Nodal Metastases Associated With Fusion Oncogenes Are Age Dependent in Young Adult Patients With Thyroid Cancer.

CONTEXT: Fusion oncogenes, especially those involving RET or NTRK, are known drivers of papillary thyroid cancer (PTC). They are prevalent in pediatric patients and correlate with aggressive tumor behavior. OBJECTIVE: We explored the age dependence of fusion oncogenes and aggressive tumor behavior in young adult PTC patients. EXPERIMENTAL DESIGN: We examined 150 tumors from 142 PTC patients aged between 17∼35 years old with established tumor-node-metastasis stages. Oncogenic drivers and the thyroid differentiation score (TDS) were determined by DNA and RNA sequencing of a target panel. Transcriptome analysis was performed in PTCs with RET fusions. RESULTS: Among 150 PTCs, we detected BRAF V600E (n = 105), RET fusions (n = 15), NTRK3 fusions (n = 8), and BRAF fusions (n = 4). We found that fusion oncogenes were associated with nodal metastasis when age was tiered into 3 groups: <25 years, 25∼29 years, and 30∼35 years. Patients under 25 years old showed a marginal increase in tumor stage compared to those over 25 years (75.00% vs 21.74%, P = .0646). Risk of lateral lymph node metastasis increased with younger age (75.00% vs 27.27% vs 8.33%, P = .0369). As with advanced tumor and node stage, patients harboring fusion oncogenes and aged under 25 years showed the lowest TDS; genes associated with immunoglobulin production and production of molecular mediators of the immune response were significantly upregulated. CONCLUSIONS: Adult PTC patients under 25 years with fusion oncogenes showed a tendency toward advanced tumor stage and lower thyroid differentiation. Integrating onset age together with oncogenic alterations is worthwhile when managing adult PTC patients.

Methotrexate plus reduced or full-dose glucocorticoids for the treatment of active, moderate-to-severe Graves' orbitopathy.

Objective: A combination of glucocorticoids with mycophenolate is recommended by current guidelines to boost response to Graves' orbitopathy (GO) therapy. This study was designed to evaluate the therapeutic effects and safety of methotrexate (MTX) plus reduced (3.0 g) or full-dose (4.5 g) i.v. methylprednisolone (MP) vs full-dose i.v. MP alone. Design and methods: This was a prospective, randomized, observer-masked, single-center clinical trial conducted in a tertiary clinical center. Ninety-seven patients with active moderate-to-severe GO were screened and 90 patients underwent randomization between April 2018 and Oct 2019. All patients completed 12 weeks of treatment and received clinical assessment. The patients received either MP 4.5 g only, MP 4.5 g plus oral MTX, or MP 3.0 g plus oral MTX. The primary outcome was the CAS response at week 12. Secondary outcomes were adverse events and other individual ophthalmic parameters. Results: At week 12, 53.3% of MP, 76.7% of reduced MP plus MTX, and 76.7% of MP plus MTX achieved a CAS response, although the difference was not significant (P = 0.1). The overall response rates of the MP group, the reduced MP plus MTX group, and the MP plus MTX group were 43.3%, 53.3%, and 60%, respectively (P = 0.5). Subgroup analysis found that smoking status interacted with marginal significance with treatment effect (P = 0.048). Importantly, adverse event incidence was significantly lower in the reduced MP + MTX group (P = 0.017). Conclusions: Our study shows that reduced MP plus MTX therapy is effective and safer in treating active and moderate-to-severe GO patients than 4.5 g MP monotherapy.

Circular RNA circPSAP functions as an efficient miR-331-3p sponge to regulate proliferation, apoptosis and bortezomib sensitivity of human multiple myeloma cells by upregulating HDAC4.

