NF-κB p65 and TCF-4 interactions are associated with LPS-stimulated IL-6 secretion of macrophages.

Proinflammatory cytokine plays a central role in host defense and acute inflammatory responses. Both positive and negative correlations of NF-κB and Wnt/ß-catenin pathways have been reported depending on cell types in response to inflammatory stimuli for IL-6 cytokine production. Macrophages are vital to the regulation of immune responses and the development of inflammation, but the crosstalk between two pathways has not been elucidated so far in macrophages. We observed a positive cross-regulation between the NF-κB and Wnt/ß-catenin pathways for IL-6 production in human macrophages. To verify the functional validity of this interaction, LY294002 or PNU74654, representative blockers of each pathway, were treated. IL-6 secretion was reduced to the basal level by both inhibitor treatments, even when stimulated by LPS. We also found that NF-κB p65 migrated to the nucleus and interacted with the transcription factor TCF-4 in macrophages upon LPS stimulation.

Induction of the BRAFV600E Mutation in Thyroid Cells Leads to Frequent Hypermethylation.

OBJECTIVES: The BRAFV600E mutation is a major driver mutation in papillary thyroid cancer. The aim of this study was to elucidate the correlation between DNA methylation and gene expression changes induced by the BRAFV600E mutation in thyroid cells. METHODS: We used Nthy/BRAF cell lines generated by transfection of Nthy/ori cells with the wild-type BRAF gene (Nthy/WT cells) and the V600E mutant-type BRAF gene (Nthy/V600E cells). We performed gene expression microarray and DNA methylation array analyses for Nthy/WT and Nthy/V600E cells. Two types of array data were integrated to identify inverse correlations between methylation and gene expression. The results were verified in silico using data from The Cancer Genome Atlas (TCGA) and in vivo through pyrosequencing and quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: In the Nthy/V600E cells, 199,821 probes were significantly hypermethylated, and 697 genes showed a "hypermethylation-downregulation" pattern in Nthy/V600E. Tumor suppressor genes and apoptosis-related genes were included. In total, 66,446 probes were significantly hypomethylated, and 227 genes showed a "hypomethylation-upregulation" pattern in Nthy/V600E cells. Protooncogenes and developmental protein-coding genes were included. In the TCGA analysis, 491/697 (70.44%) genes showed a hypermethylation-downregulation pattern, and 153/227 (67.40%) genes showed a hypomethylation-upregulation pattern. Ten selected genes showed a similar methylation-gene expression pattern in pyrosequencing and qRT-PCR. CONCLUSION: Induction of the BRAFV600E mutation in thyroid cells led to frequent hypermethylation. Anticancer genes, such as those involved in tumor suppression or apoptosis, were downregulated by upstream hypermethylation, whereas carcinogenic genes, such as protooncogenes, were upregulated by hypomethylation. Our results suggest that the BRAFV600E mutation in thyroid cells modulates DNA methylation and results in cancer-related gene expression.

BRAFV600E Mutation Enhances Estrogen-Induced Metastatic Potential of Thyroid Cancer by Regulating the Expression of Estrogen Receptors.

BACKGRUOUND: Cross-talk between mitogen-activated protein kinase and estrogen has been reported; however, the role of BRAFV600E in the estrogen responsiveness of thyroid cancer is unknown. We elucidated the effect of BRAFV600E on the estrogen-induced increase in metastatic potential in thyroid cancer. METHODS: Using a pair of cell lines, human thyroid cell lines which harbor wild type BRAF gene (Nthy/WT) and Nthy/BRAFV600E (Nthy/V600E), the expression of estrogen receptors (ERs) and estrogen-induced metastatic phenotypes were evaluated. Susceptibility to ERα- and ERß-selective agents was evaluated to confirm differential ER expression. ESR expression was analyzed according to BRAFV600E status and age (≤50 years vs. >50 years) using The Cancer Genome Atlas (TCGA) data. RESULTS: Estradiol increased the ERα/ERß expression ratio in Nthy/V600E, whereas the decreased ERα/ERß expression ratio was found in Nthy/WT. BRAFV600E-mutated cell lines showed a higher E2-induced increase in metastatic potential, including migration, invasion, and anchorage-independent growth compared with Nthy/WT. An ERα antagonist significantly inhibited migration in Nthy/V600E cells, whereas an ERß agonist was more effective in Nthy/WT. In the BRAFV600E group, ESR1/ESR2 ratio was significantly higher in younger age group (≤50 years) compared with older age group (>50 years) by TCGA data analysis. CONCLUSION: Our data show that BRAFV600E mutation plays a crucial role in the estrogen responsiveness of thyroid cancer by regulating ER expression. Therefore, BRAFV600E might be used as a biomarker when deciding future hormone therapies based on estrogen signaling in thyroid cancer patients.

