Aberrant expression of Cancer Osaka Thyroid Oncogene mitogen-activated protein kinase kinase kinase 8 (COT) (MAP3K8) is a driver of resistance to B-RAF inhibition. However, the de novo expression and clinical implications of COT in papillary thyroid cancer (PTC) have not been investigated. The aim of this study is to investigate the expression of A-, B-, CRAF, and COT in PTC (n=167) and analyze the clinical implications of aberrant expression of these genes. Quantitative polymerase chain reaction (qPCR) and immunohistochemical staining (IHC) were performed on primary thyroid cancers. Expression of COT was compared with clinicopathological characteristics including recurrence-free survival. Datasets from public repository (NCBI) were subjected to Gene Set Enrichment Analysis (GSEA). qPCR data showed that the relative mRNA expression of A-, B-, C-RAF and COT of PTCwere higher than normal tissues (all P<0.01). In addition, the expression of COT mRNA in PTC showed positive correlation with A- (r=0.4083, P<0.001), B- (r=0.2773, P=0.0003), and C-RAF (r=0.5954, P<0.001). The mRNA expressions of A-, B,- and C-RAF were also correlated with each other (all P<0.001). In IHC, the staining intensities of B-RAF andCOTwere higher in PTCthan in normal tissue (P<0.001). Interestingly, moderate-to-strong staining intensities of B-RAF and COT were more frequent in B-RAFV600E-positive PTC (P<0.001, P=0.013, respectively). In addition, aberrant expression of COT was related to old age at initial diagnosis (P=0.045) and higher recurrence rate (P=0.025). In multivariate analysis, tumor recurrence was persistently associatedwithmoderate-to-strong staining ofCOT after adjusting for age, sex, extrathyroidal extension,multifocality, T-stage, Nstage, TNM stage, and B-RAFV600E mutation (odds ratio, 4.662; 95% confidence interval 1.066-21.609; P=0.045). Moreover, moderate-tostrong COT expression in PTC was associated with shorter recurrencefree survival (mean follow-up duration, 14.2±4.1 years; P=0.0403). GSEA indicated that gene sets related to B-RAF-RAS (P<0.0001, false discovery rate [FDR] q-value=0.000) and thyroid differentiation (P=0.048, FDR q-value=0.05) scores were enriched in lower COT expression group and gene sets such as T-cell receptor signaling pathway and Toll-like receptor signaling pathway are coordinately upregulated in higher COT expression group (both, P<0.0001, FDR q-value=0.000). Aberrant expression of A-, B-, and C-RAF, and COT is frequent in PTC; increased expression of COT is correlated with recurrence of PTC.
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