Mutant MYO1F alters the mitochondrial network and induces tumor proliferation in thyroid cancer.

Familial aggregation is a significant risk factor for the development of thyroid cancer and familial non-medullary thyroid cancer (FNMTC) accounts for 5-7% of all NMTC. Whole exome sequencing analysis in the family affected by FNMTC with oncocytic features where our group previously identified a predisposing locus on chromosome 19p13.2, revealed a novel heterozygous mutation (c.400G > A, NM_012335; p.Gly134Ser) in exon 5 of MYO1F, mapping to the linkage locus. In the thyroid FRTL-5 cell model stably expressing the mutant MYO1F p.Gly134Ser protein, we observed an altered mitochondrial network, with increased mitochondrial mass and a significant increase in both intracellular and extracellular reactive oxygen species, compared to cells expressing the wild-type (wt) protein or carrying the empty vector. The mutation conferred a significant advantage in colony formation, invasion and anchorage-independent growth. These data were corroborated by in vivo studies in zebrafish, since we demonstrated that the mutant MYO1F p.Gly134Ser, when overexpressed, can induce proliferation in whole vertebrate embryos, compared to the wt one. MYO1F screening in additional 192 FNMTC families identified another variant in exon 7, which leads to exon skipping, and is predicted to alter the ATP-binding domain in MYO1F. Our study identified for the first time a role for MYO1F in NMTC.

Molecular pathology of thyroid tumours of follicular cells: a review of genetic alterations and their clinicopathological relevance.

Thyroid cancer is the most common endocrine malignancy. Knowledge of the molecular pathology of thyroid tumours originating from follicular cells has greatly advanced in the past several years. Common molecular alterations, such as BRAF p.V600E, RAS point mutations, and fusion oncogenes (RET-PTC being the prototypical example), have been, respectively, associated with conventional papillary carcinoma, follicular-patterned tumours (follicular adenoma, follicular carcinoma, and the follicular variant of papillary carcinoma/non-invasive follicular thyroid neoplasm with papillary-like nuclear features), and with papillary carcinomas from young patients and arising after exposure to ionising radiation, respectively. The remarkable correlation between genotype and phenotype shows how specific, mutually exclusive molecular changes can promote tumour development and initiate a multistep tumorigenic process that is characterised by aberrant activation of mitogen-activated protein kinase and phosphoinositide 3-kinase-PTEN-AKT signalling. Molecular alterations are becoming useful biomarkers for diagnosis and risk stratification, and as potential treatment targets for aggressive forms of thyroid carcinoma. What follows is a review of the principal genetic alterations of thyroid tumours originating from follicular cells and of their clinicopathological relevance.

Features and Progression of Potential Celiac Disease in Adults.

BACKGROUND & AIMS: Individuals with potential celiac disease have serologic and genetic markers of the disease with little or no damage to the small intestinal mucosa. We performed a prospective study to learn more about disease progression in these people. METHODS: We collected data from 77 adults (59 female; median age, 33 years) diagnosed with potential celiac disease (on the basis of serology and HLA type) at Bologna University in Italy from 2004 through 2013. The subjects had normal or slight inflammation of the small intestinal mucosa. Clinical, laboratory, and histologic parameters were evaluated at diagnosis and during a 3-year follow-up period. RESULTS: Sixty-one patients (46 female; median age, 36 years) showed intestinal and extraintestinal symptoms, whereas the remaining 16 (13 female; median age, 21 years) were completely asymptomatic at diagnosis. All subjects tested positive for immunoglobulin A endomysial antibody and tissue transglutaminase antibody, except for 1 patient with immunoglobulin A deficiency; 95% of patients were carriers of HLA-DQ2. Duodenal biopsies from 26% patients had a Marsh score of 0, and 74% had a Marsh score of 1. A higher proportion of symptomatic patients had autoimmune disorders (36%) and antinuclear antibodies (41%) than asymptomatic patients (5% and 12.5%, respectively), and symptomatic patients were of older age at diagnosis (P < .05). Gluten withdrawal led to significant clinical improvement in all 61 symptomatic patients. The 16 asymptomatic patients continued on gluten-containing diets, and only 1 developed mucosal flattening; levels of anti-endomysial and tissue transglutaminase antibodies fluctuated in 5 of these patients or became undetectable. CONCLUSIONS: In a 3-year study of adults with potential celiac disease, we found most to have symptoms, but these improved on gluten withdrawal. Conversely, we do not recommend a gluten-free diet for asymptomatic adults with potential celiac disease because they do not tend to develop villous atrophy.