BRAFV600E Transduction of an SV40-Immortalized Normal Human Thyroid Cell Line Induces Dedifferentiated Thyroid Carcinogenesis in a Mouse Xenograft Model.

Background: Despite active studies of the clinical importance of BRAFV600E, suitable research models to investigate the role of this mutation in the etiopathogenesis of human thyroid cancers are limited. Thus, we generated cell lines by transducing the simian virus (SV)-40 immortalized human thyroid cell line Nthy-ori 3-1 (Nthy) with lentiviral vectors expressing either BRAFWT (Nthy/WT) or BRAFV600E. Nthy/WT and Nthy/V600E cells were then xenografted into mice to evaluate the carcinogenic role of BRAFV600E. Methods: Each cell line was subcutaneously injected into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice, and a pathological analysis was performed. The effects of the mutation were further verified by using a BRAFV600E-selective inhibitor (PLX-4032, vemurafenib). The transcriptome was analyzed by RNA sequencing and compared with data from The Cancer Cell Line Encyclopedia and Gene Expression Omnibus. Results: While Nthy/WT was not tumorigenic in vivo, Nthy/V600E formed tumors reaching 2784.343 mm3 in 4 weeks, on average. A pathological analysis indicated that Nthy/V600E tumors were dedifferentiated thyroid cancer. We found metastases in the lung, liver, and relevant lymph nodes. A transcriptomic analysis revealed 5512 differentially expressed genes (DEGs) between the mutant and wild-type cell lines, and more DEGs were shared with anaplastic thyroid cancer than with papillary thyroid cancer. BRAFV600E activated the cell cycle mainly by regulating G1/S phases. PLX-4032 treatment significantly inhibited tumor growth and metastasis. Conclusions: Our data show that BRAFV600E plays a pivotal role in the carcinogenic transformation of an SV40-transfected immortalized normal human thyroid cell line. This xenograft model is expected to contribute to studies of the etiopathogenesis and treatment of highly malignant thyroid cancers.

Refractory healing after surgical therapy of osteonecrosis of the jaw: associated risk factors in aged patients.

Purpose: Osteonecrosis of the jaw (ONJ), both medication-related and non medication-related, mainly occurs in aged patients. It needs surgical intervention. Refractory healing after an operation of ONJ can significantly lower the quality of life of elderly patients. The purpose of this study was to determine risk factors associated with refractory healing in aged patients. Patients and methods: We performed a retrospective study of ONJ in aged patients who underwent surgical treatments in a single institute during a 12-year period. Multiple logistic regression analysis was used to determine independent risk factors associated with refractory healing. Results: A total of 122 patients were included. Of them, 25 patients were identified as the refractory group and 97 patients as the control group. Diabetes mellitus (DM) (AOR=5.03, 95% CI: 1.74-14.52) and glucocorticoid administration (AOR=7.97, 95% CI: 2.52-25.23) were found to be significant risk factors for refractory healing of ONJ. Conclusion: DM and medication of glucocorticoid might be risk factors for refractory healing of ONJ.

Expression of SLC5A5 in Circulating Tumor Cells May Distinguish Follicular Thyroid Carcinomas from Adenomas: Implications for Blood-Based Preoperative Diagnosis.

Preoperative diagnosis of thyroid nodules reduces unnecessary surgery. Circulating tumor cells (CTCs) may contain information of primary tumor(s). We asked whether the peripheral blood expression of genes specific for circulating tumor cells (CTCs) differentiates benign thyroid nodules from malignant nodules. Peripheral blood mononuclear cells from thyroid nodule patients (n = 20) were isolated preoperatively and the expression of seven CTC-associated genes was measured in patients with thyroid nodule(s) (n = 20). Among the tested genes, the expression of SLC5A5 and LGALS3 were validated in a larger number of patients (n = 64) and our results show that SLC5A5 expression differentiated follicular adenomas from follicular carcinomas (area under the curve (AUC) = 0.831). The expression of SLC5A5 in CTCs may preoperatively distinguish thyroid follicular adenomas from follicular carcinomas.

