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The Lynch syndrome (LS) is an autosomal dominant disorder characterized by a strongly increased risk of developing colorectal cancer and several extra-colonic malignancies, such as carcinomas of the endometrium, ovary, ureter, stomach, and small intestine [1]. Lynch syndrome is caused by germline mutations in mismatch repair genes (MMR)[2], mainly in MLH1 and MSH2, rarely in MSH6 and PMS2 [3,4]. Tumors usually develop at a relatively young age (G mutation (rs267607760)in MLH1gene.

Aberrant activation of the

Context Ultrasonography (US) is considered the most sensitive tool for imaging persistent or recurrent papillary thyroid cancer (PTC) in the neck. Objective To clarify the usefulness of routine neck US in low- and intermediate-risk patients with PTC with no evidence of disease 1 year after thyroidectomy. Design Retrospective analysis of prospectively recorded data. Setting Academic center. Patients Two hundred twenty-six patients with PTC with sonographically normal neck lymph nodes and unstimulated serum thyroglobulin (Tg) levels that were either undetectable ( Interventions Yearly assessment: unstimulated serum Tg level, anti-Tg-antibody (TgAb) titer, TSH levels, and ultrasound examination of neck lymph nodes. Main Outcome Measures Rates of ultrasonographic lymph node abnormalities at the 3-year and last follow-up visits. Results In patients with an undetectable Tg level at the 1-year evaluation, sonographically suspicious neck lymph nodes were found in 1.2% of patients at 3 years and in 1.8% at the last visit [negative predictive values (NPVs) of 1-year Tg < 0.2 ng/mL: 98.8% (95% CI 95.8% to 99.9%) and 98.2% (95% to 99.6%), respectively]. Similar NPVs emerged for low detectable 1-year Tg levels [98.2% (90.3% to 99.9%) and 94.5% (84.9% to 98.9%) at the 3-year and last visits, respectively]. Seventy-five percent of the nodal lesions were likely false positive; none required treatment. Conclusions Low- and intermediate-risk patients with PTC with negative ultrasound findings and unstimulated Tg levels

Purpose: Several studies have shown that different tumour types sharing a driver gene mutation do not respond uniformly to the same targeted agent. Our aim was to use an unbiased network-based approach to investigate this fundamental issue using BRAF mutant tumours and the BRAF inhibitor vemurafenib. Methods: We applied SWIM, a software able to identify putative regulatory (switch) genes involved in drastic changes to the cell phenotype, to gene expression profiles of different BRAF mutant cancers and their normal counterparts in order to identify the switch genes that could potentially explain the heterogeneity of these tumours' responses to vemurafenib. Results: We identified lung adenocarcinoma as the tumour with the highest number of switch genes (298) compared to its normal counterpart. By looking for switch genes encoding for kinases with homology sequences similar to known vemurafenib targets, we found that thyroid cancer and lung adenocarcinoma have a similar number of putative targetable switch gene kinases (5 and 6, respectively) whereas colorectal cancer has just one. Conclusions: We are persuaded that our network analysis may aid in the comprehension of molecular mechanisms underlying the different responses to vemurafenib in BRAF mutant tumours.

The SARS-CoV-2 pandemic is a worldwide public health emergency. Despite the beginning of a vaccination campaign, the search for new drugs to appropriately treat COVID-19 patients remains a priority. Drug repurposing represents a faster and cheaper method than de novo drug discovery. In this study, we examined three different network-based approaches to identify potentially repurposable drugs to treat COVID-19. We analyzed transcriptomic data from whole blood cells of patients with COVID-19 and 21 other related conditions, as compared with those of healthy subjects. In addition to conventionally used drugs (e.g., anticoagulants, antihistaminics, anti-TNFα antibodies, corticosteroids), unconventional candidate compounds, such as SCN5A inhibitors and drugs active in the central nervous system, were identified. Clinical judgment and validation through clinical trials are always mandatory before use of the identified drugs in a clinical setting., Graphical Abstract ga1

Despite impressive efforts invested in epigenetic research in the last 50 years, clinical applications are still lacking. Only a few university hospital centers currently use epigenetic biomarkers at the bedside. Moreover, the overall concept of precision medicine is not widely recognized in routine medical practice and the reductionist approach remains predominant in treating patients affected by major diseases such as cancer and cardiovascular diseases. By its’ very nature, epigenetics is integrative of genetic networks. The study of epigenetic biomarkers has led to the identification of numerous drugs with an increasingly significant role in clinical therapy especially of cancer patients. Here, we provide an overview of clinical epigenetics within the context of network analysis. We illustrate achievements to date and discuss how we can move from traditional medicine into the era of network medicine (NM), where pathway-informed molecular diagnostics will allow treatment selection following the paradigm of precision medicine.

A fundamental topic in network medicine is disease genes prioritization. The underlying hypothesis is that disease genes are organized as modules confined within the interactome. Here, we propose a novel algorithm called DiaBLE ( DIA MOnD B ackground L ocal E xpansion) which is a modified version of DIAMOnD, a successful algorithm based on the concept of connectivity significance. Instead of taking the whole interactome as the background model, DiaBLE considers as gene universe the smallest local expansion of the current seeds set at each iteration step. We show that DiaBLE significantly increases the overall DIAMOnD ranking quality of genes prioritization both in terms of cross-validation and biological consistency. Here, we focus on the two algorithms only since a comparative analysis among gene prioritization methods is beyond the scope of this study. Finally, we briefly discuss the improvement of biological insight provided by DiaBLE for two cancers (head and neck squamous cell carcinoma and kidney renal clear cell carcinoma).