BRAF V600E and risk stratification of thyroid microcarcinoma: a multicenter pathological and clinical study.

Studies from single institutions have analyzed BRAF in papillary microcarcinomas, sometimes with contradictory results. Most of them have provided limited integration of histological and clinical data. To obtain a comprehensive picture of BRAF V600E-mutated microcarcinomas and to evaluate the role of BRAF testing in risk stratification we performed a retrospective multicenter analysis integrating microscopical, pathological, and clinical information. Three hundred and sixty-five samples from 300 patients treated at six medical institutions covering different geographical regions of Italy were analyzed with central review of all cases. BRAF V600E statistical analysis was conducted on 298 microcarcinomas from 264 patients after exclusion of those that did not meet the required criteria. BRAF V600E was identified in 145/298 tumors (49%) including the following subtypes: 35/37 (95%, P<0.0001) tall cell and 72/114 (64%, P<0.0001) classic; conversely 94/129 follicular variant papillary microcarcinomas (73%, P<0.0001) were BRAF wild type. BRAF V600E-mutated microcarcinomas were characterized by markedly infiltrative contours (P<0.0001) with elongated strings of neoplastic cells departing from the tumor, and by intraglandular tumor spread (P<0.0001), typically within 5 mm of the tumor border. Multivariate analysis correlated BRAF V600E with specific microscopic features (nuclear grooves, optically clear nuclei, tall cells within the tumor, and tumor fibrosis), aggressive growth pattern (infiltrative tumor border, extension into extrathyroidal tissues, and intraglandular tumor spread), higher American Thyroid Association recurrence risk group, and non-incidental tumor discovery. The following showed the strongest link to BRAF V600E: tall cell subtype, many neoplastic cells with nuclear grooves or with optically clear nuclei, infiltrative growth, intraglandular tumor spread, and a tumor discovery that was non-incidental. BRAF V600E-mutated microcarcinomas represent a distinct biological subtype. The mutation is associated with conventional clinico-pathological features considered to be adverse prognostic factors for papillary microcarcinoma, for which it could be regarded as a surrogate marker. BRAF analysis may be useful to identify tumors (BRAF wild type) that have negligible clinical risk.

Anoctamin 1 is apically expressed on thyroid follicular cells and contributes to ATP- and calcium-activated iodide efflux.

BACKGROUND/AIMS: Iodide efflux from thyroid cells into the follicular lumen is essential for the synthesis of thyroid hormones, however, the pathways mediating this transport have only been partially identified. A calcium-activated pathway of iodide efflux has long been recognized, but its molecular identity unknown. Anoctamin 1 (ANO1) is a calcium-activated chloride channel (CaCC), and this study aims to investigate its contribution to iodide fluxes in thyroid cells. METHODS: RT-PCR, immunohistochemistry, and live cell imaging with the fluorescent halide biosensor YFP-H148Q/I152L were used to study the expression, localization and function of ANO1 in thyroid cells. RESULTS: ANO1 mRNA was detected in human thyroid tissue and FRTL-5 thyrocytes, and ANO1 protein was localized to the apical membrane of follicular cells. ATP induced a transient loss of iodide from FRTL-5 cells that was dependent on the mobilization of intracellular calcium, and was inhibited by CaCC/ANO1 inhibitors and siRNA against ANO1. Calcium-activated iodide efflux was also observed in CHO cells over-expressing the Sodium Iodide Symporter (NIS) and ANO1. CONCLUSION: ANO1 in thyrocytes functions as a calcium-activated channel mediating iodide efflux, and may contribute to the rapid delivery of iodide into the follicular lumen for the synthesis of thyroid hormones following activation by calcium-mobilizing stimuli.

Intracellular anion fluorescence assay for sodium/iodide symporter substrates.