BACKGROUND: Multiple myeloma (MM) is a malignant tumor of plasma cells in the bone marrow. Circular RNAs (circRNAs) exert important activity in the tumorigenesis and chemoresistance of MM. In the current work, we sought to identify the expression, activity, and mechanism of circPSAP activity in MM. METHODS: CircPSAP, microRNA (miR)-331-3p, and histone deacetylase 4 (HDAC4) were quantified by qRT-PCR and immunoblotting assays. Cell proliferation and survival were assessed by CCK-8 assay. Cell cycle and apoptosis were detected by flow cytometry. The direct relationship between miR-331-3p and circPSAP or HDAC4 3'UTR was validated by dual-luciferase reporter, RNA pull-down, and RNA immunoprecipitation (RIP) assays. RESULTS: CircPSAP was overexpressed in human MM and high levels of circPSAP predicted poor prognosis in MM patients. CircPSAP depletion repressed cell proliferation and promoted apoptosis and BTZ sensitivity. Mechanistically, circPSAP functioned as a miR-331-3p sponge, and circPSAP regulated cell proliferation, apoptosis and BTZ sensitivity by sponging miR-331-3p. MiR-331-3p directly targeted and inhibited HDAC4. MiR-331-3p-mediated inhibition of HDAC4 impaired cell proliferation and enhanced cell apoptosis and BTZ sensitivity. Moreover, circPSAP modulated HDAC4 expression by acting as a miR-331-3p sponge. CONCLUSION: Our findings highlight a novel mechanism, in which circPSAP functions as a miR-331-3p sponge to impact MM cell proliferation, apoptosis and BTZ sensitivity by regulating HDAC4 expression.

Safety, Pharmacokinetics, and Pharmacodynamics of Oral Insulin Administration in Healthy Subjects: A Randomized, Double-Blind, Phase 1 Trial.

Oral delivery is an ideal method of insulin administration and is currently a promising research field. Here, we evaluated the safety, pharmacokinetic, and pharmacodynamic characteristics of oral administration of an insulin capsule (ORMD-0801) with 2 different sources of recombinant human insulin. This was a single-center, randomized, double-blind, placebo-controlled, dose-escalating phase 1 trial. Single dosing of the oral insulin capsule was administered in 70 healthy Chinese subjects. In stage 1, four dose groups (8, 16, 32, and 48 mg) for capsules containing Sanofi insulin and in stage 2, three dose groups (8, 32, and 48 mg) containing Hefei Tianmai insulin were evaluated consequently. The results showed that the oral insulin formulations with either source in the dose range 8 to 48 mg were safe, and no serious adverse events were observed. After a standard breakfast 45 minutes after dosing, the area under the concentration-time curve (AUC) from time 0 to time t and AUC from time 0 to infinity for insulin in the 8-mg and 48-mg dose groups in stage 1 and for 8- to 48-mg groups in stage 2 were slightly increased compared with placebo, but no significant dose-related changes in the pharmacokinetic parameters were observed for either stage. The peak-valley difference and the change in value of the AUC for glucose from baseline showed a dose-related increase in the dose range from 8 to 48 mg in both stages. Together, this study indicated that in healthy Chinese subjects, this oral capsule containing 2 different insulin formulations was safe and well tolerated after a single-dose administration.

Knockdown of zinc finger protein 267 suppresses diffuse large B-cell lymphoma progression, metastasis, and cancer stem cell properties.

