Induction-concurrent chemoradiotherapy with or without sintilimab in patients with locoregionally advanced nasopharyngeal carcinoma in China (CONTINUUM): a multicentre, open-label, parallel-group, randomised, controlled, phase 3 trial.

BACKGROUND: Anti-PD-1 therapy and chemotherapy is a recommended first-line treatment for recurrent or metastatic nasopharyngeal carcinoma, but the role of PD-1 blockade remains unknown in patients with locoregionally advanced nasopharyngeal carcinoma. We assessed the addition of sintilimab, a PD-1 inhibitor, to standard chemoradiotherapy in this patient population. METHODS: This multicentre, open-label, parallel-group, randomised, controlled, phase 3 trial was conducted at nine hospitals in China. Adults aged 18-65 years with newly diagnosed high-risk non-metastatic stage III-IVa locoregionally advanced nasopharyngeal carcinoma (excluding T3-4N0 and T3N1) were eligible. Patients were randomly assigned (1:1) using blocks of four to receive gemcitabine and cisplatin induction chemotherapy followed by concurrent cisplatin radiotherapy (standard therapy group) or standard therapy with 200 mg sintilimab intravenously once every 3 weeks for 12 cycles (comprising three induction, three concurrent, and six adjuvant cycles to radiotherapy; sintilimab group). The primary endpoint was event-free survival from randomisation to disease recurrence (locoregional or distant) or death from any cause in the intention-to-treat population. Secondary endpoints included adverse events. This trial is registered with ClinicalTrials.gov (NCT03700476) and is now completed; follow-up is ongoing. FINDINGS: Between Dec 21, 2018, and March 31, 2020, 425 patients were enrolled and randomly assigned to the sintilimab (n=210) or standard therapy groups (n=215). At median follow-up of 41·9 months (IQR 38·0-44·8; 389 alive at primary data cutoff [Feb 28, 2023] and 366 [94%] had at least 36 months of follow-up), event-free survival was higher in the sintilimab group compared with the standard therapy group (36-month rates 86% [95% CI 81-90] vs 76% [70-81]; stratified hazard ratio 0·59 [0·38-0·92]; p=0·019). Grade 3-4 adverse events occurred in 155 (74%) in the sintilimab group versus 140 (65%) in the standard therapy group, with the most common being stomatitis (68 [33%] vs 64 [30%]), leukopenia (54 [26%] vs 48 [22%]), and neutropenia (50 [24%] vs 46 [21%]). Two (1%) patients died in the sintilimab group (both considered to be immune-related) and one (<1%) in the standard therapy group. Grade 3-4 immune-related adverse events occurred in 20 (10%) patients in the sintilimab group. INTERPRETATION: Addition of sintilimab to chemoradiotherapy improved event-free survival, albeit with higher but manageable adverse events. Longer follow-up is necessary to determine whether this regimen can be considered as the standard of care for patients with high-risk locoregionally advanced nasopharyngeal carcinoma. FUNDING: National Natural Science Foundation of China, Key-Area Research and Development Program of Guangdong Province, Natural Science Foundation of Guangdong Province, Overseas Expertise Introduction Project for Discipline Innovation, Guangzhou Municipal Health Commission, and Cancer Innovative Research Program of Sun Yat-sen University Cancer Center. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

The ERK inhibitor GDC-0994 selectively inhibits growth of BRAF mutant cancer cells.

BRAF or RAS mutation-induced aberrant activation of the mitogen-activated protein kinase (MAPK) pathway is frequently observed in human cancers. As the key downstream node of MAPK pathway, ERK1/2 is as an important therapeutic target. GDC-0994 (ravoxertinib), an orally bioavailable, highly selective small-molecule inhibitor of ERK1/2, showed acceptable safety and pharmacodynamic profile in a recent phase I clinical trial. In this study, we investigated dependence of the anti-tumor effect of ERK inhibitor GDC-0994 on genetic alterations in the MAPK pathway. The results showed that GDC-0994 sharply inhibited cell proliferation and colony formation and induced remarkable G1 phase cell-cycle arrest in cancer cells harboring BRAF mutation but had little effect on cell behaviors in most RAS mutant or wild-type cell lines. The expression of a large number of genes, particularly the genes in the cell cycle pathway, were significantly changed after GDC-0994 treatment in BRAF mutant cells, while no remarkable expression change of such genes was observed in wild-type cells. Moreover, GDC-0994 selectively inhibited tumor growth in a BRAF mutant xenograft mice model. Our findings demonstrate a BRAF mutation-dependent anti-tumor effect of GDC-0994 and provide a rational strategy for patient selection for ERK1/2 inhibitor treatment.