CRISPR RNAs trigger innate immune responses in human cells.

Here, we report that CRISPR guide RNAs (gRNAs) with a 5'-triphosphate group (5'-ppp gRNAs) produced via in vitro transcription trigger RNA-sensing innate immune responses in human and murine cells, leading to cytotoxicity. 5'-ppp gRNAs in the cytosol are recognized by DDX58, which in turn activates type I interferon responses, causing up to ∼80% cell death. We show that the triphosphate group can be removed by a phosphatase in vitro and that the resulting 5'-hydroxyl gRNAs in complex with Cas9 or Cpf1 avoid innate immune responses and can achieve targeted mutagenesis at a frequency of 95% in primary human CD4+ T cells. These results are in line with previous findings that chemically synthesized sgRNAs with a 5'-hydroxyl group are much more efficient than in vitro-transcribed (IVT) sgRNAs in human and other mammalian cells. The phosphatase treatment of IVT sgRNAs is a cost-effective method for making highly active sgRNAs, avoiding innate immune responses in human cells.

A Middle East respiratory syndrome screening clinic for health care personnel during the 2015 Middle East respiratory syndrome outbreak in South Korea: A single-center experience.

BACKGROUND: Transmission of Middle East respiratory syndrome (MERS) to health care personnel (HCP) is a major concern. This study aimed to review cases of MERS-related events, such as development of MERS-like symptoms or exposure to patients. METHODS: A MERS screening clinic (MSC) for HCP was setup in the National Medical Center during the MERS outbreak in 2015. Clinical and laboratory data from HCP who visited the MSC were retrospectively reviewed. Additionally, these data were compared with the results of postoutbreak questionnaire surveys and interviews about MERS-related symptoms and risk-related events. RESULTS: Of the 333 HCP who participated in MERS patient care, 35 HCP (10.5%) visited the MSC for MERS-like symptoms. No one was infected with MERS, and the most common symptom was fever (68.6%) followed by cough (34.3%). However, 106 of 285 postoutbreak survey participants experienced at least 1 MERS-related symptom and 26 reported exposure to patients without appropriate personal protective equipment, whereas only 4 HCP visited the MSC to report exposure events. CONCLUSIONS: Although a considerable number of HCP experienced MERS-related symptoms or unprotected exposure during MERS patient care, some did not take appropriate action. These findings imply that for infection control strategy to be properly performed, education should be strengthened so that HCP can accurately recognize the risk situation and properly notify the infection control officer.

Clinical and pathologic features related to the impacted third molars in patients of different ages: A retrospective study in the Korean population.

BACKGROUND/PURPOSE: The surgical extraction of impacted third molars (ITMs) is a common surgical procedure in dentistry. If prophylactic removal of ITMs is beneficial, however, is a still disputed issue. The aim of this study was to analysis the pathologic changes in impacted third molars (ITMs) and adjacent teeth according to patient age groups in the Korean population to determine if the prophylactic removal of ITMs is to be supported or not. MATERIALS AND METHODS: A retrospective study of patients who underwent surgical extraction of impacted third molars was performed. The patients were divided into 5 groups according to their age. Each group was analyzed with respect to patients' chief complaints, specific pathologic conditions in ITMs, and the damage to adjacent teeth due to untreated ITMs. RESULTS: In this study, 2883 impacted third molars in 1109 patients were analyzed. The most common patients' chief complaint was pain, and the frequency of pain was significantly higher in older age groups. The frequency and severity of pathologic changes in ITMs and adjacent second molars due to ITMs were increased with advancing age. CONCLUSION: Based on the results of this study, we conclude that the prophylactic removal of ITMs that have a higher probability of pathologic changes can be considered to be a reasonable treatment modality in younger patients to reduce morbidity resulting from surgical extraction compared with patients who attained advanced age.

Expression Profiling of a Human Thyroid Cell Line Stably Expressing the BRAFV600E Mutation.

