Susi Barollo

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 (131I) uptake.

Evidence of a Low Prevalence of RAS Mutations in a Large Medullary Thyroid Cancer Series

BACKGROUND Approximately 60% of sporadic medullary thyroid carcinomas (sMTC) remain orphan of a recognized genetic cause. Recently, a high percentage of RAS point mutations have been described in RET-negative sMTC. The aim of this study was to assess the prevalence of RAS point mutations in a large series of MTC collected in four Italian centers. METHODS For this purpose, we studied codons 12, 13, and 61 of H-, K-, and N-RAS genes in 188 MTC samples, either hereditary or sporadic, by direct sequencing. Correlations between the RAS mutational status and the clinical-pathological features of MTC patients as well as a meta-analysis of all published data were performed. RESULTS The prevalence of RAS mutations in the present series of MTC was 10.1%, and 17.6% when considering only RET-negative cases. RAS mutations were found in MTC tumoral tissue, but not in peripheral blood indicating their somatic origin. A novel mutation in codon 72 (M72I) was found, but with a low or null transforming potential. No association was found between the presence of RAS mutations and the clinical-pathological features of the patients. Although not statistically significant, a positive association between the presence of RAS mutations and a better outcome was observed. The meta-analysis of all published studies confirmed a prevalence of 8.8% for RAS mutations in MTC. CONCLUSIONS The prevalence of RAS mutations in our MTC series was relatively low and consistent with the meta-analysis data. Only somatic RAS mutations were found and only in RET-negative sMTC. Likewise, MTCs that harbor a RAS mutation identify a subgroup of tumors with less aggressive behavior. To our knowledge, this is the largest series of MTCs studied for the presence of mutations in RAS genes and the first meta-analysis on this specific topic.

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.

Influence of physiological dietary selenium supplementation on the natural course of autoimmune thyroiditis

Objective  Our study aimed to investigate whether physiological doses of selenium (Se) influence the natural course of autoimmune thyroiditis (AIT).

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 in primary and recurrent papillary thyroid cancers: the relationship with 131I and 2-[18F]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.

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.

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.

Abstract 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.

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.

The PDCD4/miR-21 pathway in medullary thyroid carcinoma.

Programmed cell death 4 (PDCD4) is a tumor suppressor gene involved in tumorogenesis. MicroRNA-21 (miR-21) specifically targets PDCD4, and recent studies suggest that PDCD4 is also regulated by Akt (antiapoptotic regulator within phosphatidylinositol 3-kinase). Medullary thyroid carcinoma (MTC) is a rare neuroendocrine cancer, and disease stage at diagnosis represents the main prognostic indicator. A consecutive series of 64 MTCs was considered. REarranged during Transfection (RET) and rat sarcoma (RAS) mutation status was assessed by direct sequencing. Quantitative real-time polymerase chain reaction was used to quantify mature hsa-miR-21. PDCD4 and Ki-67 immunostaining was performed with an automated platform. Immunoblot analysis of PI3K/Akt pathway was done on thyroid tissues. MTCs were consistently associated with miR-21 up-regulation (P < .0016) and featured significant PDCD4 nuclear down-regulation. An inverse correlation emerged between miR-21 overexpression and PDCD4 down-regulation (P = .0013). At enrollment, high miR-21 levels were associated with high calcitonin levels (P = .0003), lymph node metastases (P = .001), and advanced stages (P = .0003). At the end of follow-up, high miR-21 levels were associated with biochemically persistent disease (P = .0076). At enrollment, instead, PDCD4 nuclear down-regulation was associated with high calcitonin levels (P = .04), more advanced stages of disease (P < .01), and persistent disease after the follow-up (P = .02). p-Akt was more expressed in RAS-mutated MTC than in nonmutated cancers and normal tissue. This study showed, in MTCs, that miR-21 regulates PDCD4 expression and also that the miR-21/PDCD4 pathway correlates with clinicopathological variables and prognosis. Further studies should investigate the role of miR-21 as a prognostic biomarker and the feasibility of using PDCD4-restoring strategies as a therapeutic approach to MTC.