The sodium/iodide symporter (NIS) is primarily responsible for iodide accumulation in the thyroid gland for the synthesis of thyroid hormones; however, it can also transport other lyotropic anions in the thyroid gland and nonthyroid tissues. Some NIS substrates have important physiological or clinical roles, and others are environmental contaminants with health-related consequences. The aim of this study was to assess the utility of a yellow fluorescent protein variant, YFP-H148Q/I152L, as a biosensor to monitor the cellular uptake of NIS substrates, including thiocyanate (SCN(-)), nitrate (NO(3)(-)), chlorate (ClO(3)(-)), perchlorate (ClO(4)(-)), and perrhenate (ReO(4)(-)). The fluorescence of purified YFP-H148Q/I152L was suppressed by anions with an order of potency of ReO(4)(-)>ClO(4)(-)=I(-)=SCN(-)=ClO(3)(-)>NO(3)(-)≫Cl(-). Anions also suppressed the fluorescence of YFP-H148Q/I152L expressed in FRTL-5, a thyroid cell line with high NIS expression. Quantitation of intracellular concentrations revealed differences among anions in the affinity and maximal velocity of NIS-mediated uptake as well as in the rate constant for passive efflux. These results suggest that YFP-H148Q/I152L can serve as an intracellular biosensor of NIS-transported anions and may be useful to study the physiology of endogenous anions as well as the health-related consequences of environmental anions.

Perchlorate transport and inhibition of the sodium iodide symporter measured with the yellow fluorescent protein variant YFP-H148Q/I152L.

Perchlorate is an environmental contaminant that impairs thyroid function by interacting with the sodium iodide symporter (NIS), the transporter responsible for iodide uptake in the thyroid gland. Perchlorate is well known as a competitive inhibitor of iodide transport by NIS, and recent evidence demonstrates that NIS can also transport perchlorate. In this study, we evaluated the yellow fluorescent protein (YFP) variant YFP-H148Q/I152L, as a genetically encodable biosensor of intracellular perchlorate concentration monitored by real-time fluorescence microscopy. Fluorescence of recombinant YFP-H148Q/I152L was suppressed by perchlorate and iodide with similar affinities of 1.2 mM and 1.6 mM, respectively. Perchlorate suppressed YFP-H148Q/I152L fluorescence in FRTL-5 thyroid cells and NIS-expressing COS-7 cells, but had no effect on COS-7 cells lacking NIS. Fluorescence changes in FRTL-5 cells were Na+-dependent, consistent with the Na+-dependence of NIS activity. Perchlorate uptake in FRTL-5 cells resulted in 10-fold lower intracellular concentrations than iodide uptake, and was characterized by a higher affinity (K(m) 4.6 microM for perchlorate and 34.8 muM for iodide) and lower maximal velocity (V(max) 6.8 microM/s for perchlorate and 39.5 microM/s for iodide). Perchlorate also prevented iodide-induced changes in YFP-H148Q/I152L fluorescence in FRTL-5 cells, with half-maximal inhibition occurring at 1.1-1.6 muM. In conclusion, YFP-H148Q/I152L detects perchlorate accumulation by thyroid and other NIS-expressing cells, and reveals differences in the kinetics of perchlorate versus iodide transport by NIS.

Perfluorooctane sulfonic acid, a persistent organic pollutant, inhibits iodide accumulation by thyroid follicular cells in vitro.

Poly- and perfluoroalkyl substances (PFAS) are a class of endocrine disrupting chemicals (EDCs) reported to alter thyroid function. Iodide uptake by thyroid follicular cells, an early step in the synthesis of thyroid hormones, is a potential target for thyroid disruption by EDCs. The aim of the present study was to evaluate the acute effects of perfluorooctane sulfonic acid (PFOS) and perfluorooctane carboxylic acid (PFOA), two of the most abundant PFAS in the environment, on iodide transport by thyroid follicular cells in vitro. Dynamic changes in intracellular iodide concentration were monitored by live cell imaging using YFP-H148Q/I152, a genetically encoded fluorescent iodide biosensor. PFOS, but not PFOA, acutely and reversibly inhibited iodide accumulation by FRTL-5 thyrocytes, as well as by HEK-293 cells transiently expressing the Sodium Iodide Symporter (NIS). PFOS prevented NIS-mediated iodide uptake and reduced intracellular iodide concentration in iodide-containing cells, mimicking the effect of the NIS inhibitor perchlorate. PFOS did not affect iodide efflux from thyroid cells. The results of this study suggest that disruption of iodide homeostasis in thyroid cells may be a potential mechanism for anti-thyroid health effects of PFOS. The study also confirms the utility of the YFP-H148Q/I152 cell-based assay to screen environmental PFAS, and other EDCs, for anti-thyroid activity.