International audience; Tangier disease is a rare disorder of lipoprotein metabolism that presents with extremely low levels of HDL cholesterol and apoprotein A-I. It is caused by mutations in the ATP-binding cassette transporter A1 (ABCA1) gene. Clinical heterogeneity and mutational pattern of Tangier disease are poorly characterized. Moreover, also familial HDL deficiency may be caused by mutations in ABCA1 gene. ATP-binding cassette transporter A1 (ABCA1) gene mutations in a patient with Tangier disease, who presented an uncommon clinical history, and in his family were found and characterized. He was found to be compound heterozygous for two intronic mutations of ABCA1 gene, causing abnormal pre-mRNAs splicing. The novel c.1510-1G > A mutation was located in intron 12 and caused the activation of a cryptic splice site in exon 13, which determined the loss of 22 amino acids of exon 13 with the introduction of a premature stop codon. Five heterozygous carriers of this mutation were also found in proband's family, all presenting reduced HDL cholesterol and ApoAI (0.86 ± 0.16 mmol/L and 92.2 ± 10.9 mg/dL respectively), but not the typical features of Tangier disease, a phenotype compatible with the diagnosis of familial HDL deficiency. The other known mutation c.1195-27G > A was confirmed to cause aberrant retention of 25 nucleotides of intron 10 leading to the insertion of a stop codon after 20 amino acids of exon 11. Heterozygous carriers of this mutation also showed the clinical phenotype of familial HDL deficiency. Our study extends the catalog of pathogenic intronic mutations affecting ABCA1 pre-mRNA splicing. In a large family, a clear demonstration that the same mutations may cause Tangier disease (if in compound heterozygosis) or familial HDL deficiency (if in heterozygosis) is provided.

Context:The European Thyroid Association (ETA) has classified posttreatment cervical ultrasound findings in thyroid cancer patients based on their association with disease persistence/recurrence.Objective:The objective of the study was to assess this classification's ability to predict the growth and persistence of such lesions during active posttreatment surveillance of patients with differentiated thyroid cancer (DTC).Design:This was a retrospective, observational study.Setting:The study was conducted at a thyroid cancer center in a large Italian teaching hospital.Patients:Center referrals (2005–2014) were reviewed and patients selected with pathologically-confirmed DTC; total thyroidectomy, with or without neck dissection and/or radioiodine remnant ablation; abnormal findings on two or more consecutive posttreatment neck sonograms; and subsequent follow-up consisting of active surveillance. Baseline ultrasound abnormalities (thyroid bed masses, lymph nodes) were classified according to the ETA system. Patients were divided into group S (those with one or more lesions classified as suspicious) and group I (indeterminate lesions only). We recorded baseline and follow-up clinical data through June 30, 2015.Main Outcomes:The main outcomes were patients with growth (>3 mm, largest diameter) of one or more lesions during follow-up and patients with one or more persistent lesions at the final visit.Results:The cohort included 58 of the 637 DTC cases screened (9%). A total of 113 lesions were followed up (18 thyroid bed masses, 95 lymph nodes). During surveillance (median 3.7 y), group I had significantly lower rates than group S of lesion growth (8% vs 36%, P = .01) and persistence (64% vs 97%, P = .014). The median time to scan normalization was 2.9 years.Conclusions:The ETA's evidence-based classification of sonographically detected neck abnormalities can help identify papillary thyroid cancer patients eligible for more relaxed follow-up.

Objective: We report a case of a MODY 2 family: a disease frequently under-diagnosed. Patients and Methods: We analyzed the case of three brothers that we suspected as affected by Type 1 diabetes because of their low BMI without clinical or biochemical parameters for this diagnosis. Their father was diagnosed as affected from Type 2 diabetes at the age 31 years old. Results: Genetic analysis revealed the presence in all analyzed family members of non-sense Ser 383x GCK mutation mapping in exon 9 of the gene. Conclusions: We described a case of a patient misdiagnosed as T2DM. Only after the observation of a mild hyperglicemia also in his three sons, we supposed the diagnosis of MODY 2 and we confirmed it through the genetic test. These observations enforce the validity of the designed clinical algorithm for the identification of patients to be selected for the genetic diagnosis of MODY 2.

H&uuml, rthle cell carcinomas (HCC) are rare differentiated thyroid cancers that display low avidity for radioactive iodine and respond poorly to kinase inhibitors. Here, using next-generation sequencing, we analyzed the mutational status of primary tissue and poorly differentiated metastatic tissue from two HCC patients. In both cases, metastatic tissues harbored a mutation of SETD2, each resulting in loss of the SRI and WW domains of SETD2, a methyltransferase that trimethylates H3K36 (H3K36me3) and also interacts with p53 to promote its stability. Functional studies of the novel p.D1890fs6* mutation (case 1) revealed significantly reduced H3K36me3 levels in SETD2-mutated tissue and primary cell cultures and decreased levels of the active form of p53. Restoration of SETD2-wildtype expression in the SETD2-mutant cells significantly reduced the expression of four well-known stemness markers (OCT-4, SOX2, IPF1, Goosecoid). These findings suggest potential roles for SETD2 loss-of-function mutations in HCC progression, possibly involving p53 destabilization and promotion of stemness. Their prevalence and potential treatment implications in thyroid cancer, especially HCC, require further study.