Risk stratification by combining common genetic mutations and TERT promoter methylation in papillary thyroid cancer.

PURPOSE: Risk stratification based on somatic mutations in TERT promoter and BRAF/RAS has been well established for papillary thyroid cancer (PTC), and there is emerging evidence showed that TERT promoter methylation was frequently observed in thyroid cancer patients with adverse features. This study was aimed to comprehensive explore the prognostic value of BRAF/RAS mutations, TERT promoter mutations, and TERT promoter methylation in PTC. METHODS: The relationships of BRAF/RAS mutations, TERT promoter mutations, and TERT promoter methylation with clinical characteristics and outcomes of PTC were analyzed in 382 patients with PTC. RESULTS: TERT promoter mutation and hypermethylation were collectively observed in 52 (13.6%) samples and associated with BRAF/RAS mutation, aggressive clinical characteristics, and poor clinical outcomes of PTC. Coexistence of BRAF/RAS and TERT alterations was found in 45 of 382 (11.8%) PTC patients and strongly associated with old patient age, extrathyroidal extension, advanced pathologic T stage and metastasis. Importantly, patients with both BRAF/RAS and TERT alterations had higher rates of tumor recurrence (13.6% vs 1.5%, P = 0.042) and disease progression (24.4% vs 3.3%, P < 0.001) than patients without any alterations, and cox regression analysis revealed that the coexistence of BRAF/RAS and TERT alterations, but not BRAF/RAS or TERT alterations alone, increased the risk of progression-free interval with an adjusted HR of 10.35 (95% CI: 1.79-59.81, P = 0.009). CONCLUSIONS: This study suggested that comprehensively analysis of BRAF/RAS mutations, TERT promoter mutation and methylation is an effective strategy to identify high-risk patients with PTC.

Comprehensive analysis of BTNL9 as a prognostic biomarker correlated with immune infiltrations in thyroid cancer.

BACKGROUND: Thyroid cancer (THCA) is the most common type of endocrine cancers, and the disease recurrences were usually associated with the risks of metastasis and fatality. Butyrophilin-like protein 9 (BTNL9) is a member of the immunoglobulin families. This study investigated the prognostic role of BTNL9 in THCA. METHODS: Gene enhancers of BTNL9 were identified by interrogating H3K27ac ChIP-seq and RNA-seq data of papillary thyroid cancer (PTC) and benign thyroid nodule (BTN) tissues. Meanwhile, BTNL9 expression level was verified by qRT-PCR in 30 pairs of primary THCA and adjacent normal tissues. Clinicopathological and RNA sequencing data were obtained from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) to analyze the relations between BTNL9 expression and immune cell infiltration, chemokines/cytokines, immune checkpoint genes, clinical parameters and prognosis values. Besides, survival analysis combining BTNL9 expression and immune cell infiltration scores was conducted. Functional enrichment analysis was performed to investigate the potential biological mechanisms. Cox regression analyses were used to explore independent clinical indicators, and a nomogram model incorporating BTNL9 expression with clinical parameters was established. RESULTS: BTNL9 showed significantly stronger H3K27ac modifications in BTN than PTC tissues at the promoter region (chr5: 181,035,673-181,047,436) and gene body (chr5: 181,051,544-181,054,849). The expression levels of BTNL9 were significantly down-regulated in THCA samples compared to normal tissues, and were strongly associated with different tumor stages, immune cell infiltrations, chemokines/cytokines and immune checkpoint genes in THCA. Functional enrichment analyses indicated that BTNL9 was involved in immune-related and cancer-related pathways. The Kaplan-Meier analysis showed lower BTNL9 expression was associated with poorer progression-free interval (PFI). BTNL9 expression and pathologic stages were independent prognostic indicators of PFI in THCA. CONCLUSIONS: The results implied an important role of BTNL9 in the tumor progression, with the possibility of serving as a novel prognostic biomarker and a potential therapeutic target for THCA.

CircGLIS3 inhibits thyroid cancer invasion and metastasis through miR-146b-3p/AIF1L axis.

