Iodine status from childhood to adulthood in females living in North-East Italy: Iodine deficiency is still an issue.

PURPOSE: This survey aimed to assess iodine status in a female population at different ages, also investigating their eating habits. METHODS: We measured urinary iodine concentrations (UIC) in: 634 females at puberty and 361 fertile women in 246 of whom were considered also their children (134 daughters and 120 sons). All subjects completed a food frequency questionnaire. RESULTS: Median UIC decreased from childhood to adulthood (median UIC 107, 77 and 55 µg/l in the young girls, females at puberty and fertile women, respectively). Though using iodized salt improved iodine status in all groups, a significantly higher UIC was only noted in females at puberty. Milk consumption significantly increased UIC at all ages. In mother-child (both daughters and sons) pairs, the children's median UIC was nearly twice as high as their mothers' (UIC 115 vs. 57 µg/l). Milk consumption varied significantly: 56% of the mothers and 76% of their children drank milk regularly. The children (both daughters and sons) and mothers who drank milk had UIC ≥100 µg/l in 59 and 34% of cases, respectively, among the pairs who did not drink milk, 44% of the children and 19% of the mothers had UIC ≥100 µg/l. On statistical regression, 3.6% of the variability in the children's UIC depended on that of their mothers. CONCLUSIONS: Dietary iodine status declines from childhood to adulthood in females due to different eating habits. A mild iodine deficiency emerged in women of child-bearing age that could have consequences during pregnancy and lactation.

MicroRNA profiles in familial and sporadic medullary thyroid carcinoma: preliminary relationships with RET status and outcome.

BACKGROUND: MicroRNAs (miRNAs) are involved in the pathogenesis of human cancers, including medullary thyroid carcinoma (MTC). The aim of this study was to test the hypothesis that different miRNA profiles are related to RET status and prognosis in patients with hereditary MTC (hMTC) and sporadic MTC (sMTC). METHODS: We analyzed the expression of nine miRNAs (miR-21, miR-127, miR-154, miR-224, miR-323, miR-370, miR-9*, miR-183, and miR-375) by quantitative real-time-polymerase chain reaction in 34 cases of sMTC, 6 cases of hMTC, and 2 cases of C-cell hyperplasia (CCH). We also analyzed the immunohistochemical expression of PDCD4, an miR-21 gene target. sMTC (n=34) was genotyped for somatic RET and RAS mutations. Disease status was defined on the basis of the concentration of serum calcitonin at the latest follow-up and other parameters as indicated in the results. RESULTS: MTC and CCH were both characterized by a significant overexpression of the whole set of miRNAs (the increase being 4.2-fold for miR-21, 6.7-fold for miR-127, 8.8-fold for miR-154, 6.6-fold for miR-224, 5.8-fold for miR-323, 6.1-fold for miR-370, 13-fold for miR-9*, 6.7-fold for miR-183, and 10.1 for miR-375, p<0.0001). PDCD4 expression was significantly downregulated in MTC samples, consistent with miR-21 upregulation. Significantly lower miR-127 levels were observed in sMTC carrying somatic RET mutations in comparison to sMTC carrying a wild-type RET. In sMTC and familial MTC, the miR-224 upregulation correlated with the absence of node metastases, lower stages at diagnosis, and with biochemical cure during follow-up. CONCLUSIONS: miRNAs are significantly dysregulated in MTC, and this dysregulation is probably an early event in C-cell carcinogenesis. miR-224 upregulation could represent a prognostic biomarker associated with a better outcome in MTC patients.

Comparison of the diagnostic accuracy of combined elastosonography and BRAF analysis vs cytology and ultrasonography for thyroid nodule suspected of malignancy.

OBJECTIVE: Diagnosing thyroid nodules preoperatively using traditional diagnostic tools - ultrasonography (US) and cytology - still carries a considerable degree of uncertainty, and surgery is recommended for a far from negligible number of patients simply for diagnostic purposes. Thyroid elastosonography (USE) and BRAF analysis have recently proved useful in detecting thyroid malignancies. The aim of this study is to establish whether combining USE and BRAF testing ameliorates preoperative diagnosis of thyroid nodule candidates for intervention by conventional approaches, thereby avoiding the need for diagnostic surgical procedures. DESIGN AND PATIENTS: We retrospectively analysed the files of 155 consecutive patients with 164 nodules, all assessed by ultrasonography, cytology, USE and BRAF testing, who underwent thyroid surgery. RESULTS: Of the 164 nodules, 74 (45%) were benign and 90 (55%) were malignant at final histology. Combining ultrasonography and cytology identified 21 (13%) as benign, 93 (57%) as malignant or probably malignant and 50 (30%) as 'suspended' (when the combined test was not able to classify the node as benign or malignant) with a 99% sensitivity, 28% specificity, 63% PPV, 95% NPV and 67% accuracy. Combining USE and BRAF testing indicated that 59 (36%) were benign, 74 (45%) were malignant and 31 (19%) were in a 'suspended' category, with a 95% sensitivity, 74% specificity, 82% PPV, 93% NPV and 86% accuracy. CONCLUSIONS: In assessing thyroid nodules suspected of malignancy, the combined analysis of USE and BRAF is equally sensitive and more specific than conventional procedures, achieving more accurate preoperative diagnoses than US and cytology combined. USE and BRAF analysis for thyroid nodule evaluation might reduce the number of unnecessary surgical procedures.