Background. Obesity has been hypothesized to contribute to the aggressiveness of thyroid cancer through the production of abnormal levels of serum adipokines. Leptin receptor (OB-R) expression has also been documented in papillary thyroid cancer (PTC). Aim. In this translational study, we analyzed in vitro the effects of leptin on the growth and migration of thyroid cancer cells (TPC-1 and K1), the molecular mechanisms underlying leptin’s action, and the influence of prolonged leptin exposure on cell response to a protein kinase inhibitor lenvatinib. The expression levels of OB-R mRNA and protein were also investigated in vivo in a series of aggressive PTCs divided into two groups based on the presence of the BRAF mutation. Results. In TPC-1 and K1 cells, prolonged treatment with leptin (500 ng/ml for 96 h) resulted in a mild increase in the proliferation (about 20% over control only in K1 cells, p<0.05) and in the migration of both cancer cell lines. Immunoblot analysis revealed a slight increase in the phosphorylation of AKT, but no effect on β-catenin and phospho-ERK expressions. The inhibitory effects of lenvatinib on the viability of both cell lines were not influenced by the leptin treatment. OB-R transcript (in fresh tissues) and proteins (in formalin-fixed and paraffin-embedded specimens) were expressed in all PTC tissues examined, with no significant differences between BRAF-mutated and BRAF-wild-type tumors. Conclusions. These results demonstrate leptin’s role in mildly increasing the aggressive phenotype of PTC cells but without influencing the action of lenvatinib. Further studies will clarify whether it is possible to target OB-R, expressed in all aggressive PTCs, as an adjuvant treatment approach for these malignancies.

Synonymous mutations continue to be filtered out from most large-scale cancer genome studies, but several lines of evidence suggest they can play driver roles in neoplastic disease. We investigated a case of an aggressive, apparently sporadic medullary thyroid carcinoma (MTC) harboring a somatic RET p.Cys634Arg mutation (a known MTC driver). A germ-line RET substitution (p.Cys630=) had also been found but was considered clinically irrelevant because of its synonymous nature. Next generation sequencing (NGS) of the tumor tissues revealed that the RET mutations were in cis. There was no evidence of gene amplification. Expression analysis found an increase of RET transcript in p.Cys630=;p.Cys634Arg patient compared with that found in 7 MTCs harboring p.Cys634 mutations. Minigene expression assays demonstrated that the presence of the synonymous RET mutation was sufficient to explain the increased RET mRNA level. In silico analyses and RNA immunoprecipitation experiments showed that the p.Cys630 = variant created new exonic splicing enhancer motifs that enhanced SRp55 recruitment to the mutant allele, leading to more efficient maturation of its pre-mRNA and an increased abundance of mature mRNA encoding a constitutively active RET receptor. These findings document a novel mechanism by which synonymous mutations can contribute to cancer progression., Author summary Synonymous mutations—once considered “silent” because they do not alter the gene product’s amino-acid sequence—are now emerging as potential drivers of cancer. Our recent investigation of an aggressive medullary thyroid carcinoma (MTC) revealed a novel mechanism that could underlie such effects. The MTC analyzed harbored a somatic p.Cys634Arg mutation of the RET protooncogene (a well-known MTC driver). A second RET substitution (p.Cys630=) discovered in the germline had been considered clinically irrelevant because of its synonymous nature. Our next-generation sequencing analysis of the patient’s cancer tissues revealed that the RET mutations were in cis. We also found that RET mRNA levels in patient’s MTC were significantly higher than those found in seven other MTCs with various amino-acid substitutions at position 634 in RET. Subsequent experiments demonstrated that the presence of the synonymous mutation created new exonic splicing enhancer motifs in the mutant allele, which led to more efficient maturation of its transcript and increased expression of a constitutively active RET receptor.

Recent studies have revealed in normal thyroid tissue the presence of the transcript of several phosphodiesterases (PDEs), enzymes responsible for the hydrolysis of cyclic nucleotides. In this work, we analyzed the expression of PDE5 in a series of human papillary thyroid carcinomas (PTCs) presenting or not BRAF V600E mutation and classified according to ATA risk criteria. Furthermore, we tested the effects of two PDE5 inhibitors (sildenafil, tadalafil) against human thyroid cancer cells. PDE5 gene and protein expression were analyzed in two different cohorts of PTCs by real-time PCR using a TaqMan micro-fluid card system and Western blot. MTT and migration assay were used to evaluate the effects of PDE5 inhibitors on proliferation and migration of TPC-1, BCPAP, and 8505C cells. In a first series of 36 PTCs, we found higher expression levels of PDE5A in tumors versus non-tumor (normal) tissues. PTCs with BRAF mutation showed higher levels of mRNA compared with those without mutation. No significant differences were detected between subgroups with low and intermediate ATA risk. Upregulation of PDE5 was also detected in tumor tissue proteins. Similar results were obtained analyzing the second cohort of 50 PTCs. Moreover, all tumor tissues with high PDE5 levels showed reduction of Thyroglobulin, TSH receptor, Thyroperoxidase, and NIS transcripts. In thyroid cancer cells in vitro, sildenafil and tadalafil determined a reduction of proliferation and cellular migration. Our findings demonstrate for the first time an overexpression of PDE5 in PTCs, and the ability of PDE5 inhibitors to block the proliferation of thyroid cancer cells in culture, therefore, suggesting that specific inhibition of PDE5 may be proposed for the treatment of these tumors.