PURPOSE: Studies have shown that circRNA is involved in the occurrence and development of human cancers. However, it remains unclear that the contribution of circRNA in thyroid carcinoma and its role in the process of tumorigenesis. METHODS: The expression profile of circRNA-miRNA-mRNA in thyroid carcinoma was detected by RNA sequencing and verified by qRT-PCR. The characteristics of circGLIS3 were verified by RNase R and actinomycin assays, subcellular fractionation, and fluorescence in situ hybridization. The functions of circGLIS3 and AIF1L were detected by wound healing, transwell, 3D culture and Western blot. RNA Immunoprecipitation (RIP), RNA pulldown and dual-luciferase reporter assays were used to verify the target genes of circGLIS3 and downstream miRNAs. Functional rescue experiments were performed by transfecting miRNA mimics or siRNA of target genes. Finally, metastatic mouse models were used to investigate circGLIS3 function in vivo. RESULTS: In this study, we discovered a novel circRNA (has_circ_0007368, named as circGLIS3) by RNA sequencing. CircGLIS3 was down-regulated in thyroid carcinoma tissues and cells line, and was negatively associated with malignant clinical features of thyroid carcinoma. Functional studies found that circGLIS3 could inhibit the migration and invasion of thyroid carcinoma cells, and was related to the EMT process. Mechanistically, circGLIS3 can upregulate the expression of the AIF1L gene by acting as a miR-146b-3p sponge to inhibit the progression of thyroid carcinoma. CONCLUSION: Our study identified circGLIS3 as a novel tumor suppressor in thyroid cancer, indicating the potential of circGLIS3 as a promising diagnostic and prognostic marker for thyroid cancer.

Highly sensitive droplet digital PCR for detection of RET fusion in papillary thyroid cancer.

BACKGROUND: Thyroid cancer is the most frequent malignancy of the endocrine system, of which papillary thyroid cancer (PTC) is the predominant form with a rapid increasing incidence worldwide. Rearranged during transfection (RET) fusions are common genetic drivers of PTC and the potent RET inhibitor selpercatinib has been recently approved for treating advanced or metastatic RET fusion-positive thyroid cancer. In this study we aimed to develop a droplet digital PCR (ddPCR) system to accurately detect RET fusion in PTC samples. METHODS: The frequency and distribution of RET fusions in PTC were analyzed using genomic data of 402 PTC patients in The Cancer Genome Atlas (TCGA) database. To establish the ddPCR system for detecting CCDC6::RET fusion, a plasmid containing CCDC6::RET infusion fragment was constructed as standard template, the annealing temperature and concentrations of primers and probe were optimized. The analytical performance of ddPCR and quantitative reverse transcription PCR (qRT-PCR) were assessed in standard templates and tissue samples from 112 PTC patients. Sanger sequencing was performed in all the RET fusion-positive samples identified by ddPCR. RESULTS: RET fusions were observed in 25 (6.2%) of the 402 TCGA samples, and 15 (60%) of the RET fusion-positive patients had the CCDC6::RET fusion. Compared with qRT-PCR, the ddPCR method showed a lower limit of detection (128.0 and 430.7 copies/reaction for ddPCR and qRT-PCR, respectively). When applying the two methods to 112 tissue samples of PTC, eleven (9.8%) CCDC6::RET fusion-positive samples were detected by qRT-PCR, while ddPCR identified 4 additional positive samples (15/112, 13.4%). All the CCDC6::RET fusion-positive cases identified by ddPCR were confirmed by Sanger sequencing except for one case with 0.14 copies/uL of the fusion. CONCLUSION: The accurate and sensitive ddPCR method reported here is powerful to detection CCDC6::RET fusion in PTC samples, application of this method would benefit more RET fusion-positive patients in the clinic.

Cell-free DNA methylation biomarker for the diagnosis of papillary thyroid carcinoma.