Zinc finger protein 267 (ZNF267) is a member of the Kruppel-like transcription factor family, which regulates various biological processes such as cell proliferation and differentiation. However, the biological significance of ZNF267 and its potential role in diffuse large B-cell lymphoma (DLBCL) remain to be documented. Experiments were herein conducted to study the role of ZNF267 in DLBCL. real-time quantitative reverse transcription PCR and Western blotting assays were conducted to detect the expression of ZNF267 in tissues and cells. Tissue microarray and bioinformatics analyses of public data were also done to detect the expression status and clinical significance of ZNF267. Functional cell experiments including CCK8 assay, colony formation assay, 5-ethynyl-2'-deoxyuridine (EDU) assay, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay, transwell assay, and wound healing assay were conducted to study the effects of ZNF267 knockdown and overexpression on cell proliferation and mobility. Xenograft assay was also conducted to confirm the effects of ZNF267 knockdown in vivo. In the present study, we found ZNF267 was significantly upregulated in DLBCL and predicted a poor survival outcome based on the bioinformatics analysis. Functionally, the knockdown of ZNF267 resulted in less cell proliferation and mobility, whereas the overexpression led to enhanced cell proliferation and mobility. Animal experiments also confirmed that ZNF267 silence contributed to less tumor growth and less lung metastasis. Further analysis showed that ZFN267 knockdown resulted in decreased epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties. Our results suggest that ZNF267 is an oncogene in DLBCL and its silence could compromise the aggression of DLBCL, which makes ZNF267 a promising therapeutic target.

ALKBH5-mediated m6A-demethylation of USP1 regulated T-cell acute lymphoblastic leukemia cell glucocorticoid resistance by Aurora B.

Recent studies evidence that ubiquitin-specific proteases (USPs) are associated with the occurrence and chemoresistance of T-cell acute lymphoblastic leukemia (T-ALL). N6 -methyladenosine (m6A) demethylase AlkB homolog 5 (ALKBH5) exerts a carcinogenic effect in human cancers and improves the mRNA stability of USPs. Whether ubiquitin-specific protease 1 (USP1) controls chemoresistance of T-ALL is unknown. Our study demonstrated that USP1 expression was upregulated in glucocorticoid (GC)-resistant T-ALL patients and cells (CEM-C1). High expression of USP1 was correlated to the poor prognosis in T-ALL patients. Silencing USP1 increased CEM-C1 cell sensitivity to dexamethasone (Dex), reduced cell invasion, promoted cell apoptosis, and ameliorated glucocorticoid receptor (GR) expression. USP1 mediated T-ALL chemoresistance by interacting with and deubiquitination of Aurora B. Overexpression of USP1 reversed the amelioration effect of Aurora B inhibitor on CEM-C1 cell resistance to Dex. Mechanistically, ALKBH5 enhanced USP1 expression by reducing m6A level and mRNA stability in USP1 mRNA transcript. Downregulation of ALKBH5 reduced the levels of USP1 and Aurora B, facilitated CEM-C1 cell sensitivity to Dex, apoptosis, and GR expression, suppressed cell invasion. However, overexpression of USP1 reversed all the effects of ALKBH5 on CEM-C1 cells. In vivo results showed that tail vein injection of sh-USP1 resulted in a significant prolongation of mouse survival, suppressed tumor growth, maintained the normal weight of mice, reduced USP1 expression and facilitated GR expression. In conclusion, inhibition of ALKBH5-mediated m6A modification decreased USP1 expression and downregulation of USP1 ameliorated GC resistance of T-ALL through suppressing Aurora B expression and elevating GR level.

Cytology versus calcitonin assay in fine-needle aspiration biopsy wash-out fluid (FNAB-CT) in diagnosis of medullary thyroid microcarcinoma.

PURPOSE: Widespread use of sensitive ultrasound examination led to an increasing detection of medullary thyroid microcarcinoma (micro-MTC). This prospective study evaluated the diagnostic accuracy of Fine-needle Aspiration Biopsy Cytology (FNAB-C) and calcitonin assay in Fine-needle Aspiration Biopsy wash-out fluid (FNAB-CT) in thyroid nodules less than 1 cm with elevated serum calcitonin(sCT). METHODS: 87 thyroid nodules from 60 patients with elevated sCT (>10 pg/ml) were included and 51 were thyroid nodules less than 1cm. FNAB-CT and FNAB-C were performed to distinguish medullary thyroid carcinoma (MTC) lesions before surgery, histopathologic diagnoses served as main reference standards. RESULTS: FNAB-CT had a greater performance over FNAB-C for preoperative diagnosis of MTC (diagnostic accuracy: 98.85 vs 61.90%, sensitivity: 98.55 vs 55.07%, specificity: 100 vs 97.44%), especially for micro-MTC: FNAB-C established a sensitivity and diagnostic accuracy of 48.78 and 58% respectively, while FNAB-CT reached 97.56% sensitivity and 98.04% diagnostic accuracy. CONCLUSIONS: FNAB-CT demonstrated high diagnostic accuracy in diagnosing micro-MTC. Patients with microscopic thyroid nodules and elevated sCT level should perform FNAB-CT to exclude the diagnosis of MTC lesions.