Prevalence of the single-nucleotide polymorphism rs11554137 (IDH1105GGT) in brain tumors of a cohort of Italian patients.

IDH mutational status is required for proper diagnosis according to the WHO criteria revised in 2016. The single nucleotide polymorphism (SNP) rs11554137 (IDH1105GGT) at codon 105 of IDH1 has been reported in patients with several tumor types, including those with glioma. The aim of this study is to investigate the prevalence of IDH1105GGT in a cohort of brain tumors, and its association with clinicopathologic features and IDH1 and IDH2 missense mutations. Exon 4 of IDH1 and IDH2 was analyzed in a series of brain tumors classified according to current WHO criteria. DNA from control individuals was analyzed to infer the prevalence of IDH1105GGT in the reference population. Analysis was performed using next generation sequencing. IDH1105GGT was three times more frequent in patients with tumors (44/293 cases, 15.0%) vs. population controls (6/109, 5.5%) (p = 0.0102). IDH1105GGT was more frequent in grade III tumors (26.1%) compared to grade II (10.9%, p = 0.038) and grade IV tumors (13.7%, p = 0.041). IDH1 105GGT was more frequent in grade II and III tumors without an IDH tumor missense mutation (43.8%) than in those with (11.5%, p = 0.005). The IDH1105GGT SNP likely represents an important genetic marker, worthy of additional investigation to better understand the clinical and biological features of IDH-WT infiltrating gliomas.

RET/papillary thyroid cancer rearrangement in nonneoplastic thyrocytes: follicular cells of Hashimoto's thyroiditis share low-level recombination events with a subset of papillary carcinoma.

CONTEXT: RET/papillary thyroid cancer (PTC) is a marker for papillary thyroid carcinoma, but its specificity has been questioned because of the disputed identification of RET/PTC in Hashimoto's thyroiditis (HT), oncocytic tumors, and other thyroid lesions. OBJECTIVE: The objective of this study was to determine 1) whether RET/PTC occurs in nonneoplastic follicular cells of HT, and 2) its recombination rate in thyroid tumors. DESIGN/PATIENTS: Forty-three samples from 31 cases of HT were examined using interphase fluorescence in situ hybridization (FISH) with RET probes spanning the breakpoint region; real-time RT-PCR to quantify RET/PTC1, RET/PTC3, and c-RET transcripts; and RT-PCR after laser capture microdissection to enrich samples for follicular cells. The results were compared with those similarly obtained in 34 papillary carcinomas, eight thyroid oncocytic tumors, and 21 normal thyroids. RESULTS: Normal samples showed no RET rearrangement. Sixty-eight percent (15 of 22) of HT were positive by FISH; in all thyroiditis, signals were localized to rare nonneoplastic follicular cells; low-level RET/PTC was identified in 17% (five of 29) of thyroiditis cases by real-time RT-PCR and in an additional six of 11 real-time negative cases after increasing sensitivity with laser capture microdissection. Low RET/PTC1 levels were detected in 26% (nine of 34) of papillary carcinomas with an expression pattern and proportion of FISH-positive cells similar to those of the thyroiditis. Forty-seven percent (16 of 34) of papillary carcinomas and one oncocytic carcinoma expressed high RET/PTC1 mRNA levels. CONCLUSIONS: Low-level RET/PTC recombination occurs in nonneoplastic follicular cells in HT and in a subset of papillary thyroid carcinomas. RET/PTC expression variability should be taken into account for the molecular diagnosis of thyroid lesions. Overlapping molecular mechanisms may govern early stages of tumor development and inflammation in the thyroid.

Seronegative celiac disease: Shedding light on an obscure clinical entity.

