CCND1 Splice Variant as A Novel Diagnostic and Predictive Biomarker for Thyroid Cancer

Cyclin D1 protein is aberrantly overexpressed in thyroid cancers, but mutations of the CCND1 gene are rare in these tumors. We investigated the CCND1 rs9344 (G870A) polymorphism and the expression profiles of wild-type CCND1a and shortened oncogenic isoform CCND1b at the mRNA and protein levels in 286 thyroid tumors. Genotype AA of rs9344 was associated with high expression of CCND1b mRNA and was more frequently found in thyroid cancer than in benign tumors. The mRNA expression levels of CCND1b were higher in papillary thyroid carcinoma (PTC) than in benign or other malignant tumors. However, the expression of CCND1a mRNA showed no association with the parameters. Noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) was distinguished from PTC by low expression of CCND1b at mRNA and protein levels. We further observed that cyclin D1b immunostaining helped to avoid the misdiagnosis of classic PTC with predominant follicular pattern as NIFTP in a separate cohort. Nuclear cyclin D1b expression was associated with aggressive clinicopathologic features in PTC. These findings suggest that cyclin D1b overexpression can be used as a diagnostic and predictive biomarker in thyroid tumors and may be functionally involved in the development and progression of the disease

Relative expression levels of <i>EHD2</i> mRNA in TCGA dataset.

<p>Scatter dot plots (median with interquartile range) showed that the <i>EHD2</i> mRNA expression was associated with histologic subtypes (A), extrathyroidal extension (B), pT stage (C), lymph node metastasis (D), risk of recurrence (E), and molecular subtype (F). <i>EDH2</i> mRNA expression level was negatively correlated with thyroid differentiation score (G) but positively correlated with ERK score (H).</p

Prognostic implication of histological features associated with EHD2 expression in papillary thyroid carcinoma

<div><p>Papillary thyroid carcinoma (PTC) is a heterogeneous tumor with various histological and molecular subtypes. EHD2 is involved in endocytosis and endosomal recycling. This study aimed to investigate the prognostic significance of EHD2 expression in PTC and develop a new model for predicting persistent/recurrent disease after thyroidectomy. Pathologic slides of 512 consecutive patients with PTC ≥ 1 cm were retrospectively reviewed. <i>BRAF</i> mutation analysis and immunohistochemistry for EHD2 were performed. Clinical significance of <i>EHD2</i> mRNA expression was analyzed in 388 PTC patients using The Cancer Genome Atlas dataset. The presence of dyscohesive cells and psammoma bodies were found have significant association with persistent/recurrent disease (p = 0.049 and p = 0.038, respectively). The best discrimination of disease-free survival was found by dividing patients into three prognostic groups based on the following two risk factors according to the size category: psammoma bodies ≥ 4 and dyscohesive cells (≥ 1% and ≥ 20% in PTCs of < 2.0 cm and ≥ 2.0 cm, respectively). In PTCs of ≥ 2.0 cm, patients with the two risk factors had a hazard ratio of 13.303 (p = 0.005) compared to those without risk factors. High expression level of EHD2 was associated with <i>BRAF</i> V600E (p < 0.001), presence of dyscohesive cells (p = 0.010), and absence of psammoma bodies (p = 0.001). Increased <i>EHD2</i> mRNA expression level was associated with extrathyroidal extension (p < 0.001), pT3-4 (p < 0.001), lymph node metastasis (p < 0.001), higher risk of recurrence (p < 0.001), and <i>BRAF</i> V600E (p < 0.001). Our prognostic model is useful for predicting persistent/recurrent disease after surgery of PTC. <i>EHD2</i> mRNA expression could be a novel prognostic marker for PTC patients.</p></div

Kaplan-Meiyer analyses of disease-free survival of all 512 patients with papillary thyroid carcinoma (PTC) according to dyscohesive cells and psammoma bodies.

<p>Dyscohesive cells (A) and psammoma body (B) predicted disease-free survival following surgical resection of PTC. (A) p = 0.033 between 0% and 1%-10% subgroups; p = 0.329 between 0% and 11%-19% subgroups; p = 0.036 between 0% and ≥ 20% subgroups; p = 0.449 between 1%-10% and 11%-19% subgroups; p = 0.463 between 1%-10% and ≥ 20% subgroups; p = 0.307 between 11%-19% and ≥ 20% subgroups; (B) p = 0.090 between 0 and 1–3 subgroups; p = 0.005 between 0 and ≥ 4 subgroups; p = 0.381 between 1–3 and ≥ 4 subgroups. (C) Results of disease-free survival of patients who were subdivided into three groups based on risk factors of dyscohesive cells ≥ 1% and psammoma body ≥ 4. p = 0.069 between 0 risk factors and 1 risk factor; p = 0.004 between 0 risk factors and 2 risk factors; p = 0.064 between 1 risk factor and 2 risk factors.</p

Correlation between EHD2 expression levels and histological parameters.

<p>(A) Positive correlation of expression levels of EHD2 in all tumor cells with grades of dyscohesive cells in tumor periphery and invasive front. (B) Negative correlation of EHD2 expression in all tumor cells with the number of psammoma bodies in the entire tumor tissue.</p

Variables related to persistent/recurrent disease by age-and sex-adjusted binomial logistic regression analysis.

<p>Variables related to persistent/recurrent disease by age-and sex-adjusted binomial logistic regression analysis.</p

Immunohistochemical staining for EHD2 protein.

<p>The immunohistochemical results showing no expression (A), weak expression (B), moderate expression (C), and strong expression (D) in tumor cells. Endothelial cells (internal control) are positive in all staining.</p

Correlation between EHD2 expression and clinicopathologic parameters.

<p>Correlation between EHD2 expression and clinicopathologic parameters.</p

Multivariate analysis for prognostic factors predicting disease-free survival of patients with papillary thyroid carcinoma according to tumor size category.

<p>Multivariate analysis for prognostic factors predicting disease-free survival of patients with papillary thyroid carcinoma according to tumor size category.</p