Combined RET and Ki-67 assessment in sporadic medullary thyroid carcinoma: a useful tool for patient risk stratification.

OBJECTIVE: Medullary thyroid carcinoma (MTC) derives from the parafollicular C cells, being sporadic in 75% of cases and familial in 25%, due to RET proto-oncogene germinal mutations. In sporadic forms, stage at diagnosis is the most important negative prognostic factor. The aim of this study was to evaluate the prognostic impact of molecular and immunohistochemical markers in sporadic MTC. DESIGN AND METHODS: We studied 60 patients with sporadic MTC. For each case, we sought RET somatic mutations in the primary cancer and in lymph node metastases. The primary cancer also underwent immunohistochemical examination for Ki-67. RESULTS: A somatic RET mutation was found in 38% of patients, being M918T in 52% of them. We observed a statistically significant association between RET mutations and male gender (P<0.01), tumor size (P<0.05), lymph nodes (P<0.05) and distant metastases (P<0.001), advanced stage (P<0.05), increased risk of persistent disease (P=0.01), and low overall survival (P<0.01). High Ki-67 levels were similarly associated with extra-thyroid spread (P<0.05), lymph nodes (P<0.05) and distant metastases (P<0.001), advanced stage (P=0.01), and low overall survival (P=0.01). Combining somatic RET analysis with Ki-67 assessment seems to be useful for increasing the specificity of Ki-67 assessment alone and identifying patients with a more aggressive cancer: in our series, only the patients who died during the follow-up had both a somatic RET mutation and a Ki-67 expression level >50 cells/mm(2). CONCLUSIONS: The combined evaluation of RET and Ki-67 could act as an adjuvant prognostic marker useful for ameliorating the initial risk stratification of patients with sporadic MTC.

BRAF analysis by fine needle aspiration biopsy of thyroid nodules improves preoperative identification of papillary thyroid carcinoma and represents a prognostic factor. A mono-institutional experience.

BACKGROUND: The current preoperative diagnosis of a thyroid mass relies on microscopic evaluation of thyroid cells obtained by fine needle aspiration biopsy (FNAB). More recently, FNAB has been combined with molecular analysis to increase the accuracy of the cytological evaluation. In this mono-institutional prospective study, we evaluated whether the routine introduction of BRAF testing in thyroid FNAB could help ameliorate the preoperative recognition of papillary thyroid carcinoma (PTC) in "suspended" or malignant cytological categories. Moreover, we investigated the prognostic role of the BRAFV600E mutation in PTC. METHODS: BRAFV600E analysis was performed in thyroid FNAB from 270 patients classified into one of five cytological categories THY1, THY2, THY3, THY4, THY5. All subsequently underwent thyroidectomy±node dissection, from October 2008 to September 2009 in our Department. For each cytological category, we considered the definitive histological diagnosis of PTC and the presence of the BRAFV600E mutation. In 141 patients with a final tissue diagnosis of PTC, we correlated the presence of BRAFV600E with gender, age, histotype, TNM, size of the lesion, extracapsular extension, node metastases and multifocality. RESULTS: The prevalence of the BRAFV600E mutation, among PTCs at final tissue diagnosis, was 69%. It improved the FNAB diagnostic accuracy from 88% to 91%. The BRAFV600E mutation was correlated with older age, classical variant of PTC, advanced stages in patients > 45 years. CONCLUSIONS: BRAFV600E testing could play a role in improving the diagnostic accuracy of FNAB for PTC, representing a useful adjuvant tool in presurgical characterization of thyroid nodes in particular cases. There is an association between the BRAFV600E mutation and some clinico-pathological characteristics of PTC.

BRAF in primary and recurrent papillary thyroid cancers: the relationship with (131)I and 2-[(18)F]fluoro-2-deoxy-D-glucose uptake ability.

