Plasma cell free DNA to differentiate malignant from benign thyroid nodules.

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Molecular testing is increasingly used to identify malignancy in thyroid nodules (especially indeterminate category). Cell free DNA (cfDNA) levels from plasma has been useful in diagnosis of cancers of other organs/tissue. cfDNA levels would be estimated in patients with thyroid nodules to explore possibility of establishing a cut-off for identification of malignancy.Patients underwent ultrasonography (USG) and USG guided fine needle aspiration (FNA) and surgery, where indicated. Cell free DNA was extracted from plasma and quantified. In initial analysis (determination of cut-off) cfDNA levels were compared between Bethesda 2 and Bethesda 5 &6 to establish a cut-off 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.FNA (n=119) yielded, Bethesda 2 (n=69) Bethesda 5 & 6 (n=13), underwent histopathological confirmation. Cell free DNA in these two groups were 22.85±1.27 and 96.20±8.31 (ng/ml) respectively. A cfDNA cut-off 67.9 ng/ml, with AUC 0.992 (95% CI=0.97- 1.0) with 100% sensitivity and 93% specificity was established to identify malignant lesions. Indeterminate group (Bethesda 3 & 4) underwent surgery (Malignant n=24), (Benign n=13), and using the previously identified cut off cfDNA we were able to identify malignant lesions with a sensitivity of 100% and specificity of 92.3%. There was a very strong agreement between cfDNA based classification with histopathology based classification of benign and malignant nodule (Cohen's kappa 0.94, p<0.001).Plasma cfDNA estimation could help differentiate malignant from benign thyroid nodules.

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