mammalian expression plasmids and transiently expressed in HEK 293T cells to investigate the functional effects of Kir2.6 mutations. Patch-clamp and confocal laser scanning microscopy experiments were carried out, comparing the WT channel to its mutants. D252N mutation down-regulates the Kir2.6 activity, decreasing the K+ current density (∼34%) when compared to the WT channel; whereas the mutation R386C shows no significant changes from WT. The mutant D252N Kir2.6 channel also showed a substantial reduction of ∼51% in membrane abundance relative to WT channel. Our study describes the functional consequences of a single amino acid change in Kir2.6 channel. Further analysis regarding hormonal conditions and Kir channel expression are required to provide new clues about the TPP pathophysiology.

M918V RET mutation causes familial medullary thyroid carcinoma: study of 8 affected kindreds.

Germline mutations in codon 918 of exon 16 of the RET gene (M918T) are classically associated with multiple endocrine neoplasia type 2B (MEN 2B) with highly aggressive medullary thyroid cancer (MTC), pheochromocytoma and a unique phenotype. The objectives of this study are to describe the rare M918V RET mutation discovered in 8 MTC kindreds from Brazil lacking the MEN 2B phenotype classically observed in M918T patients and to investigate the presence of a founder effect for this germline mutation. Eight apparently sporadic MTC cases were diagnosed with the germline M918V RET mutation. Subsequently, their relatives underwent clinical and genetic assessment (n = 113), and M918V was found in 42 of them. Until today, 20/50 M918V carriers underwent thyroidectomy and all presented MTC/C-cell hyperplasia; the remainder carriers are on clinical follow-up. None of the M918V carriers presented clinical features of MEN 2B. Their clinical presentation was heterogeneous, and the age at tumor diagnosis ranged from 24 to 59 years. Lymph node metastases were present in 12/20 patients, and presumable distant metastases in 2/20; in contrast, we observed a carrier of up to 87 years of age without evidence of MTC. Ethnographic fieldwork and haplotype analyses suggested that the founder mutation first settled in that area fifteen generations ago and originated from Portugal. Our study is the first to demonstrate the RET M918V mutation co-segregating in 8 familial MTC kindreds with validated evidence of a founder effect. We suggest that M918V MTC should be clinically considered an American Thyroid Association (ATA) moderate-risk category.

Multifocality in Sporadic Medullary Thyroid Carcinoma: An International Multicenter Study.

BACKGROUND: Current surgical standard of care in sporadic medullary thyroid carcinoma (sMTC) consists of a minimum of total thyroidectomy with central neck dissection. Some have suggested thyroid lobectomy with isthmusectomy and central neck dissection for patients with sMTC, given their lower frequency of bilateral disease, although this topic has not been thoroughly studied. This study assessed the prevalence of multifocality in sMTC via a large international multi-institutional retrospective review to quantify this prevalence, including the impact of geography, to assess more accurately the risks associated with alternative surgical approaches. METHODS: A retrospective chart review of sMTC patients from 11 institutions over 29 years (1983-2011) was undertaken. Data regarding focality, extent of disease, RET germline analysis plus family and clinical history for multiple endocrine neoplasia type 2 (MEN2), and demographic data were collected and analyzed. RESULTS: Patients from four continents and seven countries were included in the sample. Data for 313 patients with documented sMTC were collected. Of these, 81.2% were confirmed with negative RET germline testing, while the remaining 18.8% demonstrated a negative family history and no manifestations of MEN2 syndromes other than MTC. Bilateral disease was identified in 17/306 (5.6%) patients, while multifocal disease was noted in 50/312 (16.0%) sMTC patients. When only accounting for germline negative patients, these rates were not significantly different (5.6% and 17%, respectively). Among them, when disease was unifocal in the ipsilateral lobe and isthmus, bilateral disease was present in 6/212 (2.8%) cases. When disease was multifocal in the ipsilateral lobe or isthmus, then bilateral disease was present in 8/37 (21.6%) cases (p < 0.001). No geographic differences in focality were identified. CONCLUSIONS: The 5.6% prevalence of bilateral foci in sMTC suggests that total thyroidectomy should remain the standard of care for initial surgery, as less complete thyroid surgery may fail to address fully the primary site of disease. Whether ipsilateral tumor focality should be an independent factor determining the need for completion thyroidectomy when sMTC is diagnosed after hemithyroidectomy remains to be determined.

Analysis of somatic mutations in BRAF, CDKN2A/p16 and PI3KCA in patients with medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC), a neuroendocrine tumor originating from thyroid parafollicular cells, has been demonstrated to be associated with mutations in RET, HRAS, KRAS and NRAS. However, the role of other genes involved in the oncogenesis of neural crest tumors remains to be fully investigated in MTC. The current study aimed to investigate the presence of somatic mutations in BRAF, CDKN2A and PI3KCA in MTC, and to investigate the correlation with disease progression. DNA was isolated from paraffin­embedded tumors and blood samples from patients with MTC, and the hotspot somatic mutations were sequenced. A total of 2 novel HRAS mutations, p.Asp33Asn and p.His94Tyr, and polymorphisms within the 3' untranslated region (UTR) of CDKN2A (rs11515 and rs3088440) were identified, however, no mutations were observed in other genes. It was suggested that somatic point mutations in BRAF, CDKN2A and PI3KCA do not participate in the oncogenesis of MTC. Further studies are required in order to clarify the contribution of the polymorphisms identified in the 3'UTR of CDKN2A in MTC.

Macrocalcitonin Is a Novel Pitfall in the Routine of Serum Calcitonin Immunoassay.

CONTEXT: Calcitonin (CT) is a sensitive marker of medullary thyroid carcinoma (MTC) and is used for primary diagnosis and follow-up after thyroidectomy. However, persistently elevated CT is observed even after complete surgical removal without evidence of a recurrent or persistent tumor. OBJECTIVE: To investigate the presence of assay interference in the serum CT of MTC patients who are apparently without a structural disease. PATIENTS AND METHODS: We studied three index MTC cases for CT assay interference and 14 patients with metastatic MTC. The CT level was measured using an immunofluorometric assay. Screening for assay interference was performed by determination of CT levels before and after serum treatment with polyethylene glycol. Additionally, samples were analyzed by chromatography on ultra-performance liquid chromatography and protein A-Sepharose. RESULTS: Patients with biochemical and structural disease showed CT mean recovery of 84.1% after polyethylene glycol treatment, whereas patients suspected of interference showed recovery from 2-7%. The elution profile on UPLC showed that the immunometric CT from these three patients behaved like a high molecular mass aggregate (>300 kDa). Additionally, when these samples were applied to the protein A-Sepharose, CT immunoreactivity was retained on the column and was only released after lowering the pH. CONCLUSIONS: For the first time, our results show the presence of a novel pitfall in the CT immunoassay: "macrocalcitonin." Its etiology, frequency, and meaning remain to be defined, but its recognition is of interest and can help clinicians avoid unnecessary diagnostic investigations and treatment during the follow-up of MTC.