Marina Muzza

BRAF mutations in an Italian cohort of thyroid cancers

objective  Recently, a somatic point mutation of the BRAF gene (V599E) has been identified as the most common genetic event in papillary thyroid carcinoma (PTC) with a variable frequency (about 25–70%) in different series from USA, Japan, Portugal and Ukraine.

The tight relationship between papillary thyroid cancer, autoimmunity and inflammation: clinical and molecular studies

Objective  The recent concept that oncogenes responsible for thyroid neoplastic transformation are able to elicit an inflammatory protumourigenic microenvironment raises interest in further studies on papillary thyroid cancer (PTC) associated with thyroid autoimmunity.

Telomerase in differentiated thyroid cancer: Promoter mutations, expression and localization

Telomerase-reverse-transcriptase (TERT) promoter mutations have been recently described in tumors. In the present large series, TERT mutations were found in 12% of papillary thyroid cancers (PTCs) and in 14% of follicular thyroid cancers (FTCs), and were found to significantly correlate with older age at diagnosis and poorer outcome. Interestingly, the prognostic value of TERT mutations resulted to be significantly stronger than that of BRAF(V600E). Moreover, the outcome was not different among tumors with isolated TERT mutation and those with coexistent mutations (TERT/BRAF in PTCs or TERT/RAS in FTCs). TERT rs2853669 polymorphism was found in 44.4% of tumors. At WB, TERT was significantly more expressed in tumors than in normal samples, being the highest levels of expression recorded in TERT mutated cases. At IHC, in tumors and in metastatic lymph-nodes TERT staining was significantly higher in the cytoplasm than in the nucleus, whereas in normal tissue the degree of staining did not differ in the two cellular compartments. In conclusion, TERT mutations were shown to strongly correlate with a poorer outcome in differentiated thyroid tumors, and neither BRAF nor RAS mutation were found to confer an additional effect in the disease persistence. TERT protein was found to be more expressed in neoplastic than in normal tissues, and to display a different cellular localization, suggesting that it could contribute to thyroid cancer progression by mechanisms taking place in the cytoplasm.

Modifications in the Papillary Thyroid Cancer Gene Profile Over the Last 15 Years

BACKGROUND Evidence for an increased prevalence of BRAF(V600E) mutations has been documented in recent decades. The aim of this study was to evaluate the prevalence of both RET/PTC rearrangements and BRAF(V600E) mutations in an Italian cohort of papillary thyroid carcinoma (PTC) patients followed at the Endocrine Units of Pisa, Milano, and Perugia from 1996-2010. PATIENTS AND METHODS In total, 401 PTC patients were examined and grouped according to the time of surgery: group 1, 1996-2000; group 2, 2001-2005; and group 3, 2006-2010. Patients were analyzed for clinical, pathological, and molecular features. In parallel, the molecular characteristics of 459 PTC from Sicily were studied. RESULTS The genetic profiles of the three groups were significantly different (P < 0.0001). In particular, the frequency of RET/PTC rearrangements decreased from 1996-2010, occurring in 33 of 100 (33%) of the patients in group 1, 26 of 148 (17%) in group 2, and 15 of 153 (9.8%) in group 3. The incidence of BRAF(V600E) mutations increased over the same period, from 28% in group 1 (28 of 100) to 48.9% in group 2 (73 of 148) and 58.1% in group 3 (89 of 153). A consistent increase in BRAF(V600E) prevalence was observed in the Sicilian group (P < 0.0001). Moreover, a statistically significant increase in the mean age at diagnosis and decrease in tumor size over the study period was observed. CONCLUSION The genetic profile of PTC changed over the last 15 yr, with a significant decrease in the prevalence of RET/PTC rearrangements and an increase in BRAF(V600E) mutations. In addition, the mean age at diagnosis increased and tumor size decreased over the study period.