m6A methyltransferase Wilms' tumor 1-associated protein facilitates cell proliferation and cisplatin resistance in NK/T cell lymphoma by regulating dual-specificity phosphatases 6 expression via m6A RNA methylation.

Nasal-type natural killer/T-cell lymphoma (NKTCL) is an aggressive malignancy with poor survival outcomes that is relatively resistant to chemotherapy. N6-Methyladenosine (m6A) modification, the most prevalent modification of eukaryotic messenger RNA, is involved in the progression of various tumors. However, it is unclear whether it has a physiological role in NKTCL development. To address this question, we probed its function and molecular mechanisms in NKTCL. Initially, we demonstrated that Wilms' tumor 1-associated protein (WTAP), a major RNA N6-adenosine methyltransferase, was obviously upregulated in human NKTCL cell lines (YTS and SNK-6 cells), compared with normal NK cells. Functionally, depletion of WTAP noticeably repressed proliferation and facilitated apoptosis in YTS and SNK-6 cells. Moreover, intervention of WTAP evidently prohibited NKTCL cell chemotherapy resistance to cisplatin, as reflected by a lower inhibition of cell viability and decreased expression of drug resistance-associated protein expression MRP-1 and P-gp in YTS and SNK-6 cells. With regard to the mechanism, we revealed that WTAP enhanced dual-specificity phosphatases 6 (DUSP6) expression by increasing m6A levels of DUSP6 mRNA transcript, leading to oncogenic functions in NKTCL. Interestingly, WTAP contributed to the progression and chemotherapy sensitivity of NKTCL by stabilizing DUSP6 mRNA in an m6A-dependent manner. Taken together, these findings uncovered a critical function for WTAP-guided m6A methylation and identified DUSP6 as an important target of m6A modification in the regulation of chemotherapy resistance in NKTCL oncogenesis. This study highlights WTAP as a potential therapeutic target of NKTCL treatment.

Decreased SIRT1 expression in the peripheral blood of patients with Graves' disease.

SIRT1, a class III histone/protein deacetylase (HDAC), has been associated with autoimmune diseases. There is a paucity of data about the role of SIRT1 in Graves' disease. The aim of this study was to investigate the role of SIRT1 in the pathogenesis of GD. Here, we showed that SIRT1 expression and activity were significantly decreased in GD patients compared with healthy controls. The NF-κB pathway was activated in the peripheral blood of GD patients. The reduced SIRT1 levels correlated strongly with clinical parameters. In euthyroid patients, SIRT1 expression was markedly upregulated and NF-κB downstream target gene expression was significantly reduced. SIRT1 inhibited the NF-κB pathway activity by deacetylating P65. These results demonstrate that reduced SIRT1 expression and activity contribute to the activation of the NF-κB pathway and may be involved in the pathogenesis of GD.

Diagnosing Thyrotropin-Secreting Pituitary Adenomas by Short-Term Somatostatin Analogue Test.