MicroRNA (miRNA) expression is dysregulated in many human malignancies, and a growing number of studies are focused on their potential use as tumor biomarkers. To identify a miRNA signature for papillary thyroid carcinomas (PTC), we investigated miRNA expression profiles in two independent cohorts of PTCs, which included major histological subtypes [classical-type (PTC‑CT), follicular-variant (PTC‑FV), and tall-cell variant (PTC‑TCV)] and cases with low or intermediate risk of recurrence. Using TaqMan® Array Human MicroRNA A+B Cards v3.0, we first performed microRNA profiling of normal and neoplastic thyroid tissues from 29 PTC patients. Promising candidates were then investigated in a second, independent cohort of 76 PTCs using Custom TaqMan® Array MicroRNA Cards. We identified a molecular signature of 11 miRNAs that were significantly upregulated (miR‑146b-5p, miR‑146b-3p, miR‑221-3p, miR‑222‑5p, miR‑222‑3p) or downregulated (miR‑1179, miR‑486‑5p, miR‑204-5p, miR‑7-2-3p, miR‑144-5p, miR‑140-3p) in PTC tissues vs. normal thyroid tissue. Upregulation of miR‑146b-5p and miR‑222‑3p was also significantly associated with an increased risk of recurrence. Higher than normal expression of miR‑146b-5p and miR‑146b-3p characterized PTC‑CT and PTC‑TCV but not PTC‑FV, whereas miR‑21-5p was significantly upregulated only in PTC‑TCV. When PTC‑FV were subclassified as encapsulated (PTC‑EFV) or infiltrative (PTC‑IFV), miR‑204-5p was downregulated in all histological subtypes except PTC‑EFV, which displayed expression levels similar to those of normal thyroid tissues. These findings provide new insights into the molecular classification of PTC, showing that different miRNA expression profiles are associated with different histological types of PTC and different risks of recurrence.

Context: Trends toward more conservative management of papillary thyroid cancer (PTC) diminish the primacy of serum thyroglobulin (Tg) assays as a posttreatment surveillance tool. Objective: To identify thyroid tumor-associated microRNAs (miRNAs) in the serum with potential for development as unique biomarkers of PTC recurrence. Methods: We measured expression of 754 miRNAs in serum samples collected from 11 patients with PTC before and 30 days after thyroidectomy. Major candidates were then re-evaluated by absolute quantitative polymerase chain reaction analysis in an independent cohort of patients with PTC (n = 44) or benign nodules and 20 healthy controls (HCs). The 2 miRNAs most significantly associated with thyroid tumors were then assessed in matched serum samples (before and 30 days and 1 to 2 years after surgery) from the 20 PTC patients with complete follow-up datasets and results correlated with American Thyroid Association (ATA) responses to therapy. Results: Eight miRNAs (miR-221-3p, miR-222-3p, miR-146a-5p, miR-24-3p, miR-146b-5p, miR-191-5p, miR-103a-3p, and miR-28-3p) displayed levels in prethyroidectomy serum samples from patients with PTC that significantly exceeded those measured after thyroidectomy and those found in samples from HCs. The 2 most promising candidates—miR-146a-5p and miR-221-3p —were further analyzed in the 20 PTC patients mentioned earlier. Serum levels of both miRNAs after 1 to 2 years of follow-up were consistent with ATA responses to therapy in all patients, including 2 with structural evidence of disease whose Tg assays remained negative (, Precis: Analysis of miRNA levels in pre- and postoperative serum samples from PTC patients reveals possible associations between postoperative changes in miR-146a-5p and miR-221-3p and clinical outcome.

Mutations in the hTERT promoter responsible for constitutive telomerase activity are the most frequent genetic alteration detected in anaplastic thyroid cancer (ATC), and proposed as diagnostic and prognostic biomarker in these tumours. In this study we analyzed hTERT expression in a series of human ATCs and investigated the effects of small-interfering RNA-mediated silencing of hTERT on viability and migration and invasive properties of three human ATC cell lines. Expression of hTERT mRNA resulted increased in 8/10 ATCs compared to normal thyroid tissues. Silencing of hTERT in CAL-62, 8505C and SW1736 cells did not modify telomere length but determined a significant decrease (about 50%) of cell proliferation in all cell lines and a great reduction (about 50%) of migration and invasion capacity. These finding demonstrate that hTERT may be considered as a molecular target for ATC treatment.

