Germ cell tumors (GCT) are the most common solid tumor of adolescents and young adults. After resection of the primary tumor, many patients do not have evidence of disease elsewhere and are categorized as “clinical stage I (CSI) patients.” The current standard of care for CSI patients is “active surveillance” with serum tumor markers and imaging. However, between 20-50% of these patients will relapse. Moreover, the serum tumor markers that are currently available, AFP and HCG, are present in only 50% of the cases. A biomarker that could predict the likelihood of relapse in the immediate post-op period and detect relapse accurately in all patients on surveillance would rationalize medical decision-making, allowing for immediate initiation of chemotherapy in those at high likelihood of relapse and earlier detection of relapse of those on surveillance. An accurate serum biomarker would also obviate the need for most of the serial surveillance CT scans, reducing radiation exposure and health care costs. Our group has identified a panel of 4 serum miRNAs (miR-371a-3p, miR-372-3p, miR-373-3p and miR-367-3p) that, in over 1500 cases analyzed retrospectively to date, appear to be universally elevated regardless of age, gender, site of primary, or the principal histology of the GCT. We have developed a rigorous pipeline to quantify the levels of these 4 miRNAs using quantitative reverse transcription PCR. Each step in the pipeline maximizes sensitivity and specificity. During the UH2 portion of this application, we propose to conduct the final set of experiments necessary to “lock-down” the analytic process, establish a normal range of these miRNAs by which to quantify the degree of elevation of the miRNAS in cases, demonstrate that this technology is transferrable and that concordant results can be obtained across laboratories, and make final recommendations going into the UH3 portion of the grant on cutpoints for sensitivity and specificity. In the UH3 portion of the application, the serum miRNA test will be prospectively evaluated in the context of an ongoing clinical trial, AGCT1531, that is currently accruing pediatric and adult patients in the United States and Canada and will be opening in the United Kingdom in 2019. The prevalence of the elevated serum miRNAs will be determined in the immediate post-op period and at relapse, and a prospective assessment of the sensitivity, specificity, negative and positive predictive value of the test will be undertaken. With the values gathered prospectively during this clinical trial, the optimal cutpoints for sensitivity and specificity will be recommended, using receiver operating curve methodology.