ORF40-01 - Utilization of Liquid Biopsy to Identify Circulating Long Non-Coding RNA XIST as A Non-Invasive Marker for Differentiated Thyroid Cancer.

Background:
Indeterminate thyroid nodules present a significant diagnostic challenge, as cytology alone is often insufficient. The use of modern molecular techniques to detect driver mutations from fine-needle aspiration cytology (FNAC) material has helped address this limitation. Long non-coding RNAs (lncRNAs) have emerged as pivotal regulators of cancer biology which can be detected by liquid biopsy. Aim of this study was to determine the feasibility of lncRNA XIST as marker for differentiating benign and malignant thyroid nodules.
Methodology:
In-silico selection of lncRNA: Using a systematic in-silico pipeline that integrates expression profiling, clinical association, network biology, and literature validation, XIST was identified as one of the key long non-coding RNAs (lncRNAs) in differentiated thyroid cancer (DTC). Differential expression analysis (adjusted p< 0.05, |log2FC| ≥ 1) consistently demonstrated XIST to be significantly upregulated in tumor tissue compared to normal thyroid tissue, which was further corroborated across multiple datasets. Network and co-expression analyses (WGCNA and ceRNA modeling) identified XIST as a hub lncRNA that interacts with multiple microRNAs (miRNAs) and cancer-related messenger RNAs (mRNAs).
Patients presenting with thyroid nodules underwent ultrasonography (USG), ultrasound-guided fine-needle aspiration cytology (FNAC), and surgery where indicated. Patients with biopsy-proven differentiated thyroid carcinoma (DTC) were compared with those having benign thyroid nodules and with age- and sex-matched healthy controls. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure XIST expression in a discovery cohort comprising 25 DTC patients, 40 patients with benign thyroid nodules, and 40 healthy controls. Receiver operating characteristic (ROC) curve analysis was performed to determine the optimal ΔCT cut-off for distinguishing benign from malignant lesions. XIST was significantly overexpressed in the plasma of DTC patients compared with benign nodules and healthy controls . This cut-off was subsequently tested in a validation cohort of indeterminate thyroid nodules (Bethesda categories III and IV; N = 61).

When the ROC-derived ΔCT cut-off was applied to the validation cohort, circulating XIST demonstrated a sensitivity of 62% and a specificity of 78% for differentiating malignant from benign cases. Cross-validation using fine-needle aspiration and tissue samples confirmed the upregulation trends observed in plasma.

Conclusion:
This pilot study has demonstrated that circulating XIST, selected through a robust in-silico pipeline and clinically validated in patient cohorts, holds promise as a non-invasive, plasma-based molecular marker for differentiating benign from malignant thyroid nodules. This finding needs to be further validated in a larger multicentric cohort.