Advanced medullary thyroid cancers (MTCs) are now being treated with drugs that inhibit receptor tyrosine kinases, many of which involved in angiogenesis. Response rates vary widely, and toxic effects are common, so treatment should be reserved for MTCs likely to be responsive to these drugs.RETmutations are common in MTCs, but it is unclear how they influence the microvascularization of these tumors. We examined 45 MTCs with germ-line or somaticRETmutations (RETmut group) and 34 with wild-typeRET(RETwt). Taqman Low-Density Arrays were used to assess proangiogenic gene expression. Immunohistochemistry was used to assess intratumoral, peritumoral and nontumoral expression levels of VEGFR1, R2, R3, PDGFRa, PDGFB and NOTCH3. We also assessed microvessel density (MVD) and lymphatic vessel density (LVD) based on CD31-positive and podoplanin-positive vessel counts, respectively, and vascular pericyte density based on staining for a-smooth muscle actin (a-SMA), a pericyte marker. Compared withRETwt tumors,RETmut tumors exhibited upregulated expression of proangiogenic genes (mRNA and protein), especially VEGFR1, PDGFB and NOTCH3. MVDs and LVDs were similar in the two groups. However, microvessels inRETmut tumors were more likely to be a-SMA positive, indicating enhanced coverage by pericytes, which play key roles in vessel sprouting, maturation and stabilization. These data suggest that angiogenesis inRETmut MTCs may be more intense and complete than that found inRETwt tumors, a feature that might increase their susceptibility to antiangiogenic therapy. Given their increased vascular pericyte density,RETmut MTCs might also benefit from combined or preliminary treatment with PDGF inhibitors.

Background: Agents capable of increasing radioiodine concentration by stimulating the sodium/iodide symporter (NIS) expression have been extensively investigated for the treatment of certain well-differentiated breast cancers. Aim: In this study, we analyzed the regulation of the NIS and lactoperoxidase (LPO) gene expression in 4 different human breast cancer cell lines, representative of different histotypes of breast cancer. Methods: MCF-7, T-47D, MDA-MB231, and HCC-1937 (the latter carrying the BRCA-1 mutation) were exposed to different stimulators and the levels of NIS and LPO mRNA measured by a quantitative RT-PCR. Results: AII-trans-Retinoic Acid (RA), Dexamethasone (DEX), Trichostatin A (TSA), and Sodium Butyrate (NaB) induced the expression of NIS mRNA in MCF-7 and T-47D cell lines, whereas HCC-1937 and MBA-MB231 were slightly responsive only to the histone-deacetylase inhibitors TSA and NaB. Minor stimulatory effects were detected on LPO mRNA in MCF-7 and T-47D treated with TSA and NaB or RA only in MCF-7, while no effect was detectable in the other two cell lines. Conclusions: These data indicate that retinoic acid, alone or in combination with DEX, as well as HDAC-inhibitors are very promising agents for a radioiodine-based therapy in a large spectrum of breast cancers, including neoplasms from both basal and ductal cells, especially for the well-differentiated estrogen-dependent tumors. Other molecules or other drug combinations should be tested to extend the same strategy to the less differentiated and more aggressive tumor cells, including those carrying the BRCA mutation.

Context: Type 2 deiodinase (D2) converts T4 in T3 in several human tissues, including hypothalamus and pituitary, and, therefore, plays a pivotal role in the negative feedback regulation of TSH secretion. A common variant of the gene, threonine (Thr) 92 alanine (Ala), has been identified and associated with decreased D2 enzymatic activity. Objective: Our objective was to investigate whether this polymorphism predicts the T4 dosage needed to obtain target TSH levels in thyroidectomized patients. Setting: Ambulatory patients were included in the study. Patients: A total of 191 consecutive thyroid cancer patients, previously treated by near total thyroidectomy and radioiodine ablation, were studied. They were on stable T4 dose treatment aimed at obtaining either suppressed (supp) (n = 117, < 0.1 mU/liter) or near-supp (n = 74, ≥ 0.1 < 0.5 mU/liter) serum TSH levels. Main Outcome Measures: DNA genotyping for D2 Thr92Ala variant and evaluation of T4 dose (μg/kg) needed to obtain target TSH levels were determined. Results: Ala/Ala homozygous patients needed a higher T4 dose as compared with patients carrying the Thr92 variant (X/Thr patients) according to a recessive genetic model (2.08 ± 0.43 vs. 1.90 ± 0.35 μg/kg; P < 0.05). This difference was observable in the near-supp group (P = 0.002), but not in the supp group (P = 0.4). Conclusions: D2 Thr92Ala polymorphism seems to predict the need for higher T4 intake in thyroidectomized patients. If this finding is confirmed in additional studies, it may predict the T4 requirement to suppress TSH on the basis of the individual genetic background.

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

Context: RET mutation analysis provides useful information on the clinical outcome of medullary thyroid carcinomas (MTCs) and the risk of disease in the family members. Objective: The objective of this study was to document genotype-phenotype relationships in an Italian family with a novel RET mutation. Design/Setting: RET gene alterations were investigated in a patient with unifocal MTC and her relatives. The identified mutation was subjected to in vitro functional testing. Patients: Patients included a female proband who developed MTC at age 60, her five children, and three grandchildren. Main Outcome Measures: DNA extracted from the blood and the proband’s tumor were analyzed for RET alterations. The transforming potential and mitogenic properties of the identified mutation were investigated. Results: A novel heterozygous germline RET mutation at codon 777 (AAC→AGC, N→S) (RET/N777S) was identified in the proband and three of her relatives. Two of the latter presented thyroid nodules, but none had MTC o...