BACKGROUND: Cell-free DNA (cfDNA) is being explored as biomarker for non-invasive diagnosis of cancer. We aimed to establish a cfDNA-based DNA methylation marker panel to differentially diagnose papillary thyroid carcinoma (PTC) from benign thyroid nodule (BTN). METHODS: 220 PTC- and 188 BTN patients were enrolled. Methylation markers of PTC were identified from patients' tissue and plasma by reduced representation bisulfite sequencing and methylation haplotype analyses. They were combined with PTC markers from literatures and were tested on additional PTC and BTN samples to verify PTC-detecting ability using targeted methylation sequencing. Top markers were developed into ThyMet and were tested in 113 PTC and 88 BTN cases to train and validate a PTC-plasma classifier. Integration of ThyMet and thyroid ultrasonography was explored to improve accuracy. FINDINGS: From 859 potential PTC plasma-discriminating markers that include 81 markers identified by us, the top 98 most PTC plasma-discriminating markers were selected for ThyMet. A 6-marker ThyMet classifier for PTC plasma was trained. In validation it achieved an Area Under the Curve (AUC) of 0.828, similar to thyroid ultrasonography (0.833) but at higher specificity (0.722 and 0.625 for ThyMet and ultrasonography, respectively). A combinatorial classifier by them, ThyMet-US, improved AUC to 0.923 (sensitivity = 0.957, specificity = 0.708). INTERPRETATION: The ThyMet classifier improved the specificity of differentiating PTC from BTN over ultrasonography. The combinatorial ThyMet-US classifier may be effective in preoperative diagnosis of PTC. FUNDING: This work was supported by the grants from National Natural Science Foundation of China (82072956 and 81772850).

STRA6 regulates tumor immune microenvironment and is a prognostic marker in BRAF-mutant papillary thyroid carcinoma.

Background: BRAF mutation is one of the most common genetic alterations contributing to the initiation and progression of papillary thyroid carcinoma (PTC). However, the prognostic value of BRAF mutation for PTC is limited. Novel markers are needed to identify BRAF-mutant patients with poor prognosis. Methods: Transcriptional expression data were downloaded from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. Pathway enrichment was performed by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and gene set enrichment analysis (GSEA). Protein-protein interaction networks were predicted by the GeneMANIA. The correlation between STRA6 expression and immune infiltration was analyzed by tumor immune estimation resource (TIMER) and tumor-immune system interaction database (TISIDB). Immunohistochemistry was used to detect the STRA6 protein expression level of PTC. Infiltration of regulatory T cells (Tregs) and CD8+ T cells in tumor samples were analyzed by fluorescent multiplex immunohistochemistry. Results: In BRAF-mutant PTC, STRA6 was extremely upregulated and predicted unfavorable survival, which was an independent risk factor for increased mortality risk. Bioinformatic analyses indicated that STRA6 might activate the MAPK pathway synergistically with BRAFV600E. The expression of STRA6 was associated with immune infiltrates and T cell exhaustion. Fluorescent multiplex immunohistochemistry showed that STRA6 increased Tregs abundance and decreased CD8+ T cells infiltration in PTC. Moreover, STRA6 promoted epithelial-mesenchymal transition via increased cancer-associated fibroblasts infiltration. Conclusions: Our study demonstrates STRA6 may serve as a prognostic marker for BRAF-mutated PTC, which may drive thyroid carcinogenesis via activation of oncogenic pathway and regulation of tumor immunosuppressive microenvironment.

STRA6 Promotes Thyroid Carcinoma Progression via Activation of the ILK/AKT/mTOR Axis in Cells and Female Nude Mice.

BACKGROUND: Metastasis has emerged to be an important cause for poor prognosis of thyroid carcinoma (TC) and its molecular mechanisms are not fully understood. STRA6 is a multifunctional membrane protein widely expressed in embryonic and adult tissues. The function and mechanism of STRA6 in TC remain elusive. OBJECTIVE: We aimed to explore the role of STRA6 in TC progression and provide a therapeutic target for TC. METHODS: The expression and clinicopathological relevance of STRA6 were explored in TC. Stable STRA6-knockdown TC cells were established and used to determine the biological function of STRA6 in vitro and in vivo. RNA sequencing and co-immunoprecipitation were performed to unveil the molecular mechanism of STRA6 in TC progression. The potential of STRA6 as a therapeutic target was evaluated by lipid nanoparticles (LNPs) containing siRNA. RESULTS: STRA6 was upregulated in TC and correlated with aggressive clinicopathological features, including extrathyroidal extension and lymph node metastasis, which contributed to the poor prognosis of TC. STRA6 facilitated TC progression by enhancing proliferation and metastasis in vitro and in vivo. Mechanistically, STRA6 could interact with integrin-linked kinase (ILK) and subsequently activate the protein kinase B/mechanistic target of rapamycin (AKT/mTOR) signaling pathway. We further unveiled that STRA6 reprogrammed lipid metabolism through SREBP1, which was crucial for the metastasis of TC. Moreover, STRA6 siRNA delivered by LNPs significantly inhibited cell growth in xenograft tumor models. CONCLUSIONS: Our study demonstrates the critical roles of STRA6 contributing to TC progression via the ILK/AKT/mTOR axis, which may provide a novel prognostic marker as well as a promising therapeutic target for aggressive TC.