BACKGROUND: Although serological tests are useful for identifying celiac disease, it is well established that a minority of celiacs are seronegative. AIM: To define the prevalence and features of seronegative compared to seropositive celiac disease, and to establish whether celiac disease is a common cause of seronegative villous atrophy. METHODS: Starting from 810 celiac disease diagnoses, seronegative patients were retrospectively characterized for clinical, histological and laboratory findings. RESULTS: Of the 810 patients, fourteen fulfilled the diagnostic criteria for seronegative celiac disease based on antibody negativity, villous atrophy, HLA-DQ2/-DQ8 positivity and clinical/histological improvement after gluten free diet. Compared to seropositive, seronegative celiac disease showed a significantly higher median age at diagnosis and a higher prevalence of classical phenotype (i.e., malabsorption), autoimmune disorders and severe villous atrophy. The most frequent diagnosis in the 31 cases with seronegative flat mucosa was celiac disease (45%), whereas other diagnoses were Giardiasis (20%), common variable immunodeficiency (16%) and autoimmune enteropathy (10%). CONCLUSIONS: Although rare seronegative celiac disease can be regarded as the most frequent cause of seronegative villous atrophy being characterized by a high median age at diagnosis; a close association with malabsorption and flat mucosa; and a high prevalence of autoimmune disorders.

BRAF mutation predicts a poorer clinical prognosis for papillary thyroid cancer.

CONTEXT: Use of BRAF mutation in papillary thyroid cancer (PTC) has the potential to improve risk stratification of this cancer. OBJECTIVE: The objective of the study was to investigate the prognostic value of BRAF mutation in patients with PTC. DESIGN, SETTING, AND SUBJECTS: In a multicenter study of 219 PTC patients, data on their clinicopathological characteristics and clinical courses between 1990 and 2004 were retrospectively collected, and their tumor BRAF mutation status was determined. Associations of BRAF mutation with initial tumor characteristics and subsequent recurrence were analyzed. MAIN OUTCOME MEASURE: Relationships between the BRAF mutation status and clinicopathological outcomes, including recurrence, were measured. RESULTS: We found a significant association between BRAF mutation and extrathyroidal invasion (P < 0.001), lymph node metastasis (P < 0.001), and advanced tumor stage III/IV (P = 0.007) at initial surgery. This association remained significant on multivariate analysis, adjusting for conventional clinicopathological predictors of recurrence excluding the histological PTC subtype, but was lost when the tumor subtype was included in the model. BRAF mutation was also significantly associated with tumor recurrence, 25 vs. 9% with and without mutation, respectively (P = 0.004), during a median of 15 (interquartile range, 3-29) months of follow-up. This association remained significant on multivariate analysis adjusting for conventional clinicopathological predictors of recurrence, even including the PTC subtype (odds ratio, 4.0; 95% confidence interval, 1.1-14.1; P = 0.03). BRAF mutation was even an independent predictor of recurrence in patients with stage I/II disease, 22 vs. 5% with and without BRAF mutation, respectively (P = 0.002). BRAF mutation was also more frequently associated with absence of tumor I-131 avidity and treatment failure of recurrent disease. CONCLUSIONS: In patients with PTC, BRAF mutation is associated with poorer clinicopathological outcomes and independently predicts recurrence. Therefore, BRAF mutation may be a useful molecular marker to assist in risk stratification for patients with PTC.

A novel missense mutation in ANO5/TMEM16E is causative for gnathodiaphyseal dyplasia in a large Italian pedigree.

Gnathodiaphyseal dysplasia (GDD) is an autosomal dominant syndrome characterized by frequent bone fractures at a young age, bowing of tubular bones and cemento-osseus lesions of the jawbones. Anoctamin 5 (ANO5) belongs to the anoctamin protein family that includes calcium-activated chloride channels. However, recent data together with our own experiments reported here add weight to the hypothesis that ANO5 may not function as calcium-activated chloride channel. By sequencing the entire ANO5 gene coding region and untranslated regions in a large Italian GDD family, we found a novel missense mutation causing the p.Thr513Ile substitution. The mutation segregates with the disease in the family and has never been described in any database as a polymorphism. To date, only two mutations on the same cysteine residue at position 356 of ANO5 amino-acid sequence have been described in GDD families. As ANO5 has also been found to be mutated in two different forms of muscular dystrophy, the finding of this third mutation in GDD adds clues to the role of ANO5 in these disorders.