Background: Diagnosis of thyrotropin (TSH)-secreting pituitary adenomas (TSHoma) before surgery remains a challenge, especially for microadenomas. We aimed to establish a short-term somatostatin analogue (SSA) test to differentiate TSHomas from other causes of syndromes of inappropriate secretion of TSH (IST), mainly resistance to thyroid hormone ß (RTHß). Materials and Methods: We first evaluated the sensitivity and specificity of SSA test in a training cohort (TSHoma, n = 32; RTHß, n = 20). The test was then validated in an independent cohort (TSHoma, n = 9; RTHß, n = 2). We finally applied the SSA test in 12 perceptively enrolled IST cases with negative imaging findings and absent thyroid hormone receptor beta (THRB) mutations or mixed hormone imbalances. Results: Both TSHoma and RTHß patients showed a decrease of TSH at the start of the SSA test, but the velocity of the TSH suppression slowly decreased in RTHß patients after 2 hours. The suppression ratio of TSH at 24 hours versus 2 and 0 hours was significantly greater in TSHoma patients compared with RTHß patients (70.58% ± 18.6% vs. 6.01% ± 25.41%, p < 0.0001, 79.83% ± 12.79% vs. 51.16% ± 13.62%, p < 0.0001, respectively). The 24- versus 2-hour suppression ratio showed the best diagnostic accuracy at a cut point of 44.46% in the training cohort, with a sensitivity of 95.00%, a specificity of 93.75%, and a positive predictive value (PPV) of 88.89%. The accuracy was confirmed in the validation cohort. Three out of 12 patients in the prospective cohort showed a TSH suppression ratio greater than 44.46% and all developed microadenomas during follow-up. Conclusions: A short-term SSA test provides an alternative diagnostic approach for TSHomas. A positive SSA test result is suggestive for a TSHoma even before positive findings become apparent on pituitary imaging. However, studies including larger number of patients, especially those with RTHß, are needed to confirm our findings.

Mild TPO deficiency characterized by progressive goiter and normal serum TSH level.

PURPOSE: Mild thyroid peroxidase (TPO) deficiency is rare and can be extremely occult. This study aimed to replenish the phenotypic and genetic spectrum of mild TPO deficiency. METHODS: Four unrelated patients with progressive goiter were described in this study. Genes associated with congenital hypothyroidism were analyzed and in vitro functional experiments were conducted to evaluate the residual TPO enzyme activities of each mutant. RESULTS: The four patients (age: 5-27 years old) were characterized by progressive goiter, discordant alteration in thyroid hormones with free triiodothyronine (FT3) to free thyroxine (FT4) ratio ranging from 0.557 to 1.012, two with slightly elevated TSH level and two with normal TSH level. Six different mutations of TPO gene were identified including three novel mutations (p.Glu337Lys, p.Ala544Val, and p.Glu641Lysfs∗21). Two mutants (p.Asp224del and p.Ala544Val) with residual TPO activity of 41 and 65% may explain the mild TPO-deficient picture in our study. After levothyroxine (L-T4) therapy, three patients showed gradual decline of FT3 to FT4 ratio and two patients showed reduced thyroid size. CONCLUSION: Patients with mild TPO deficiency can present with progressive goiter, normal TSH level, and largely reserved TPO activities.

ROLE OF MAGNETIC RESONANCE IMAGING IN THE ASSESSMENT OF ACTIVE THYROID-ASSOCIATED OPHTHALMOPATHY PATIENTS WITH LONG DISEASE DURATION.