Graves Disease and Inflammatory Bowel Disease: A Bidirectional Mendelian Randomization.

CONTEXT: Both Graves disease (GD) and inflammatory bowel disease (IBD) are common autoimmune diseases that severely damage a patient's quality of life. Previous epidemiological studies have suggested associations between GD and IBD. However, whether a causal relationship exists between these 2 diseases remains unknown. OBJECTIVE: To infer a causal relationship between GD and IBD using bidirectional 2-sample Mendelian randomization (MR). METHODS: We performed bidirectional 2-sample MR to infer a causal relationship between GD and IBD using genome-wide association study summary data obtained from Biobank Japan and the International Inflammatory Bowel Disease Genetic Consortium. Several methods (random-effect inverse variance weighted, weighted median, MR-Egger regression, and MR-PRESSO) were used to ensure the robustness of the causal effect. Heterogeneity was measured based on Cochran's Q value. Horizontal pleiotropy was evaluated by MR-Egger regression and leave-one-out analysis. RESULTS: Genetically predicted IBD may increase the risk of GD by 24% (odds ratio [OR] 1.24, 95% CI 1.01-1.52, P = .041). Crohn disease (CD) may increase the risk of GD, whereas ulcerative colitis (UC) may prevent patients from developing GD. Conversely, genetically predicted GD may slightly increase the risk of CD, although evidence indicating that the presence of GD increased the risk of UC or IBD was lacking. Outlier-corrected results were consistent with raw causal estimates. CONCLUSION: Our study revealed a potentially higher comorbidity rate for GD and CD. However, UC might represent a protective factor for GD. The underlying mechanism and potential common pathways await discovery.

Distinct molecular subtypes of papillary thyroid carcinoma and gene signature with diagnostic capability.

Papillary thyroid carcinoma (PTC) is heterogeneous and its molecular characteristics remain elusive. We integrated transcriptomic sequencing, genomic analysis and clinicopathologic information from 582 tissue samples of 216 PTC and 75 benign thyroid nodule (BTN) patients. We discovered four subtypes of PTC including Immune-enriched Subtype, BRAF-enriched Subtype, Stromal Subtype and CNV-enriched Subtype. Molecular subtypes were validated in an external cohort of 497 PTC cases from the TCGA. Tumors in the Immune-enriched Subtype showed higher immune infiltration and overexpression of immune checkpoints, whilst BRAF-enriched Subtype showed a higher tendency for extrathyroidal extension and more advanced TNM stage. Key oncogenes including LRRK2, SLC34A2, MUC1, FOXQ1 and KRT19 were overexpressed and enriched in oncogenic MAPK and PI3K/AKT signaling pathways in BRAF-enriched subtype. Further analysis of BRAF-enriched Subtype identified three subclasses with different degrees of malignancies. We also uncovered the molecular link of the initiation and progression from BTN to subtypes of PTC using trajectory analysis. Moreover, a 20-gene expression signature was generated for differential diagnosis of PTC from BTN patients. Together, our work identified previously unreported molecular subtypes of PTC, offering opportunities to stratify patients into optimal treatment plans based on molecular subtyping.

Integrative metabolomic characterization identifies plasma metabolomic signature in the diagnosis of papillary thyroid cancer.

Discrimination of malignancy from thyroid nodules poses challenges in clinical practice. We aimed to identify the plasma metabolomic biomarkers in discriminating papillary thyroid cancer (PTC) from benign thyroid nodule (BTN). Metabolomics profiling of plasma was performed in two independent cohorts of 651 subjects of PTC (n = 215), BTN (n = 230), and healthy controls (n = 206). In addition, 132 patients with thyroid micronodules (<1 cm) and 44 patients with BTN suspected malignancy by ultrasound were used for biomarker validation. Recursive feature elimination algorithm was used for metabolic biomarkers selecting. Significant differential metabolites were demonstrated in patients with thyroid nodules (PTC and BTN) from healthy controls (P = 0.0001). A metabolic biomarker panel (17 differential metabolites) was identified to discriminate PTC from BTN with an AUC of 97.03% (95% CI: 95.28-98.79%), 91.89% sensitivity, and 92.63% specificity in discovery cohort. The panel had an AUC of 92.72% (95% CI: 87.46-97.99%), 86.57% sensitivity, and 92.50% specificity in validation cohort. The metabolic biomarker signature could correctly identify 84.09% patients whose nodules were suspected malignant by ultrasonography but finally histological benign. Moreover, high accuracy of 87.88% for diagnosis of papillary thyroid microcarcinoma was displayed by this panel and showed significant improvement in accuracy, AUC and specificity when compared with ultrasound. We identified a novel metabolic biomarker signature to discriminate PTC from BTN. The clinical use of this biomarker panel would have improved diagnosis stratification of thyroid microcarcinoma in comparison to ultrasound.