BRAF(V600E) mutation and expression of proangiogenic molecular markers in papillary thyroid carcinomas.

OBJECTIVE: Tyrosine kinase inhibitors (TKIs) are evaluated for treatment of radioiodine refractory thyroid cancer. Their effects in this setting are based on blockade of proangiogenic signaling mediated by receptors for vascular endothelial growth factors (VEGFs) and platelet-derived growth factors (PDGF). Most TKIs also block other cancer-relevant kinases, such as B-type Raf kinase (BRAF), which are constitutively activated in approximately half of papillary thyroid carcinomas (PTCs), but the impact of these effects is not clear. DESIGN: The aim of our study was to investigate the impact of BRAF(V600E) on proangiogenic gene expression and microvascular features of PTCs. METHODS: mRNA levels for VEGFA, VEGF receptors, and coreceptors (VEGFRs 1, 2, and 3, neuropilin-1), and PDGF receptor ß (PDGFRß or PDGFRB) were measured with real-time PCR in BRAF(V600E) (n=55) and wild-type BRAF (BRAF-wt; n=35) PTCs. VEGF and VEGFR protein expression and microvessel densities (MVD) and lymphatic vessel densities (LVDs) were assessed by immunohistochemistry in 22 of the 90 PTCs (including 11 BRAF(V600E) cases). Angiogenic gene expression was also studied in vitro after induction/silencing of the BRAF(V600E) mutation in thyrocyte lines. RESULTS: Transcript levels of proangiogenic factors were significantly lower in BRAF(V600E) PTCs versus BRAF-wt PTCs (P<0.0001), but MVD and LVDs were not significantly different. VEGFA mRNA levels in thyroid cell lines decreased when BRAF(V600E) mutation was induced (P=0.01) and increased when it was silenced (P=0.01). CONCLUSIONS: Compared with BRAF-wt PTCs, those harboring BRAF(V600E) exhibit downregulated VEGFA, VEGFR, and PDGFRß expression, suggesting that the presence of BRAF mutation does not imply a stronger prediction of response to drugs targeting VEGF and PDGFB signaling pathways.

Fluorescence quantitation of thyrocyte iodide accumulation with the yellow fluorescent protein variant YFP-H148Q/I152L.

The thyroid gland accumulates iodide for the synthesis of thyroid hormones. The aim of the current study was to quantify iodide accumulation in cultured thyroid cells by live cell imaging using the halide-sensitive yellow fluorescent protein (YFP) variant YFP-H148Q/I152L. In vivo calibrations were performed in FRTL-5 thyrocytes to determine the sensitivity of YFP-H148Q/I152L to iodide. In the presence of ion-selective ionophores, YFP-H148Q/I152L fluorescence was suppressed by halides in a pH-dependent manner with 20-fold selectivity for iodide versus chloride and competition between the two halides. At a physiological pH of 7 and a chloride concentration of 15mM, the affinity constant of YFP-H148Q/I152L for iodide was 3.5mM. In intact FRTL-5 cells, iodide induced a reversible decrease in YFP-H148Q/I152L fluorescence. FRTL-5 cells concentrated iodide to 60 times the extracellular concentration. Iodide influx exhibited saturation kinetics with respect to extracellular iodide with a K(m) of 35 microM and a V(max) of 55 microM/s. Iodide efflux exhibited saturation kinetics with respect to intracellular iodide concentration with a K(m) of 2.2mM and a V(max) of 43 microM/s. The results of this study demonstrate the utility of YFP-H148Q/I152L as a sensitive and selective biosensor for the quantification of iodide accumulation in thyroid cells.

Cell-based imaging of sodium iodide symporter activity with the yellow fluorescent protein variant YFP-H148Q/I152L.