Carotid intima-media thickness (CIMT) is an intermediate phenotype for early atherosclerosis and a strong predictor of myocardial infarction and ischemic stroke [1]. Several studies have described the effects of candidate genes on CIMT, being unclear, however, the relative contribution of genetic variation to the development of the early stages of atherosclerosis [2]. The ectoenzyme pyrophosphatase phosphodiesterase 1 (ENPP-1) inhibits insulin receptor signaling [3]. A non synonymous polymorphism (K121Q, rs1044498) of the ENPP-1 gene is responsible for a gain of function of the protein [3], resulting in an increased ability to inhibit insulin receptor signaling. This variant has been associated with insulin resistance and type 2 diabetes in most studies [4, 5]. In agreement with the link between insulin resistance and cardiovascular disease (CVD), the Q121 variant has been shown to modulate susceptibility to premature myocardial infarction [6] and ischemic stroke [7] and to increase pulse pressure, a marker of arterial stiffness [8]. Some of these associations have been shown to be stronger in obese subjects, suggesting a gene-byobesity interaction in facilitating insulin resistance and atherogenic process [4, 9]. Up to date no results on ENPP-1 function on CIMT are available. Thus, we tested whether the ENPP-1 Q121 variant exerts an effect on CIMT measured at the common carotid artery (CCA) and at bulb and interacts with overweight–obesity in modulating CIMT.

The case reported the rapid remission of disease recurrence achieved adding foscarnet, a DNA polymerase inhibitor that interacts with fibroblast growth factor 2, to low molecular weight heparin and sunitinib for the first time in a patient with an anaplastic thyroid cancer (ATC). A 65-year-old woman with a multinodular goiter referred for a rapid enlargement of a nodule. Histological examination revealed an ATC with a little area of papillary thyroid cancer (PTC). The patient was resistant to selective single-target treatment. Immunophenotyping and gene analyses found a significant increase in FGF2 and FGFR1 expression in the primary ATC area (FGF2 = 38.2 ± 6.2% in ATC vs 34.6 ± 6.0% in the differentiated area of PTC, P

Abnormal expression of non-coding micro RNA (miRNA) has been described in medullary thyroid carcinoma (MTC). Expression of genes encoding factors involved in miRNA biogenesis results often deregulated in human cancer and correlates with aggressive clinical behavior. In this study, expression of four genes involved in miRNA biogenesis (DICER, DROSHA, DCGR8, and XPO5) was investigated in 54 specimens of MTC. Among them, 33 and 13 harbored RET and RAS mutations, respectively. DICER, DGCR8, and XPO5 mRNA levels were significantly overexpressed in MTC harboring RET mutations, in particular, in the presence of RET634 mutation. When MTCs with RET and RAS mutations were compared, only DGCR8 displayed a significant difference, while MTCs with RAS mutations did not show significant differences with respect to non-mutated tumors. We then attempted to correlate expression of miRNA biogenesis genes with tumor aggressiveness. According to the TNM status, MTCs were divided in two groups and compared (N0 M0 vs. N1 and/or M1): for all four genes no significant difference was detected. Cell line experiments, in which expression of a RET mutation is silenced by siRNA, suggest the existence of a causal relationship between RET mutation and overexpression of DICER, DGCR8, and XPO5 genes. These findings demonstrate that RET- but not RAS-driven tumorigenic alterations include abnormalities in the expression of some important genes involved in miRNA biogenesis that could represent new potential markers for targeted therapies in the treatment of RET-mutated MTCs aimed to restore the normal miRNA expression profile.

Epigenetic control of gene expression plays a major influence in the development and progression of many cancer types. Aim of the present study was to investigate the expression of epigenetic regulators in a large cohort of medullary thyroid carcinomas (MTC), correlating the data with the clinical outcome and mutational status of the patients. Taqman Low Density Arrays (TLDAs) were used to analyze expression levels of several genes involved in the epigenetic control of transcription in a series of 54 MTCs. The patients cohort included 13 familial MTCs and 41 sporadic forms; 33 hosted a RET mutation and 13 a RAS somatic mutation. The expression profiling revealed in the more aggressive diseases (i.e. occurrence of metastases; persistent disease; disease-related death) a significant increase of EZH2 and SMYD3 gene expression. The increased levels of EZH2 and SMYD3 did not correlate significantly with mutational status of RET or RAS genes. Thus, the histone methyltransferases EZH2 and SMYD3 mRNA expression may represent useful prognostic biomarkers tailoring the most appropriate follow-up and timing of therapeutic approaches.

In Western countries, thyrotoxic periodic paralysis (TPP) is a rare condition with only sporadic cases described so far. Here, we describe a 29-year-old Italian man who presented with leg weakness and hypokalaemia. Treatment with intravenous potassium resulted in a rapid resolution of symptoms. TPP as the underlying cause was suggested by suppressed thyroid-stimulating hormone (TSH), elevated free T3 and free T4, and the presence of TSH-receptor antibodies (TRAB). Genetic analysis showed no mutations in the candidate exons of calcium ( CACN1AS), potassium ( KCNE3) and sodium ( SCN4A) channel genes. However, we identified the presence of two single nucleotide polymorphisms (SNPs), 1491C > T and 1551 T > C, in exon 11 of the CACN1AS gene. Although the 1491C > T SNP is not apparently involved in the pathogenesis of the disease, the 1551 T > C SNP has been associated with TPP in Asians and reported in only one case in European Caucasians. Further investigations are needed to clarify whether such polymorphisms have a role in the disease pathogenesis in Caucasians.