Association of Total Thyroidectomy or Thyroid Lobectomy With the Quality of Life in Patients With Differentiated Thyroid Cancer With Low to Intermediate Risk of Recurrence.

IMPORTANCE: Owing to the good prognosis of differentiated thyroid cancer (DTC), guidelines recommend total thyroidectomy (TT) or thyroid lobectomy (TL) as surgical treatment for DTC with low to intermediate risk of recurrence. However, the association of these surgeries with the health-related quality of life (HRQOL) of patients with DTC with low to intermediate risk of recurrence is unclear. OBJECTIVE: To longitudinally compare the HRQOL of patients with DTC undergoing different surgeries. DESIGN, SETTING, AND PARTICIPANTS: This prospective observational longitudinal cohort study enrolled patients diagnosed with DTC with low to intermediate risk of recurrence at the First Affiliated Hospital, Sun Yat-sen University, China, from October 1, 2018, to September 31, 2019. Eligible patients were categorized into TL and TT groups according to the surgery they underwent. They were evaluated preoperatively and followed up at 1, 3, 6, and 12 months postoperatively using 3 HRQOL-related questionnaires (European Organization for Research and Treatment of Cancer Quality of Life Questionnaire, version 3.0; Hospital Anxiety and Depression Scale; and Thyroid Cancer-Specific Quality of Life Questionnaire); serum thyrotropin levels, complications, and patient satisfaction were also monitored. Data were analyzed to compare the HRQOL of patients undergoing different surgeries at different time points. EXPOSURES: Total thyroidectomy or TL. MAIN OUTCOMES AND MEASURES: The primary end point was HRQOL (European Organization for Research and Treatment of Cancer Quality of Life Questionnaire, version 3.0; Hospital Anxiety and Depression Scale; and Thyroid Cancer-Specific Quality of Life Questionnaire) at different time points, and the secondary end points were postoperative complications, thyrotropin level, and patient satisfaction. RESULTS: Of the 1060 eligible patients, 563 underwent TL (438 women [77.8%]; median [IQR] age, 38 [31-45] years), and 497 underwent TT (390 women [78.5%]; median [IQR] age, 38 [32-48] years). Compared with the TL group, including the 1- to 4-cm tumor subgroup, the TT group experienced more postoperative HRQOL problems at 1 and 3 months postoperatively. However, nearly all the differences disappeared at 6 and 12 months postoperatively. CONCLUSIONS AND RELEVANCE: Results of this study suggest that HRQOL of patients with DTC with low to intermediate risk of recurrence is not associated with the extent of surgery, and HRQOL may not be an important consideration when making surgical decisions. If better HRQOL is requested in the short term, TL may be preferred.

The ETS Inhibitor YK-4-279 Suppresses Thyroid Cancer Progression Independent of TERT Promoter Mutations.

Hotspot mutations in the core promoter region of the telomerase reverse transcriptase (TERT) gene have been well established to associate with aggressive clinical characteristics, radioiodine refractory, tumor recurrence, and mortality in thyroid cancer. Several E-twenty-six (ETS) transcription factors were reported to selectively bound to the mutant TERT promoter and activated TERT expression. In this study we aimed to investigate whether TERT promoter mutations confer sensitivity to ETS inhibitor YK-4-279 in thyroid cancer cells and whether this inhibitor could be served as a potential therapeutic agent for thyroid cancer. In vitro assays showed that YK-4-279 treatment sharply suppressed cell viability, colony formation, migration, and invasion, as well as induced cell cycle arrest and apoptosis in a panel of thyroid cancer cells. The cell viability after YK-4-279 treatment was similar between cell lines harboring mutant and wild-type TERT promoters. Furthermore, YK-4-279 treatment reduced both luciferase activity and mRNA expression of TERT independent of TERT promoter mutation status. Data from RNA-seq further revealed that YK-4-279 significantly affected biological processes including DNA replication and cell cycle. Reduced DNA helicase activity and decreased expression of several helicase genes were observed after YK-4-279 treatment. Moreover, YK-4-279 significantly inhibited tumor growth and induced apoptosis in a xenograft mice model. Thus, ETS inhibitor YK-4-279 suppressed TERT expression and conferred anti-tumor activity in a TERT promoter mutation-independent manner, and it could be a potential agent for the treatment of advanced thyroid cancers.

