Solid organ transplant recipients are an ideal population in which to study the link between oncogenic viral infections and cancer due to the deep immunosuppression required to prevent allograft rejection, which increases their risk of developing clinical complications such as infections and cancer. Our long-term goal is to study the relations among immunosuppression, infections, and cancer using transplantation as a model system. Our central hypothesis is that novel biomarkers of cancer risk such as detection of circulating tumor DNA, sequencing of circulating cell-free DNA, and detailed immune profiling can be used for early cancer detection, to identify changes in the virome that precede malignant transformation, and to quantify overall immunosuppression. We will test our hypothesis via three specific aims: (1) To evaluate circulating tumor DNA for early detection of post-transplant malignancies, focusing on post-transplant lymphoproliferative disorders (PTLDs). We will evaluate the performance of CAPP-Seq, an ultra-sensitive assay for early cancer detection, in existing cohorts of over 2000 heart and lung transplant recipients followed at Stanford University and 6 collaborating sites. We will study patients with PTLDs to (a) determine the kinetics of emerging somatic variants preceding tumor development, (b) define the window for accurate early prediction of cancer risk via circulating tumor DNA, and (c) relate these findings to oncotropic viral expansion and immune system suppression. Similar exploratory analyses will be performed in patients with post-transplant lung and colorectal cancers. (2) To profile oncoviruses in cell-free DNA and evaluate integration sites as cancer risk predictors. To distinguish features in the oncotropic virome preceding malignant transformation, we will enrich oncoviral cell-free DNA to enable identification of human:virus gene fusion by deep sequencing, and will determine whether read coverage is consistent with genome integration or with free DNA. We will then profile DNA from primary tumors and cell-free DNA, and will compare integration site coverage in tumor subtypes. (3) To quantify associations among immunosuppression, viral infection and cancer development. We will perform novel immune profiling assays at defined time points following transplantation and will correlate results with development of acute rejection, opportunistic infections, and cancer. Specifically, we will measure circulating Anellovirus load, will infer immune cell subsets from RNA-seq, and will sequence the B-cell antibody heavy chain. We will determine how these results relate to administered immunosuppression, and will build mathematical models to predict risk of clinical complications. This contribution is significant because knowledge of the molecular signatures associated with cancer risk and early detection may lead to novel ways to prevent, monitor, and treat malignant disease. Our innovative approach, in which we will employ novel methods developed by our group to study a very high-risk transplant patient cohort, will lay the foundation for studies aimed at prevention and early detection of cancer as a means of improving clinical outcomes.