Fetal cell microchimerism in papillary thyroid cancer: studies in peripheral blood and tissues

Fetal cell microchimerism (FCM) is defined as the persistence, for decades after pregnancy, of fetal cells in maternal organs and circulation without any apparent rejection. We recently reported evidence, in papillary thyroid cancer (PTC) tissues, supporting a possible role of FCM in tumor damage and repair. To extend those data at the peripheral level, 106 women with a previous male pregnancy, comprising 57 with PTC and 49 healthy controls were enrolled. The presence of circulating male DNA was assessed by the amplification of the Y chromosome‐specific gene SRY, with a sensitivity of 1 male cell per 1 million female cells. Moreover, to compare the microchimeric status in blood and in tumors, the neoplastic tissues of 19 women were studied. At the blood level, a significantly lower frequency of FCM was found in parous women with PTC with respect to controls (49.1% vs. 77.6%; p = 0.002). By PCR, male DNA was identified in the tumor tissues of 6 patients, and FISH analyses confirmed the presence of microchimeric cells (range 2.1–6.9 cells/section). In some patients, FCM was negative in the blood, whereas microchimeric cells were identified in the tumor. In conclusion, the prevalence of FCM in peripheral blood was found to be significantly lower in patients than in healthy controls. The presence of microchimeric cells in the tumors, but not at the peripheral level, supports the hypothesis that fetal cells could reside in maternal niches and could be recruited to diseased areas, where they could differentiate to regenerate damaged tissues.

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.

Histopathological and molecular studies in patients with goiter and hypercalcitoninemia: reactive or neoplastic C-cell hyperplasia?

The cut-off values able to differentiate between reactive or neoplastic C-cell hyperplasia (CCH) or to predict sporadic medullary thyroid cancer (MTC) are still debated both for basal and stimulated calcitonin (bCT and sCT). In the present study, the prevalence and the histological patterns of CCH in 15 patients with multinodular goiter (MNG), bCT>10 pg/ml and sCT levels >50 pg/ml were studied. As controls, 16 patients with MNG and bCT levels <10 pg/ml and 4 patients with familial (FMTC) were included. For each case, calcitonin (CT) immunoreactive cells were counted in 60 consecutive high-power fields (400x) and CCH classified as focal, diffuse, nodular, or neoplastic. RET genetic analyses were performed at the germline and tissue levels in MTC and CCH cases. In patients with MNG, sCT levels >50 pg/ml were associated with CCH or MTC, being the total number of C-cells/60 fields significantly higher than that found in MNG with normal bCT (P = 0.0008) and comparable with that detected in FMTCs. In the group with sCT>50 pg/ml, the C-cells displayed a neoplastic phenotype. Neither germline nor somatic RET mutations were found. In conclusion, sCT levels >50 pg/ml were always associated with CCH, without correlation between CT levels and the number of C-cells or the final diagnosis. The C-cells had a morphology and distribution pattern similar to those observed in FMTC. Thus, sCT levels >50 pg/ml indicate the presence of CCH with a possible preneoplastic potential, suggesting the opportunity to perform a prophylactic surgical treatment.

The Clinical and Molecular Characterization of Patients With Dyshormonogenic Congenital Hypothyroidism Reveals Specific Diagnostic Clues for DUOX2 Defects

CONTEXT Mutations in the DUOX2 gene have been associated with transient or permanent congenital hypothyroidism due to a dyshormonogenic defect. OBJECTIVE This study aimed to verify the prevalence of DUOX2 mutations and the associated clinical features in children selected by criteria supporting a partial iodide organification defect (PIOD). PATIENTS AND METHODS Thirty children with PIOD-like criteria were enrolled and genotyped. A detailed clinical characterization was undertaken together with the functional analysis of the DUOX2 variations and the revision of the clinical and molecular data of the literature. RESULTS In this large selected series, the prevalence of the DUOX2 mutations was high (37%). We identified 12 missense variants, one splice site, and three frameshift DUOX2 mutations. Functional analyses showed significant impairment of H2O2 generation with five missense variants. Stop-codon mutants were shown to totally abolish DUOX2 activity by nonsense-mediated RNA decay, exon skipping, or protein truncation. DUOX2 mutations, either mono- or biallelic, were most frequently associated with permanent congenital hypothyroidism. Moreover, the present data suggested that, together with goiter and PIOD, the most significant features to select patients for the DUOX2 analysis are the low free T4 and the high TSH concentrations at the first postnatal serum sampling, despite borderline blood spot TSH. Interestingly, the analysis of previously described DUOX2 mutated cases confirmed the validity of these findings. CONCLUSIONS The defects in the peroxide generation system are common among congenital hypothyroidism patients with PIOD. The most robust clinical parameters for selecting patients for DUOX2 analysis have been identified, and several DUOX2 variants have been functionally characterized.