Genome-Wide Histone H3K27 Acetylation Profiling Identified Genes Correlated With Prognosis in Papillary Thyroid Carcinoma.

Thyroid carcinoma (TC) is the most common endocrine malignancy, and papillary TC (PTC) is the most frequent subtype of TC, accounting for 85-90% of all the cases. Aberrant histone acetylation contributes to carcinogenesis by inducing the dysregulation of certain cancer-related genes. However, the histone acetylation landscape in PTC remains elusive. Here, we interrogated the epigenomes of PTC and benign thyroid nodule (BTN) tissues by applying H3K27ac chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) along with RNA-sequencing. By comparing the epigenomic features between PTC and BTN, we detected changes in H3K27ac levels at active regulatory regions, identified PTC-specific super-enhancer-associated genes involving immune-response and cancer-related pathways, and uncovered several genes that associated with disease-free survival of PTC. In summary, our data provided a genome-wide landscape of histone modification in PTC and demonstrated the role of enhancers in transcriptional regulations associated with prognosis of PTC.

Selenite Induces Cell Cycle Arrest and Apoptosis via Reactive Oxygen Species-Dependent Inhibition of the AKT/mTOR Pathway in Thyroid Cancer.

Thyroid cancer is the most common endocrine malignancy, and its incidence has increased in the past decades. Selenium has been shown to have therapeutic effects against several tumors. However, its role in thyroid cancer and its underlying molecular mechanism remains to be explored. In the present study, we demonstrated that sodium selenite significantly decreased cell viability and induced G0/G1 cell cycle arrest and apoptosis in thyroid cancer cells in a dose-dependent manner. Transcriptomics revealed that sodium selenite induced intracellular reactive oxygen species (ROS) by promoting oxidative phosphorylation. Increased intracellular ROS levels inhibited the AKT/mTOR signaling pathway and upregulated EIF4EBP3. Intracellular ROS inhibition by N-acetylcysteine (NAC) ameliorated the cellular effects of sodium selenite. The in vitro findings were reproduced in xenograft thyroid tumor models. Our data demonstrated that sodium selenite exhibits strong anticancer effects against thyroid cancer cells, which involved ROS-mediated inhibition of the AKT/mTOR pathway. This suggests that sodium selenite may serve as a therapeutic option for advanced thyroid cancer.

Genetic alterations in cfDNA of benign and malignant thyroid nodules based on amplicon-based next-generation sequencing.

BACKGROUND: Circulating cell-free DNA (cfDNA) serves as a biomarker in multiple malignant diseases. However, controversy still surrounds the role of cfDNA detection in the diagnosis and monitoring of papillary thyroid carcinoma (PTC). This study set out to identify the role of cfDNA detection in distinguishing between benign and malignant thyroid nodules. METHODS: Tissue, blood cell, and plasma samples were collected from 10 patients with benign nodules and 10 patients with malignant nodules. The DNA isolated from these samples was subject to PCR-based amplification using primers designed for 50 proto-oncogenes and tumor suppressor genes. PCR products were sequenced using Illumina technology, and the mutations were detected with varScan among sequencing data for each sample and comparative analysis was carried out. RESULTS: Through amplicon sequencing, we found one non-synonymous somatic mutation in the benign nodules and three in the malignant nodules. Among these four mutations, BRAFV600E mutation was detected in the tissue samples of 8 out of the 10 PTC patients, but it was not detected in the benign nodules. However, no BRAFV600E mutation was detected in cfDNA. Further differential analysis of cfDNA indicated that some genes had more mutations in benign patients than in malignant patients, such as MET and IDH, and some genes had more mutations in malignant patients, such as PIK3CA and EZH2. CONCLUSIONS: We found that BRAFV600E mutation was a credible disease-related mutation in PTC; however, it could not be detected in cfDNA. Moreover, there was a large difference in mutation gene distribution between benign and malignant thyroid nodules.

TET1 is a Tumor Suppressor That Inhibits Papillary Thyroid Carcinoma Cell Migration and Invasion.