OBJECTIVE: BRAF V600E is a potential marker of poor prognosis in papillary thyroid cancers (PTC). In a previous report, we showed that recurrent PTC with no radioiodine ((131)I) uptake are frequently associated with BRAF mutations, a low expression of thyroid-related genes and a high expression of glucose type-1 transporter gene. AIM: The aim of the present study was to assess BRAF status in a large series of recurrent PTC patients, considering paired primary and recurrent cancers. The BRAF genotype was correlated with the ability to concentrate (131)I and/or 2-[(18)F]fluoro-2-deoxi-d-glucose ((18)F-FDG) in the recurrent cancers, serum markers of recurrence, and patient outcome. DESIGN AND METHODS: We studied 50 PTC patients with recurrent cervical disease submitted to a re-intervention, followed up in median for 9 years. BRAF analysis was conducted by direct sequencing and mutant allele-specific PCR amplification. In 18 cases, molecular analysis was also assessed in the primary cancer. Out of 50 patients, 30 underwent (18)F-FDG-positron emission tomography-computed tomography. RESULTS: BRAF V600E-positive recurrent patients were found (131)I-negative in 94% of cases (P<0.001); 73% of the cancers carrying BRAF V600E were both (131)I-negative and (18)F-FDG positive. In paired primary and recurrent PTC, BRAF V600E was observed in 79% of the primary cancers and 84% of their recurrences. Three patients with (131)I-negative and BRAF V600E-positive recurrent cancers deceased during follow-up. CONCLUSIONS: BRAF mutations are more common in thyroid recurrences with no (131)I uptake than in (131)I-positive cases. They are correlated with the ability to concentrate (18)F-FDG, and they can appear, albeit rarely, as a de novo event in the course of PTC recurrences.

Galectin-3 cytotest in thyroid follicular neoplasia: a prospective, monoinstitutional study.

OBJECTIVE: To analyze the utility of galectin-3 (Gal3) immunohistochemistry (IHC) on preoperatively obtained fine needle aspiration biopsy (FNAB) in identifying the subgroup of follicular neoplasia (FN) patients who were candidates for thyroidectomy. STUDY DESIGN: This prospective, monoinstitutional study applied a standardized Gal3 immunostaining protocol (cell block specimens; Gal3 scores: G0 [no Gal3+ve IHC reaction], G1 [Gal3+ve thyrocytes < or = 10%], G2 [Gal3+ve thyrocytes > 10%) in 100 consecutive cytologically assessed FN. All patients underwent thyroidectomy, and the FNs were always histologically categorized (World Health Organization criteria). RESULTS: The overall malignancy rate was 15%. Gal3 expression in presurgical samples significantly correlated with the postoperative diagnosis (p < 0.0001). When all positive Gal3 cases were pooled together (G1+G2), the IHC test performed as follows: sensitivity = 80%; specificity = 86%; positive predictive value = 50%; negative predictive value = 96%. All the Gal3-G2 cases (presurgical cell block) showed postoperative evidence of malignancy. All 9 cases of papillary tumor expressed Gal3 in both cell block and postoperative histology. Among the 6 follicular cancers, the prevalence of Gal3 overexpression in the cell block was 50%. CONCLUSION: The cell block procedure applied to thyroid FNAB samples enables the Gal3 cytotest to be implemented usefully in the preoperative identification of those cases of FN postoperatively found to be malignant (also significantly reducing the number of inappropriate thyroidectomies). Strong Gal3 expression should prompt immediate surgical treatment.

Characterization of the largest kindred with MEN2A due to a Cys609Ser RET mutation.

RET codon 609 point mutations are rare and may predispose to aggressive medullary thyroid carcinoma (MTC). In a kindred with 15 carriers of the Cys609Ser RET mutation we observed no MTC before 17 years of age, no lymph node metastases before 30 years and no distant metastases before 60 years. Two patients developed pheochromocytoma and one had primary hyperparathyroidism as the first sign of the syndrome. In conclusion, at variance from what already known, in this large kindred the Cys609Ser RET mutation predispose to a scarcely aggressive, highly penetrant MTC and a low penetrance of pheochromocytoma and primary hyperparathyroidism.

Iodine status in pregnancy: role of dietary habits and geographical origin.