The sodium iodide symporter (NIS) mediates iodide (I(-)) transport in the thyroid gland and other tissues and is of increasing importance as a therapeutic target and nuclear imaging reporter. NIS activity in vitro is currently measured with radiotracers and electrophysiological techniques. We report on the development of a novel live cell imaging assay of NIS activity using the I(-)-sensitive and genetically encodable yellow fluorescent protein (YFP) variant YFP-H148Q/I152L. In FRTL-5 thyrocytes stably expressing YFP-H148Q/I152L, I(-) induced a rapid and reversible decrease in cellular fluorescence characterized by 1) high affinity for extracellular I(-) (35 muM), 2) inhibition by the NIS inhibitor perchlorate, 3) extracellular Na(+) dependence, and 4) TSH dependence, suggesting that fluorescence changes are due to I(-) influx via NIS. Individual cells within a population of FRTL-5 cells exhibited a 3.5-fold variation in the rate of NIS-mediated I(-) influx, illustrating the utility of YFP-H148Q/I152L to detect cell-to-cell difference in NIS activity. I(-) also caused a perchlorate-sensitive decrease in YFP-H148Q/I152L fluorescence in COS-7 cells expressing NIS but not in cells lacking NIS. These results demonstrate that YFP-H148Q/I152L is a sensitive biosensor of NIS-mediated I(-) uptake in thyroid cells and in nonthyroidal cells following gene transfer and suggest that fluorescence detection of cellular I(-) may be a useful tool by which to study the pathophysiology and pharmacology of NIS.

Association of aberrant methylation of tumor suppressor genes with tumor aggressiveness and BRAF mutation in papillary thyroid cancer.

The role of aberrant tumor suppressor gene methylation in the aggressiveness of papillary thyroid cancer (PTC) has not been documented. By showing promoter methylation-induced gene silencing in PTC-derived cell lines, we first demonstrated the functional consequence of methylation of several recently identified tumor suppressor genes, including those for tissue inhibitor of metalloproteinase-3 (TIMP3), SLC5A8, death-associated protein kinase (DAPK) and retinoic acid receptor beta2 (RARbeta2). We then investigated the role of methylation of these genes in the aggressiveness of PTC by examining the relationship of their aberrant methylation to clinicopathological characteristics and BRAF mutation in 231 primary PTC tumors. Methylation of TIMP3, SLC5A8 and DAPK was significantly associated with several aggressive features of PTC, including extrathyroidal invasion, lymph node metastasis, multifocality and advanced tumor stages. Methylation of these genes was also significantly associated with BRAF mutation in PTC, either individually or collectively in various combinations. Methylation of these genes, either individually or collectively, occurred more frequently in more aggressive classical and tall-cell PTC subtypes than in less aggressive follicular-variant PTC, with the latter known to infrequently harbor BRAF mutation. Several other tumor suppressor genes investigated were not methylated. These results suggest that aberrant methylation and hence silencing of TIMP3, SLC5A8, DAPK and RARbeta2, in association with BRAF mutation, may be an important step in PTC tumorigenesis and progression.

Real-time quantitative RT-PCR identifies distinct c-RET, RET/PTC1 and RET/PTC3 expression patterns in papillary thyroid carcinoma.

RET/PTC1 and RET/PTC3 are the markers for papillary thyroid carcinoma. Their reported prevalence varies broadly. Nonrearranged c-RET has also been detected in a variable proportion of papillary carcinomas. The published data suggest that a wide range in expression levels may contribute to the different frequency of c-RET and, particularly, of RET/PTC detection. However, quantitative expression analysis has never been systematically carried out. We have analyzed by real-time RT-PCR 25 papillary carcinoma and 12 normal thyroid samples for RET/PTC1, RET/PTC3 and for RET exons 10-11 and 12-13, which are adjacent to the rearrangement site. The variability in mRNA levels was marked and four carcinoma groups were identified: one lacking RET/PTC rearrangement with balanced RET exon levels similar to those of the normal samples (7/25 cases, 28%), the second (6/25 cases, 24%) with balanced RET expression and very low levels of RET/PTC1, the third with unbalanced RET exons 10-11 and 12-13 expression, high RET/PTC1 levels but no RET/PTC3 (7/25 cases, 28%), and the fourth with unbalanced RET expression, high RET/PTC1 levels and low levels of RET/PTC3 (5/25 cases, 20%). Papillary carcinomas with high RET/PTC1 expression showed an association trend for large tumor size (P=0.063). Our results indicate that the variability in c-RET and RET/PTC mRNA levels contributes to the apparent inconsistencies in their reported detection rates and should be taken into account not only for diagnostic purposes but also to better understand the role of c-RET activation in thyroid tumorigenesis.