Real-world, Prospective, and Multicenter Validation of a microRNA-based Thyroid Molecular Classifier

Background The diagnosis of cancer in thyroid nodules with indeterminate cytology (Bethesda III/IV) is challenging as fine-needle aspiration (FNA), the gold standard method, has limitations, and these cases usually require diagnostic surgery. As approximately 77% of these nodules are not malignant, a diagnostic test accurately identifying benign thyroid nodules can reduce surgery rates. We have previously reported the development and validation of a microRNA-based thyroid molecular classifier for precision endocrinology (mir-THYpe) with high sensitivity and specificity, which could be performed directly from readily available cytological smear slides without the need for a new dedicated FNA. We sought to evaluate whether the use of this test in real-world clinical routine can reduce the rates of surgeries for Bethesda III/IV thyroid nodules and analyze the test performance. Methods We designed a real-world, prospective, multicenter cohort study. Molecular tests were performed in a real-world clinical routine with samples (FNA smear slides) prepared at 128 cytopathology laboratories. Patients were followed-up from March 2018 until surgery or until March 2020 (for those patients not recommended for surgery). The final diagnosis of thyroid tissue samples was retrieved from postsurgical anatomopathological reports. Results After applying the exclusion criteria, 435 patients (440 nodules) classified as Bethesda III/IV were followed-up. The rate of avoided surgeries was 52.5% for all surgeries and 74.6% for “potentially unnecessary” surgeries. After the statistical treatment of non-resected test-negative samples, the test achieved 89.3% sensitivity (95% CI 82–94.3), 81.65% specificity (95% CI 76.6–86), 66.2% positive predictive value (95% CI 60.3–71.7), and 95% negative predictive value (95% CI 91.7–97) at 28.7% (95% CI 24.3–33.5) cancer prevalence. The test influenced 92.3% of clinical decisions. Conclusions The reported data demonstrate that the use of the microRNA-based classifier in the real-world can reduce the rate of thyroid surgery with robust performance and significantly influence clinical decision-making. ### Competing Interest Statement MTS holds equity at ONKOS Molecular Diagnostics. BMR and SS are formal employees at ONKOS Molecular Diagnostics. The other authors declare no competing interests. ### Funding Statement This study was funded by Onkos Molecular Diagnostics and FAPESP - Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (Project 2017/16417-9) ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study was approved by the board of the investigational ethics committee from Midwestern State University in Brazil, following the National Committee of Ethics in Research (IRB/CONEP) process and listed under CAAE 39107520.0.0000.0106 report #4352267 All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Due to the nature of this research, to protect participants' privacy and confidentiality, raw data are not shared publicly. Relevant data supporting the findings and conclusions of this study are available within the article and its supplementary materials. Upon a justifiable request, the share of de-identified data should be approved by the board of an investigational ethics committee.