Comprehensive Gene Expression Analysis in Papillary Thyroid Carcinoma Reveals a Transcriptional Profile Associated with Reduced Radioiodine Avidity

Abstract

Papillary thyroid carcinoma (PTC) is the most common form of well-differentiated thyroid cancer (WDTC) and generally has a favorable prognosis. However, subsets of these tumors can metastasize, leading to aggressive disease progression and poorer clinical outcomes. Radioactive iodine (RAI) therapy is routinely given in the adjuvant setting following thyroidectomy and lymph node dissection for WDTC. Nevertheless, its therapeutic efficacy is limited to tumors with high iodine avidity. Early post-surgical classification of thyroid cancers as either iodine-avid or refractory is crucial for enabling more personalized and effective treatment strategies. In this study, we aimed to identify transcriptomic determinants associated with RAI refractoriness (RAI-R) to improve prognostication. We collected clinicopathologic data and conducted RNA-seq on 36 tissue samples (18 high-avidity and 18 low-avidity), each uniquely characterized by ex vivo iodine concentration measurements taken directly from surgical specimens. Whole-transcriptomic analysis identified 63 differentially expressed genes, with six (S100A4, CRTC2, ANO1, WWTR1, DEPTOR, MT1G) showing consistent deregulation. The expression of ANO1, an established iodine transporter at the apical membrane of the thyroid follicular cells, correlated significantly with iodine avidity (r = 0.54). Validation via RT-qPCR confirmed differential expression trends. Gene ontology and pathway enrichment analyses highlighted thyroid hormone synthesis, PI3K-AKT, and MAPK signaling pathways as key regulators of RAI avidity. A refined multivariate predictive model incorporating ANO1 mRNA expression, histological subtypes, and sample type demonstrated strong predictive performance (adjusted R² = 0.55). These findings suggest ANO1 as a promising biomarker for predicting iodine avidity in thyroid cancer.

Keywords: ANO1; Papillary thyroid carcinoma; RNA-sequencing; Radioiodine avidity.

Fig. 1

Histology of high- and low-avidity papillary thyroid carcinoma. A Hematoxylin and eosin (H&E) stained tall cell subtype of papillary thyroid carcinoma with BRAF p.V600E and C228T TERT promoter mutations. This tumor displayed low radioiodine avidity as measured ex vivo. B Clinical routine BRAF VE1 immunohistochemistry with a positive cytoplasmic signal in the same case. C H&E stained minimally invasive encapsulated follicular variant papillary thyroid carcinoma with high radioiodine avidity. D As expected, no BRAF VE1 immunoreactivity was noted. All images are magnified × 400

Fig. 2

Gene expression analysis of high-avidity and low-avidity cases. A Principal component analysis (PCA) plot based on high-avidity (n = 16) and low-avidity (n = 16) tumors. Each dot corresponds to an individual tumor case. B Volcano plot showing the most dysregulated genes between high- and low-avidity tumor cases. C Heatmap of the 63 differentially expressed genes (DEGs) between the two groups of cases analyzed. All the low-avidity cases are clustered together on the left part of the graph, while the high-avidity cases are clustered on the right. Three high-avidity cases were recognized as low-avidity. D Bar plot of representative gene ontology (GO) terms categorized into three main functional groups: biological process (BP) in orange, cellular component (CC) in green, and molecular function (MF) in blue. Within each group, the top ten significative (p-value < 0.05) GO terms are displayed on the y-axis and ranked by their enrichment score (x-axis), with higher scores indicating greater enrichment significance. E Expression level of the most consistent deregulated genes between high- and low-avidity cases detected by RNA-seq. CRTC2 and S100A4 were found to be upregulated in low-avidity cases compared with high-avidity, while ANO1, WWTR1, DEPTOR, and MT1G were downregulated in low-avidity cases compared with high-avidity

Fig. 3

Correlation between ANO1 mRNA expression and iodine avidity in tumor tissue. The correlation coefficient was r = 0.54, CI 0.23–0.75, p < 0.01, with 83-fold higher avidity for a tumor with tenfold higher ANO1 expression. Levels of avidity observed in normal thyroid tissue (green), the lower detection limit for avidity in the experiment (orange), and the linear correlation line with confidence intervals (grey) are also displayed for clarity

Fig. 4

ANO1 immunoreactivity in papillary thyroid carcinoma (PTC) with variations in radioiodine avidity. All photomicrographs were captured at × 400 magnification. The top row shows control experiments for each slide, demonstrating strong cytosolic ANO1 expression in a gastrointestinal stromal tumor (GIST) and weaker membranous staining in a de-identified case of Graves’ diffuse hyperplasia as well as negative immunoreactivity in normal colon. The bottom row illustrates weak and focal ANO1 expression in adjacent normal thyroid tissue followed by two PTC cases, one with high iodine avidity (HA) and one with low (LA). The H scores shown are 150 for the avid case and 0 for the non-avid case. Note that in the thyroid samples, ANO1 expression is localized to the apical membrane