Objective: In thyroid-associated ophthalmopathy (TAO), long disease duration is negatively correlated with the response to immunosuppression treatment. The current treatment decision-making process does not involve magnetic resonance imaging (MRI); thus, we investigated the predictive value of MRI parameters for the immunosuppressive response in active moderate to severe TAO patients with different disease durations. Methods: We retrospectively analyzed the baseline MRI parameters of active TAO patients treated with guideline-recommended weekly glucocorticoid therapy in our center. Data were stratified by the quartile of disease duration. The signal intensity ratio (SIR) of T2-weighted images was used to describe the activity of extraocular muscles (EOMs). Results: Compared to the lowest quartile of disease duration, SIR values of EOMs were significantly lower in quartile 3 (Q3) and quartile 4 (Q4). Meanwhile, the clinical activity score (CAS) curve did not change in parallel and was not correlated with the SIR curve. In the highest quartile of disease duration, nonresponders had significantly lower SIR values of the most inflamed muscle (P = .03) and the medial rectus (P = .004) than did the responders, while no such significance was observed in patients within the lower 3 quartiles. A multivariable predictive model (including CAS, TAO duration, and SIR value) was established in each quartile. The fit of the model was better than CAS with regard to prognostic prediction and showed a high positive predictive value (Model 1: 86.67%; Model 2: 92.86%) and negative predictive value (Model 1: 88.89%; Model 2: 90%) in the top quartile. Conclusion: The anterior manifestation assessed by CAS is not always consistent with retro-orbital activity in long-term TAO patients. CAS is sufficient to reflect disease activity in short-term TAO patients. The supplementation of CAS with orbital MRI would be valuable in selecting appropriate active patients with a long disease duration. Abbreviations: AUC = area under the curve; CAS = clinical activity score; EOM = extraocular muscle; FT3 = free triiodothyronine; FT4 = free thyroxine; GC = glucocorticoid; ivGC = intravenous glucocorticoids; MRI = magnetic resonance imaging; NPV = negative predictive value; PPV = positive predictive value; SIR = signal intensity ratio; TAO = thyroid-associated ophthalmopathy; TRAb = thyroid-stimulating hormone receptor antibody; TSH = thyroid-stimulating hormone.

Corrigendum: The genetic landscape of benign thyroid nodules revealed by whole exome and transcriptome sequencing.

This corrects the article DOI: 10.1038/ncomms15533.

The genetic landscape of benign thyroid nodules revealed by whole exome and transcriptome sequencing.

The genomic alterations for benign thyroid nodule, especially adenomatoid nodule, one of the most common types of hyperplasia lesion, are ill-studied. Here, we show whole-exome sequencing and/or transcriptome sequencing data on adenomatoid nodules with or without coincidental papillary thyroid carcinoma (PTC). Somatic mutation of BRAF (22/32) is only detected in PTC, while mutations in SPOP (4/38), ZNF148 (6/38) and EZH1 (3/38) are found enriched in adenomatoid nodule. In an expanded cohort of adenomatoid nodule (n=259) mutually exclusive SPOPP94R, EZH1Q571R and ZNF148 mutations are identified in 24.3% of them. Adenomatoid nodules show very few overlapped mutations and distinct gene expression patterns with their coincidental PTC. Phylogenetic tree analysis uncovers that PTCs evolved independently from their matched benign nodules. Our findings reveal that benign nodules possess a unique molecular signature that differs from PTC and provide genomic evidence for the conventional belief that PTC and benign nodules have independent origin.

miR-1193 Suppresses the Proliferation and Invasion of Human T-Cell Leukemia Cells Through Directly Targeting the Transmembrane 9 Superfamily 3 (TM9SF3).

miRNAs have been involved in various types of cancer, including T-cell leukemia. In this study, the role of miR-1193 in the proliferation and invasion of T-cell leukemia cells was explored. First, we found that miR-1193 was sharply downregulated in T-cell leukemia cells when compared with normal T cells. miR-1193 markedly decreased the proliferation and invasion in Jurkat human T-cell leukemia cells. Transmembrane 9 superfamily 3 (TM9SF3) was then predicted to be a potential target gene of miR-1193, the levels of which displayed a strongly negative correlation with miR-1193 levels in T-cell leukemia patients. We confirmed that TM9SF3 was a target gene of miR-1193 by luciferase reporter gene assay. Finally, gene overexpression and knockdown experiments in Jurkat cells revealed that TM9SF3 positively regulated cell proliferation and invasion.

Prostaglandin E2 blockade enhances the pulmonary anti-Cryptococcus neoformans immune reaction via the induction of TLR-4.