BACKGROUND/AIM: The BRAFV600E mutation acts as an initiator of cancer development in papillary thyroid carcinoma (PTC). Gene expression changes caused by the BRAFV600E mutation may have an important role in thyroid cancer development. MATERIALS AND METHODS: To study genomic alterations caused by the BRAFV600E mutation, we made human thyroid cell lines that harbor the wild-type BRAF gene (Nthy/WT) and the V600E mutant-type BRAF gene (Nthy/V600E). RESULTS: Flow cytometry and western blotting showed stable transfection of the BRAF gene. In functional experiments, Nthy/V600E showed increased anchorage-independent growth and invasion through Matrigel, compared to Nthy/WT. Microarray analysis revealed that 2,441 genes were up-regulated in Nthy/V600E compared to Nthy/WT. Gene ontology analysis showed that the up-regulated genes were associated with cell adhesion, migration, and the ERK and MAPK cascade, and pathway analysis showed enrichment in cancer-related pathways. CONCLUSION: Our Nthy/WT and Nthy/V600E cell line pair could be a suitable model to study the molecular characteristics of BRAFV600E PTC.

Viral RNA in Blood as Indicator of Severe Outcome in Middle East Respiratory Syndrome Coronavirus Infection.

We evaluated the diagnostic and clinical usefulness of blood specimens to detect Middle East respiratory syndrome coronavirus infection in 21 patients from the 2015 outbreak in South Korea. Viral RNA was detected in blood from 33% of patients at initial diagnosis, and the detection preceded a worse clinical course.

Comprehensive Analysis of the Transcriptional and Mutational Landscape of Follicular and Papillary Thyroid Cancers.

Follicular thyroid carcinoma (FTC) and benign follicular adenoma (FA) are indistinguishable by preoperative diagnosis due to their similar histological features. Here we report the first RNA sequencing study of these tumors, with data for 30 minimally invasive FTCs (miFTCs) and 25 FAs. We also compared 77 classical papillary thyroid carcinomas (cPTCs) and 48 follicular variant of PTCs (FVPTCs) to observe the differences in their molecular properties. Mutations in H/K/NRAS, DICER1, EIF1AX, IDH1, PTEN, SOS1, and SPOP were identified in miFTC or FA. We identified a low frequency of fusion genes in miFTC (only one, PAX8-PPARG), but a high frequency of that in PTC (17.60%). The frequencies of BRAFV600E and H/K/NRAS mutations were substantially different in miFTC and cPTC, and those of FVPTC were intermediate between miFTC and cPTC. Gene expression analysis demonstrated three molecular subtypes regardless of their histological features, including Non-BRAF-Non-RAS (NBNR), as well as BRAF-like and RAS-like. The novel molecular subtype, NBNR, was associated with DICER1, EIF1AX, IDH1, PTEN, SOS1, SPOP, and PAX8-PPARG. The transcriptome of miFTC or encapsulated FVPTC was indistinguishable from that of FA, providing a molecular explanation for the similarly indolent behavior of these tumors. We identified upregulation of genes that are related to mitochondrial biogenesis including ESRRA and PPARGC1A in oncocytic follicular thyroid neoplasm. Arm-level copy number variations were correlated to histological and molecular characteristics. These results expanded the current molecular understanding of thyroid cancer and may lead to new diagnostic and therapeutic approaches to the disease.

Significance of the BRAF mRNA Expression Level in Papillary Thyroid Carcinoma: An Analysis of The Cancer Genome Atlas Data.

BACKGROUND: BRAFV600E is the most common mutation in papillary thyroid carcinoma (PTC), and it is associated with high-risk prognostic factors. However, the significance of the BRAF mRNA level in PTC remains unknown. We evaluated the significance of BRAF mRNA expression level by analyzing PTC data from The Cancer Genome Atlas (TCGA) database. METHODS: Data from 499 patients were downloaded from the TCGA database. After excluding other PTC variants, we selected 353 cases of classic PTC, including 193 cases with BRAFV600E and 160 cases with the wild-type BRAF. mRNA abundances were measured using RNA-Seq with the Expectation Maximization algorithm. RESULTS: The mean BRAF mRNA level was significantly higher in BRAFV600E patients than in patients with wild-type BRAF (197.6 vs. 179.3, p = 0.031). In wild-type BRAF patients, the mean BRAF mRNA level was higher in cases with a tumor > 2 cm than those with a tumor ≤ 2.0 cm (189.4 vs. 163.8, p = 0.046), and was also higher in cases with lymph node metastasis than in those without lymph node metastasis (188.5 vs. 157.9, p = 0.040). Within BRAFV600E patients, higher BRAF mRNA expression was associated with extrathyroidal extension (186.4 vs. 216.4, p = 0.001) and higher T stage (188.1 vs. 210.2, p = 0.016). CONCLUSIONS: A higher BRAF mRNA expression level was associated with tumor aggressiveness in classic PTC regardless of BRAF mutational status. Evaluation of BRAF mRNA level may be helpful in prognostic risk stratification of PTC.