OBJECTIVES: A study was conducted on iodine status during pregnancy and its dependence on dietary habits, racial and geographical origin, and time since arrival in Italy. DESIGN AND METHODS: We enrolled 322 consecutive pregnant women: 217 Italians, 62 Eastern Europeans and 43 from Northern and Central Africa. All women completed a food frequency questionnaire on their dietary habits. The urinary iodide concentration (UIC) was determined in spot morning urine samples. RESULTS: In the group as a whole, the median UIC was 83 microg/l; the UIC was < 50 in 33% and of 150 microg/l or more in 27%; it was significantly lower in Africans and Eastern Europeans than in Italians (medians 45 and 46 vs. 100 microg/l, respectively, P = 0.005). For the foreign women, there was a significant correlation between UIC and time since arrival in Italy (r = 0.22, P = 0.02). A significant link emerged between UIC and cow's milk intake (P = 0.0001). Iodine supplements were used by 40% of the women, and UIC were higher in those who did so than in those who did not (median 103 vs. 75 microg/l, P = 0.03), particularly if the latter did not drink milk (median 98 vs. 42 microg/l, P = 0.01). Multivariate analysis showed that milk was the only variable influencing UIC (OR 1.29, P = 0.0005). CONCLUSIONS: (i) Iodine levels are too low among pregnant women in our region, and particularly in foreign women. (ii) Cow's milk intake is their main source of iodine. (iii) Iodine supplementation is mandatory during pregnancy, particularly for women do not drink milk.

RET genotypes in sporadic medullary thyroid cancer: studies in a large Italian series.

BACKGROUND: Highly discrepant data about the different distribution of RET germline single nucleotide polymorphisms (SNPs) among patients with sporadic medullary thyroid cancer (sMTC) and controls are available. DESIGN AND PATIENTS: In the present case-control study, a wide panel of seven RET SNPs has been tested in the largest sMTC series and in a matched control group. RESULTS: None of the investigated polymorphisms show a significantly different distribution in patients with sMTC when compared to controls. Twenty haplotypes and 57 genotypes were generated, and their association with the disease and with the clinical features were statistically evaluated. Interestingly, 14 genotypes were found to be unique to sMTC patients and 25 to controls. Two haplotypes and three genotypes, all including the intronic variants IVS1-126 and IVS14-24, were significantly associated with sMTC patients and with a higher tumour aggression. The functional activity of the only nonsynonymous RET variant (c.2071C > A, G691S) was tested for the first time. Interestingly, Western blot analyses showed that the fraction of Ret9-G691S protein located at the plasma membrane level was overrepresented when compared to Ret9-WT, suggesting facilitated targeting at the cell membrane for this variant. However, no transforming activity was shown in a focus formation assay on cells carrying the Ret9-G691S, against a possible oncogenic role of G691S variant. CONCLUSIONS: RET genotypes including two intronic RET variants were associated with the risk of developing sMTC and to more aggressive behaviour. Further studies are warranted to elucidate whether these RET genotypes are in linkage disequilibrium with another susceptibility gene or whether these variants could play a role in the genesis of sMTC per se.

Molecular characteristics in papillary thyroid cancers (PTCs) with no 131I uptake.

OBJECTIVE: Papillary thyroid cancers (PTCs) with no iodine uptake have an aggressive behaviour and a poor prognosis. The aim of our study was to characterize, at molecular level, a subset of PTC with no 131 iodine ((131)I) uptake. DESIGN AND METHODS: Forty-eight cancer tissues were divided into three groups: Group 1, 28 primary cancers; Group 2, 7 recurrences capable of trapping (131)I; and Group 3, 13 recurrences incapable of trapping (131)I. mRNA levels of thyroid genes (sodium/iodide symporter NIS, thyroglobulin, thyroperoxidase and pendrin) and glycolytic metabolism genes (GLUT-1, hexokinase I and II) and BRAF mutations were evaluated in the different groups. RESULTS: Cancers with no (131)I uptake had slightly reduced NIS, significantly reduced thyroglobulin (P < 0.01), thyroperoxidase (P = 0.01) and pendrin (P = 0.03) and significantly increased GLUT-1 (P = 0.01) gene expression levels; and a high frequency of BRAF mutations (77%). BRAF(V600E) mutation, in both primary and metastatic thyroid cancers, is associated with a marked drop in thyroperoxidase (29-fold) and pendrin (20-fold) expression and a considerable increase (five-fold) in GLUT-1 expression. CONCLUSIONS: (1) The loss of (131)I uptake in recurrences depends not only on a decrease in NIS gene, but possibly on a reduction in the molecules regulating its intracellular metabolism; (2) the high GLUT-1 gene expression supports the use of positron emission tomography with specific tracers in clinical management of such cancers; and (3) BRAF(V600E) point mutations may lead to less differentiated phenotypes, suggesting a worse prognosis.