Several studies have reported that the ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) K121Q polymorphism (rs1044498) interacts with increased adiposity in affecting glucose homeostasis and insulin sensitivity. Conversely, one would expect that the amelioration of glucose homeostasis observed after weight loss is modulated by the ENPP1 K121Q polymorphism. The aim of our study was to test such hypothesis, in non-diabetic overweight-obese individuals.Two hundred eleven non-diabetic overweight-obese individuals were studied. Body mass index (BMI), fasting glucose, homeostasis model assessment of insulin resistance (HOMA-IR index) and lipid levels were obtained before and after 6-week lifestyle intervention (LI; diet and exercise) and their changes calculated as baseline minus 6-week values. LI decreased BMI, glucose, HOMA-IR and triglyceride levels (p0.001 for all). No difference across genotype groups (160 KK and 51 KQ or QQ - named as XQ - individuals) was observed in these changes. In a multivariate model, BMI changes predicted fasting glucose changes (β = 0.139 mmol/L (2.50 mg/dl) for 1 unit BMI change, p = 0.005). This correlation was not significant among KK individuals (β = 0.082; p = 0.15), while much steeper and highly significant among XQ individuals (β = 0.336; p = 0.00008) (p-value for Q121-by-weight loss interaction = 0.047).Individuals carrying the ENPP1 Q121 variant are highly responsive to the effect of weight loss on fasting glucose. This reinforces the previously suggested hypothesis that the Q121 variant interacts with adiposity in modulating glucose homeostasis.

SummaryContext Germline mutations in four genes (RET, VHL, SDHB and SDHD) are detected in about 17% of patients with apparently sporadic pheochromocytoma. Thus, genetic screening of all patients with this disease is suggested for a rational diagnostic approach and management. Objective To report the clinical, biochemical and genetic analysis of three unrelated patients affected by pheochromocytoma. Design and patients All the coding regions and exon–intron boundaries of RET, VHL, SDHB and SDHD genes were sequenced in three unrelated patients with intra-adrenal pheochromocytoma: a 17-year-old girl, a 15-year-old boy and a 73-year-old man. The family history of all three cases was negative for von Hippel–Lindau lesions or other types of endocrine tumours. Structural modelling of the VHL protein was then performed. Results We identified a novel germline VHL gene point mutation, a G to A nucleotide substitution in exon 3, leading to an aspartate to asparagine amino acid change in codon 197 (D197N). No mutations were found in RET, SDHB and SDHD genes. Structural modelling of the VHL protein suggests that the D197N mutation could have a functional role. Conclusions Our study expands the number of VHL gene known mutations and indicates the usefulness of performing the genetic analysis in all patients with apparently sporadic pheochromocytoma.

Novel molecularly targeted drugs are undergoing preclinical and clinical testing to assess their efficacy against refractory thyroid carcinomas. The multikinase inhibitor Sunitinib has been shown to inhibit the kinase activity of the RET oncogene and reduce proliferation in differentiated thyroid cancer cells harboring the RET/PTC rearrangement. In this study, we evaluated its effects in human cell lines derived from differentiated (TPC-1) and anaplastic (8505C, CAL-62, and C643) thyroid cancers.The cells exposed to various concentrations of Sunitinib were examined for: (1) cell viability and presence of apoptosis, analyzed by cell counts, MTT assay, trypan blue exclusion assay, western blotting, and immunofluorescence; (2) expression of cyclin D1 and phosphorylated and nonphosphorylated extracellular signal-regulated kinase (ERK) and Akt proteins, analyzed by western blotting; and (3) transcription of genes encoding thyrocyte differentiation markers (thyroid-stimulating hormone receptor, sodium/iodide symporter, thyroglobulin, and thyroperoxidase) and proangiogenic factors (vascular endothelial growth factor A, platelet-derived growth factors A and B), measured by quantitative reverse transcriptase-polymerase chain reaction.Exposure to nanomolar concentrations of Sunitinib significantly reduced cell viability in only TPC-1 cells, and this effect was paralleled by reduction of cyclin D1 levels. Western blotting revealed reduced phosphorylation of ERK and Akt after 3 and 6 hours of drug exposure. In contrast, the growth of 8505C, CAL-62, and C-643 cells was significantly reduced only by micromolar concentrations of Sunitinib, mainly due to induced necrotic rather than apoptotic death. In these cells, Sunitinib exerted a few significant effects on the transcription of angiogenic factors or thyrocyte differentiation markers.Sunitinib has little or no effect on the growth or differentiation of anaplastic thyroid cancer cells, thus suggesting that it is unlikely to be effective in the treatment of anaplastic thyroid cancer.

Intrathyroidal medullary thyroid carcinoma (MTC) can generally be cured by surgery, but distant metastases are often already present at diagnosis.Currently, there is no effective treatment for metastatic MTC. In these cases, consensus treatment guidelines explicitly recommend new experimental drugs. Several kinase inhibitors are now being tested for treatment of MTC in clinical trials and XL184, an oral, small-molecule multi-kinase inhibitor, seems to be one of the most promising of these compounds.We review preliminary data on the safety and efficacy of XL184 in metastatic MTC based on an extensive search of the literature, which included published articles, abstracts and website information. In particular,the review focuses on the rationale for using XL184 in advanced MTC. The compound has been specifically designed to target multiple signaling pathways,and this is expected to produce synergistic antitumor effects superior to those achieved by single-kinase inhibition. Preliminary results from the Phase I study of XL184 seem to support this hypothesis.Multiple receptor tyrosine kinases (RTKs) are concomitantly activated in the same tumor. The blockade of a single RTK may engage compensatory signaling that maintains cell growth. Targeting multiple kinases might overcome both intrinsic and acquired resistance to antitumoral drugs.