Natural Head Postures of Patients With Facial Asymmetry in Frontal View Are Corrected After Orthognathic Surgeries.

PURPOSE: Although orthognathic surgeries focus on adjustment of facial asymmetry (FA), many clinicians know by experience that the natural head posture (NHP) also is corrected after the surgery. The authors examined whether this was indeed the case by the measuring the NHP during the course of orthognathic treatment. Factors associated with NHP correction also were evaluated. PATIENTS AND METHODS: In this retrospective study, clinical features, including the NHP, of patients with FA and those with facial symmetry (FS) were compared. They were outpatients of a private orthodontic dental clinic from December 2008 to March 2012. The degree of NHP tilt was evaluated using an interpupillary (IP) horizontal angle. The NHP of patients with FA were analyzed further before presurgical orthodontic treatment, after presurgical orthodontic treatment, after orthognathic surgery and postsurgical orthodontic treatment, and 1 year after completion of postsurgical orthodontic treatment. The NHP difference at each time point was analyzed using 1-way analysis of variance. An analysis of factors that influence NHP tilt correction was performed by linear regression. RESULTS: Thirty-one patients with FA and 27 with FS were evaluated. The NHP tilt was more profound in the FA group compared with the FS group. There were more patients with skeletal Class III in the FA group. The degree of NHP tilt in the FA group was decreased after orthognathic surgery and postsurgical orthodontic treatment and remained when measured 1 year later. Women were less prone than men to NHP tilt correction by orthognathic surgery. CONCLUSION: Patients with FA have a tilted NHP compared with those with FS. Orthognathic surgery for FA might correct a tilted NHP to a lesser degree in women.

Combined effect of Hashimoto's thyroiditis and BRAF(V600E) mutation status on aggressiveness in papillary thyroid cancer.

BACKGROUND: The purpose of this study was to evaluate the association between Hashimoto's thyroiditis and BRAF(V600E) mutation status in patients with papillary thyroid cancer (PTC) and to determine their combined association with tumor aggressiveness in PTC. METHODS: A total of 1780 patients with PTC who underwent surgery were enrolled in this study. Simple and multiple analyses were performed to determine the association between Hashimoto's thyroiditis and the BRAF(V600E) mutation in PTC. RESULTS: Hashimoto's thyroiditis was present in 11.5% of patients (204/1780) with PTC. Multiple logistic regressions showed that BRAF(V600E) (odds ratio [OR] = 0.493; 95% confidence interval [CI] = 0.360-0.678) and the female sex (OR = 7.146; 95% CI = 3.408-18.347) were independent factors associated with Hashimoto's thyroiditis in PTC. BRAF(V600E) mutation and the Hashimoto's thyroiditis-negative PTC group were associated with aggressive disease (OR = 3.069; 95% CI = 1.654-5.916). CONCLUSION: Hashimoto's thyroiditis was associated less frequently with BRAF(V600E) , and frequently with the female sex in patients with PTC. Hashimoto's thyroiditis and BRAF(V600E) status may help to predict clinical outcome of PTC.

Immune parameters differentiating active from latent tuberculosis infection in humans.

Tuberculosis remains a highly prevalent infectious disease worldwide. Identification of the immune parameters that differentiate active disease from latent infection will facilitate the development of efficient control measures as well as new diagnostic modalities for tuberculosis. Here, we investigated the cytokine production profiles of monocytes and CD4(+) T lymphocytes upon encountering mycobacterial antigens. In addition, cytokines and lipid mediators with immune-modulating activities were examined in plasma samples ex vivo. Comparison of these parameters in active tuberculosis patients and healthy subjects with latent infection revealed that, active tuberculosis was associated with diminished Th1-type cytokine secretion from CD4(+) T cells and less augmented inflammatory cytokine secretion from monocytes induced by IFN-γ than that in latent tuberculosis infection. In addition, a higher plasma concentration of lipoxin A4 and lower ratio of prostaglandin E2 to lipoxin A4 were observed in active cases than in latent infections. These findings have implications for preparing new therapeutic strategies and for differential diagnosis of the two types of tuberculosis infection.