BACKGROUND: Ten-eleven translocation (TET) enzymes catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) promoting demethylation in cells. However, the expression pattern and biologic significance of TET in papillary thyroid carcinoma (PTC) remain unclear. This study aimed to elucidate the biological functions of TET1 and the miRNA and mRNA expression levels in PTC cells with downregulated TET1. METHODS: The expression of the TET family in 49 PTC tissues and corresponding tumor-adjacent tissues, as well as PTC cell lines (BCPAP, K1, and TPC-1) and the normal thyroid epithelial cell line (Nthy-ori 3-1), were detected using quantitative real-time polymerase chain reaction. The 5hmC level was detected in PTC tissues and cell lines using immunohistochemistry and dot blot assay, respectively. After silencing the TET1 gene with siRNAs in BCPAP and TPC-1 cells, cell proliferation was detected using EdU assay. Transwell assay was used to investigate cell migration and invasion. miRNA and mRNA expression arrays were conducted in TET1-depleted BCPAP cells. RESULTS: The expression level of TET1 decreased in PTC tissues and cell lines and was consistent with the reduction in the 5hmC level. The knockdown of the TET1 gene with siRNAs in BCPAP and TPC-1 cells, cell proliferation was detected using EdU assay. Transwell assay was used to investigate cell migration and invasion. miRNA and mRNA expression arrays were conducted in TET1-depleted BCPAP cells. WNT4, FZD4, CDK6, MCF2L, and EDN1 was upregulated as potential target genes of dysregulated miRNAs. CONCLUSION: The study showed that TET1 dysfunction inhibited the migration and invasion of BCPAP cells and might have a potential role in the pathogenesis of PTC.

The potential targets for metastases: a study on altered circular RNA profile in breast cancer liver metastases.

Aim: To export the expression profile of circRNAs in breast cancer liver metastases (BCLM). Materials & methods: RNA sequences were used to identify the altered expression profile of circRNAs in BCLM tissues and 231 liver metastasis cells. Quantitative PCR verified the expression of circRNAs in both BCLM tissues and 231 liver metastasis cells. Results: The altered expressed circRNAs were found in BCLM tissues, compared with primary tumor tissues. These circRNAs involved in metastatic related signaling pathways. Hsa_circ_21439 and hsa_circ_11783 were verified to be the most significantly up- and downregulated circRNAs in both BCLM tissues and cells. In silico circRNA/miRNA analysis revealed that has_circ_21439 and has_circ_11783 could act as 'miRNA sponge'. Conclusion: This study provides a potential diagnosis and therapy targets for BCLM.

miR-3619-3p promotes papillary thyroid carcinoma progression via Wnt/ß-catenin pathway.

BACKGROUND: It is well known that the dysregulation of microRNAs (miRNAs) has been identified in papillary thyroid carcinoma (PTC), but their roles in the progression and metastasis of PTC remain unclear. MicroRNA-3619-3p (miR-3619-3p) is associated with cancer progression as an oncogene which is predicted to target at the Wnt/ß-catenin signaling pathway. Our study aimed to investigate the role of miR-3619-3p on PTC cell migration and invasion, as well as the underlying mechanisms. METHODS: The expression of miR-3619-3p in 36 PTC tissues and corresponding tumor-adjacent tissues, as well as 3 PTC cell lines (BCPAP, K1, TPC-1) and the normal thyroid epithelial cell line (N-thy-ori 3-1) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between miR-3619-3p expression and clinicopathologic status of PTC patients was analyzed. Migration, invasion, and wound healing, were used to evaluate the role of miR-3619-3p in PTC. The activation of ß-catenin and the possible molecular pathway were detected by western blotting. RESULTS: The expression of miR-3619-3p in PTC tissues was significantly higher than the corresponding tumor-adjacent tissues (P<0.01), and its high expression positively correlated with extrathyroidal invasion, multicentricity, and cervical lymph node metastasis. Moreover, the miR-3619-3p was also up-regulated in PTC cell lines when compared to N-thy-ori 3-1. MiR-3619-3p enhanced the capabilities of migration and invasion in PTC cell lines. Furthermore, miR-3619-3p activated Wnt/ß-catenin pathway via maintaining the mRNA stability of ß-catenin. CONCLUSIONS: miR-3619-3p promoted PTC cell migration and invasion as an oncogene via activating the Wnt/ß-catenin pathway through increasing the stability of ß-catenin.