To describe the coexistence of mutations of both the multiple endocrine neoplasia type 1 (MEN1) and type 2 (MEN2) genes in a large Italian family and evaluate if it could be associated with more aggressive clinical manifestations of the two syndromes. Blood samples were obtained for genetic and biochemical analyses. The RET gene exons (8, 10, 11, 13, 14, 15, 16, 18) and the MEN1 coding regions, including the exon–intron boundaries, were amplified by PCR and directly sequenced. We identified two germline mutations in the proband: the first one, K666M, located at the exon 11 of RET proto-oncogene and the second one, IVS4+1G>T, located in the MEN1 gene. The functional characterization of IVS4+1G>T variation, located in the splicing donor site of exon 4 of MEN1 gene, caused the in-frame junction of exon 3 to exon 5, thus obtaining a shorter protein. The same proband’s germline mutations were found in 16 relatives out of 21 screened subjects: 8 carried IVS4+1G>T, 4 RET K666M, and 4 both the mutations. This is the second report in literature of coexistence in the same family of germline mutations of both RET proto-oncogene and MEN1 gene. The simultaneous presence of the two mutations was not apparently associated with more aggressive diseases, since at last follow-up all patients appeared to be disease-free or well compensated by medical therapy; finally, no one exhibited metastatic diseases.

Administration of radioiodine (I(131)) is currently exploited for both diagnostic and therapeutic treatment of thyroid cancer. Few data are available on the sodium/iodide symporter (NIS) expression in human testis, a particular important prerequisite to predict radioiodine accumulation in the gonads of males with thyroid cancer exposed to such a treatment. In this study, we analyzed the expression of NIS in mouse, rat and human normal testis in different stages of development. By using a quantitative RT-PCR, Western blot and immunohistochemical analysis, NIS mRNA and protein were measured in both fetal and adult testicular tissues. NIS transcript was detected in both fetal and adult testis, although its expression levels were approximately 10-fold less than in thyroid gland. Western blot analysis and immunohistochemistry showed the presence of NIS protein in germinal and Leydig cells, but not in Sertoli cells with prevalent expression in the cytosol compartment of the cells. Our study demonstrates that NIS transcript and protein are expressed in normal testis. Further studies will demonstrate whether it may act as the transporter of radioiodine in normal testis of male patients with thyroid cancer.

ObjectiveTyrosine 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.DesignThe aim of our study was to investigate the impact of BRAFV600E on proangiogenic gene expression and microvascular features of PTCs.MethodsmRNA levels for VEGFA, VEGF receptors, and coreceptors (VEGFRs 1, 2, and 3, neuropilin-1), and PDGF receptor β (PDGFRβ or PDGFRB) were measured with real-time PCR in BRAFV600E (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 BRAFV600E cases). Angiogenic gene expression was also studied in vitro after induction/silencing of the BRAFV600E mutation in thyrocyte lines.ResultsTranscript levels of proangiogenic factors were significantly lower in BRAFV600E PTCs versus BRAF-wt PTCs (PVEGFA mRNA levels in thyroid cell lines decreased when BRAFV600E mutation was induced (P=0.01) and increased when it was silenced (P=0.01).ConclusionsCompared with BRAF-wt PTCs, those harboring BRAFV600E exhibit downregulated VEGFA, VEGFR, and PDGFRβ expression, suggesting that the presence of BRAF mutation does not imply a stronger prediction of response to drugs targeting VEGF and PDGFB signaling pathways.

Levothyroxine (L-T4)-based suppression of thyrotropin (TSH) secretion is widely used to prevent the growth of benign thyroid nodules, although the effectiveness of this approach has been demonstrated only in a subset of patients. In this study, we analyzed the in vivo effects of L-T4-mediated TSH suppression on elements of insulin/IGF-1-dependent growth-regulating pathways in tissues from patients with benign thyroid nodules. Nodular and non-nodular tissue specimens were collected from 63 patients undergoing thyroidectomy. 32 had received preoperative TSH suppressive therapy with TSH levels consistently below 0.5 mU/l (L-T4 group). TSH suppression had not been used in the other 31, and their TSH levels were normal (0.8-4 mU/l (control group). Quantitative RT-PCR was used to measure mRNA levels for TSH receptor, IGF1, IGF-1 receptor, insulin receptor, insulin receptor substrate 1 in nodular and non-nodular tissues from the 2 groups. Akt and phosphorylated Akt protein levels were detected by Western blot. Mean levels of mRNA for all genes tested were similar in the 2 groups, in both nodular and non-nodular tissues. The 2 groups were also similar in terms of phosphorylated Akt protein levels (measured by densitometric scan in 10 randomly selected nodules from each group). This is the first demonstration based on the study of human thyroid tissues that TSH suppression does not affect the expression of components of the insulin/IGF-1-dependent signaling pathways regulating thyrocyte growth. This may explain the lack of effectiveness of TSH-suppressive therapy in a substantial percentage of benign thyroid nodules.