Differential expression of CD57 in antigen-reactive CD4+ T cells between active and latent tuberculosis infection.

The development of diagnostic tests that predict the progression of latent tuberculosis infection to active disease is pivotal for the eradication of tuberculosis. As an initial step to achieve this goal, our study's aim was to identify biomarkers that differentiate active from latent tuberculosis infection. We compared active and latent tuberculosis infection groups in terms of the precursor frequency, functional subset differentiation, and senescence/exhaustion surface marker expression of antigen-specific CD4(+) T cells, which were defined as dividing cells upon their encountering with Mycobacterium (M.) tuberculosis antigens. Among several parameters shown to have statistically significant differences between the two groups, the frequency of CD57-expressing cells could differentiate effectively between active disease and latent infection. Our results suggest that the expression of CD57 in M. tuberculosis-reactive CD4(+) T cells could be a promising candidate biomarker with which to identify individuals with latent tuberculosis infection prone to progression to active disease.

JNK signaling in hepatocarcinoma cells is associated with the side population upon treatment with anticancer drugs.

Liver cancer is one of the most drug-resistant cancer types, and cancer stem cells are related to drug resistance. c-Jun-N-terminal kinase (JNK) signaling is involved in drug resistance, and the side population of cells (SP cells) can be used as a model to study liver cancer stem cells. We sought to evaluate the relationship between SP cells and JNK signaling in hepatocarcinoma cells. For this purpose, we examined cell proliferation and the SP cell ratio following treatment of Huh7 cells with the anticancer drugs 5-fluorouracil (5-FU) and paclitaxel. The expression of phospho-stress-activated protein kinase (SAPK)/JNK in the treated cells was evaluated using immunoblotting. 5-FU and paclitaxel treatment increased the number of SP cells and JNK phosphorylation, and decreased cell survival. Huh7 and HepG2 cells were also treated with SP600125, a JNK inhibitor, to study the relationship between SP cells and JNK signaling. The increase in the number of SP cells and the SAPK/JNK and c-Jun phosphorylation was reverted by SP600125 treatment in these cells. We also used immunohistochemistry and showed that SAPK/JNK and c-Jun phosphorylation are increased in hepatocarcinoma tissues. In conclusion, our results demonstrate that the number of SP cells and SAPK/JNK phosphorylation are increased upon treatment with anticancer drugs, and that this increase is blocked by inhibition of JNK signaling. These findings suggest that drug resistance in liver cancer may involve an increase in the number of SP cells following JNK activation.

Expression of the embryonic morphogen Nodal in differentiated thyroid carcinomas: Immunohistochemistry assay in tissue microarray and The Cancer Genome Atlas data analysis.

BACKGROUND: Nodal, an embryonic morphogen, plays a role in tumorigenesis of melanoma, breast, and prostate cancer; however, its role in thyroid carcinoma is unknown. We examined Nodal expression in thyroid tumors by immunohistochemistry assay and The Cancer Genome Atlas (TCGA) analysis. METHODS: An immunohistochemistry assay was performed in a tissue microarray comprising 128 classic papillary thyroid carcinomas (PTC), 58 follicular thyroid carcinomas (FTC), 19 follicular variants of PTC (FVPTC), 57 follicular adenomas (FA), 54 adenomatous goiters (AG), and 5 normal thyroid tissues. The TCGA database was examined to evaluate the expression of Nodal mRNA in normal thyroid and PTC. RESULTS: The proportion of tumors showing negative Nodal expression in PTC, FTC, FVPTC, FA, and AG was 0%, 1.7%, 0%, 14%, and 41%, respectively. For the diagnosis of malignant tumors, the sensitivity, specificity, positive predictive value, and negative predictive value of positive Nodal staining was 99%, 27%, 72%, and 97%, respectively. High Nodal expression was associated with older age and BRAF mutation in PTC. TCGA analysis revealed PTC had greater Nodal mRNA expression than normal thyroid (P = .012). CONCLUSION: Nodal staining might be useful "rule-out test" for the diagnosis of malignant thyroid tumor. Nodal may be associated with the tumorigenesis of thyroid malignancy.