High phenotypic intrafamilial variability in patients with Pendred syndrome and a novel duplication in the SLC26A4 gene: clinical characterization and functional studies of the mutated SLC26A4 protein

OBJECTIVE Pendred syndrome (PS) is characterized by the association of sensorineural hearing loss (SNHL) and a partial iodide organification defect at the thyroid level. It is caused by mutations in the SLC26A4 gene. The encoded transmembrane protein, called pendrin, has been found to be able to transport chloride and other anions. DESIGN The aim of the present study was to characterize a family with PS, which shows a strong intrafamilial phenotypic variability, including kidney atrophy in one member. The age of disease-onset was significantly different in all three affected siblings, ranging from 2 to 21 years for thyroid alterations and from 1.5 to 11 years for SNHL. METHODS Clinical and genetic studies were carried out in affected siblings. The functional activity of the novel duplication found was studied by a fluorimetric method in a human renal cell line (HEK293 Phoenix) in which the protein was overexpressed. RESULTS All three siblings were found to be compound heterozygotes for the missense mutation (1226G>A, R409H) and for a novel 11 bp duplication (1561_1571CTTGGAATGGC, S523fsX548). The latter mutation creates a frame shift leading to the loss of the entire carboxy-terminus domain. Functional studies of this mutant demonstrated impaired transport of chloride and iodide when expressed in HEK 293 Phoenix cells, when compared with wild type pendrin. CONCLUSIONS A novel 11 bp duplication was found in a family with Pendred syndrome, showing a high intrafamilial phenotypic variability. An impaired transmembrane anionic transport of the mutated SLC26A4 protein was demonstrated in functional studies using a heterologous cell system.

Four novel RET germline variants in exons 8 and 11 display an oncogenic potential in vitro

CONTEXT Most germline-activating mutations of the RET proto-oncogene associated with inherited medullary thyroid cancer (MTC) are localized in exons 10, 11 and 13-15. Four novel RET variants, located in the extracellular domain (p.A510V, p.E511K and p.C531R) coded by exon 8 and in the intracellular juxtamembrane region (p.K666N) coded by exon 11, were identified on the leukocyte DNA from apparently sporadic cases. METHODS Plasmids carrying Ret9-wild-type (Ret9-WT), Ret9-C634R and all Ret9 variants were transfected, and the phosphorylation levels of RET and ERK were evaluated by western blot analyses. The transforming potentials were assessed by the focus formation assay. RESULTS The p.A510V, p.E511K and p.C531R variants were found to generate RET and ERK phosphorylation levels and to have a transforming activity higher than that of Ret9-WT variant, but lower than that of Ret9-C634R variant. Differently, the p.K666N variant, located immediately downstream of the transmembrane domain, and involving a conserved residue, displayed high kinase and transforming activities. Computational analysis predicted non-conservative alterations in the mutant proteins consistent with putative modifications of the receptor conformation. CONCLUSIONS The molecular analyses revealed an oncogenic potential for all the novel germline RET variants. Therefore, the prevalence of exon 8 genomic variations with an oncogenic potential may be higher than previously thought, and the analysis of this exon should be considered after the exclusion of mutations in the classical hotspots. In addition, on the basis of these functional data, it is advisable to extend the genetic screening to all the first-degree relatives of the MTC patients, and to perform a strict follow-up of familial carriers.