PROJECT SUMMARY/ABSTRACT Each year, up to 50,000 patients in the United States receive unnecessary thyroidectomies. These unnecessary thyroidectomies are due to difficulties in preoperatively distinguishing benign thyroid nodules from thyroid cancers. In preliminary data, we developed an epigenetic test that shows promise to distinguish benign versus malignant thyroid nodules. Here, we will rigorously test and validate the ability of our epigenetic biomarkers to evaluate the biologic aggressiveness of thyroid nodules and determine whether the new epigenetic testing will improve thyroid nodule management towards the eradication of unnecessary thyroidectomies. Current molecular diagnostics for indeterminate thyroid nodules, while providing some improvement, have not eliminated the unnecessary thyroidectomies. Current molecular diagnostics are based on molecular differences between normal thyroid tissue and thyroid cancer. However, benign thyroid nodules can contain many molecular alterations including gene fusions and mutations. As a result, over half of thyroid nodules with a significant cancer risk according to the current molecular classifiers are found to be benign after thyroidectomy. In our published preliminary data, we performed a genome-wide DNA methylation analysis of 109 surgically excised thyroid nodules and adjacent benign tissue. We found that the DNA methylation pattern in benign nodules is different from thyroid cancer and normal thyroid. Based on the DNA methylation pattern specific to benign nodules and the DNA methylation pattern specific to thyroid cancer, we developed the Diagnostic DNA Methylation Signature (DDMS) approach to distinguish between benign versus malignant nodules. In a retrospective pilot study performed under 1R21CA223367, we developed DDMS further (DDMS-2). We tested the ability of the DDMS-2 assay to distinguish benign from malignant surgically excised thyroid nodules (n=121). In this Pilot study, DDMS- 2 had an estimated positive predictive value (PPV) of 96% and a negative predictive value (NPV) of 98%. Guided by our preliminary data, we hypothesize that DDMS (i) can be successfully used for molecular thyroid cancer diagnostics of pre-operative thyroid nodule aspirations; (ii) will have superior performance in comparison to current thyroid cancer molecular testing and (iii) can affect physician decision-making towards elimination of unnecessary thyroidectomies. We will accomplish our overall objective by pursuing the following specific aims: Aim 1: To perform analytical validation of the DDMS-2 assay. Aim 2: To determine the DDMS-2 accuracy in a prospective cohort obtained from 7 medical centers and containing 1450 thyroid nodule aspirations including 800 aspirations with indeterminate cytopathology. Aim 3: To compare the diagnostic accuracy between DDMS- 2 and two current thyroid cancer molecular diagnostic approaches and to evaluate how the knowledge of the DDMS-2 results impacts clinical management of thy...