The present study aimed to explore whether the inhibition of prostaglandin E2 enhances pulmonary anti-Cryptococcus neoformans immunity. Lung colony forming unit (CFU) assays demonstrated that the cryptococcal infection was dramatically depressed in mice given EP2 and EP4 or single EP antagonist treatment compared to the untreated wild type mice (p<0.05), leading to the increased survival of the infected mice by 8-9 days or 2-4 days, respectively. RT-PCR and flow cytometry assays showed that the expression of IFN-γ, IL-17, IL-22 in M1 macrophages and IL-10 in M2 macrophages increased significantly at 1 week post-infection in mice with either EP2 or EP4 blockade (p<0.05). The polarization of alveolar macrophages showed that, at 1 week post infection, the alveolar macrophages in untreated wild type mice, TLR4(-/-) mice and TLR4(-/-) mice with EP2 and EP4 blockade were strongly M2 polarized, whereas the alveolar macrophages in wild type mice with EP2 and EP4 blockade were M1 polarized. In conclusion, the blockade of EP2 and EP4 promotes mouse survival after cryptococcus infection by promoting the production of cytokines via TLR4, as well as the enhanced M1 polarization of alveolar macrophages.

Smoking was associated with poor response to intravenous steroids therapy in Graves' ophthalmopathy.

BACKGROUND: Previous studies have shown that smoking is closely related to the occurrence, severity and response to orbital radiation in Graves' ophthalmopathy (GO). The aim of this study was to investigate whether smoking impacts the response to intravenous 4.5 g methylprednisolone therapy in patients with active moderate-to-severe GO. METHODS: Ninety-two individuals with active moderate-to-severe GO who were treated with cumulative doses of 4.5 g intravenous methylprednisolone within 3 months were recruited. The patients were grouped as never smokers, active smokers (including smokers and quit smokers) and passive smokers. RESULTS: We observed significantly greater response rate in never smokers compared with active smokers (73.9% vs 29.0%, p=0.001). After adjusting the confounding factors such as age, sex, body mass index, clinical activity score, thyroid-stimulating hormone receptor antibody and the duration of GO, smoking was independently associated with poor intravenous glucocorticoid (GC) response (OR 12.40, 95% CI 1.20 to 128.14, p=0.035). We also found the response rate was significantly higher in never smokers than in quit smokers (73.9% vs 16.7%, p=0.001), while no statistical significance between current smokers and quit smokers (36.8% vs 16.7%, p=0.228). There was a trend of poor response for passive smokers compared with never smokers (64.7% vs 72.2%, p=0.583). CONCLUSIONS: Smoking, even past smoking, was an independent risk factor associated with impaired response to intravenous corticosteroids in patients with GO. Smokers with GO should be given optimised treatment strategy such as higher dose of GC or combined radiation therapy.

Circulating microRNA predicts insensitivity to glucocorticoid therapy in Graves' ophthalmopathy.

Glucocorticoid (GC) insensitivity occurs commonly in Graves' ophthalmopathy (GO), and GC therapy is associated with major adverse effects. A reliable and easily accessible biomarker is required to predict the outcome of GC therapy. This study aimed to evaluate the performance of circulating microRNA (miRNA) to predict GC insensitivity in GO patients. A total of 35 consecutive patients were included in this study. A cumulative dose of 4.5 g of methylprednisolone (MP) was administered intravenously for 12 weeks. Pretreatment serum miRNAs from the best- (N = 5) and worst- (N = 4) responding patients were profiled using miScript PCR arrays and validated by quantitative PCR in all patients. We calculated the predictive value of pretreatment assays of serum miRNAs with regard to GC insensitivity. We further investigated the roles of target miRNAs in modulating NF-κB activity and restoring transrepression of an NF-κB reporter by dexamethasone. Nine miRNAs displayed significant differences between responsive and resistant patients by miScript PCR arrays. Validation of the top two miRNAs in all 35 patients confirmed a significantly lower serum level of miR-224-5p (p = 0.0048) in resistant patients. A multivariate logistic regression model identified a composite biomarker combining baseline serum miR-224-5p and TRAb was independently associated with GC response (OR: 2.565, 95 % CI 1.011-6.505, p = 0.047). Receiver operating characteristic (ROC) curves analysis revealed the composite marker combining miR-224-5p and TRAb led to a 91.67 % positive prediction value (PPV) and a 69.56 % negative prediction value (NPV) with regard to GC resistance. Overexpression of miR-224-5p restored transrepression of the NF-κB reporter by dexamethasone under induced resistance, which may be via targeting GSK-3ß to increase GR protein level. Our study demonstrated baseline serum miR-224-5p was associated with GC sensitivity in GO and in vitro overexpression of miR-224-5p restored GC sensitivity in a resistant cell model. A parameter combined serum miR-224-5p and TRAb could effectively predict GC sensitivity in GO patients.

