Endocrine Abstracts

Background: Indeterminate thyroid nodules pose a diagnostic dilemma and the patients often undergo unnecessary surgeries or repeat surgery. Currently different molecular methods for detection of driver mutations are being used for better characterisation of these nodules. These methods are costly and not widely available all over the world. Currently use of liquid biopsy by measurement of cell-free DNA (cfDNA) levels from plasma has been useful in diagnosis and follow up of cancers of other organs/tissues. We have analysed cfDNA levels in patients with thyroid nodules to explore the possibility of establishing a cut-off for identification of malignancy and its application in the indeterminate category of nodules.

Methods: Patients underwent ultrasonography (USG) and USG-guided fine needle aspiration as well as surgery, where indicated. CfDNA was extracted from plasma by using a commercially available kit. Quantification and purity of the isolated cfDNA was measured by determining absorbance at 260 nm and 280 nm in duplicate using a Nano Drop Spectrophotometer. Surgical biopsy and histopathology were taken as gold standard for diagnosis. In initial analysis (determination of cut-off), cfDNA levels were compared between Bethesda 2 and Bethesda 5 &6 to establish a cut-off value that could differentiate malignant from benign nodules. In the subsequent analysis, the aforementioned cut-off was applied (validation of cut-off) to those with indeterminate nodules to check ability to predict malignancy.

Results: Fine needle aspiration (n=207) yielded patients with Bethesda 2 (n=112) Bethesda 5 & 6 (n=34) who underwent histopathological confirmation. Cell-free DNA levels in these 2 groups were 23.09 ± 8.47 and 90.26 ± 9.00 (ng/ml) respectively. A cfDNA cut-off of 64.05 ng/ml, with area under the curve of 0.993 (95% CI, 0.98-1.0) with 100% sensitivity and 96.4% specificity was established to identify malignant lesions. Indeterminate group (Bethesda 3 & 4 n=61) underwent surgery (malignant n=33), (benign n=28), and using the previously identified cut-off for cfDNA, we were able to identify malignant lesions with a sensitivity of 100% and specificity of 96.43%. There was a very strong agreement between cfDNA-based classification with histopathology-based classification of benign and malignant nodules (Cohen’s kappa 0.96; P < 0.001).

Conclusion: Liquid biopsy by using plasma cfDNA could be a useful test in differentiating benign and malignant nodules in indeterminate category and help in better management.