Exome sequencing reveals mutant genes with low penetrance involved in MEN2A-associated tumorigenesis.

Activating rearranged during transfection (RET) mutations function as the initiating causative mutation for multiple endocrine neoplasia type 2A (MEN2A). However, no conclusive findings regarding the non-RET genetic events have been reported. This is the first study, to our knowledge, examining genomic alterations in matched MEN2A-associated tumors. We performed exome sequencing and SNP array analysis of matched MEN2A tumors and germline DNA. Somatic alterations were validated in an independent set of patients using Sanger sequencing. Genes of functional interest were further evaluated. The germline RET mutation was found in all MEN2A-component tumors. Thirty-two somatic mutations were identified in the nine MEN2A-associated tumors, of which 28 (87.5%) were point mutations and 4 (12.5%) were small insertions, duplications, or deletions. We sequenced all the mutations as well as coding sequence regions of the 12 genes in an independent sample set including 35 medullary thyroid cancers (20 MEN2A) and 34 PCCs (22 MEN2A), but found no recurrent mutations. Recurrent alterations were found in 13 genes with either mutations or alterations in copy number, including an EIF4G1 mutation (p. E1147V). Mutation of EIF4G1 led to increased cell proliferation and RET/MAPK phosphorylation, while knockdown of EIF4G1 led to reduced cell proliferation and RET/MAPK phosphorylation in TT, MZ-CRC1, and PC-12 cells. We found fewer somatic mutations in endocrine tumors compared with non-endocrine tumors. RET was the primary driver in MEN2A-associated tumors. However, low-frequency alterations such as EIF4G1 might participate in MEN2A-associated tumorigenesis, possibly by regulating the activity of the RET pathway.

Tumor-associated macrophages promote the metastatic potential of thyroid papillary cancer by releasing CXCL8.

Tumor-associated macrophages (TAMs) can promote cancer initiation and progression by releasing cytokines. Previously, we have found the density of TAMs correlated with lymph node metastasis in papillary thyroid carcinoma (PTC). However, the mechanisms of how TAMs promote PTC progression remain unclear. In this study, we first showed that the TAMs density in the tumor core was associated with progressive PTC features and TAMs conditioned medium enhanced PTC cells invasion. Cytokine profiling identified a mixed M1/M2 phenotype and CXCL8 was the most consistently abundant cytokine in PTC-derived TAMs. CXCL8 receptors, CXCR1 and CXCR2, were positively stained in PTC cell lines and tissues, though no association with lymph node metastasis or extrathyroid extension. PTC cell invasion was abrogated by anti-CXCL8-neutralizing antibody, whereas addition of exogenous recombinant human CXCL8 enhanced the invasiveness. More importantly, CXCL8 promoted PTC metastasis in vivo. No difference was found for TAMs-derived CXCL8 expression in patients with and without lymph node metastasis or extrathyroid extension. These findings indicated that TAMs may facilitate PTC cell metastasis through CXCL8 and its paracrine interaction with CXCR1/2.