Project Summary Ablative radiotherapy is an effective way to non-invasively destroy a tumor. This high-dose treatment, however, requires precise targeting, which is complicated by the fact that tumors in the body move with respiration. There are a variety of technologies that implement tumor tracking to achieve ablative radiotherapy in the body. However, these are limited to treating just a few lesions in a patient due to the complexity and invasiveness of the necessary motion management techniques. Therefore, patients that present with later-stage disease with more than a few metastatic sites (i.e., more than 3 lesions) are not feasibly treatable with ablative radiotherapy. RefleXion Medical is developing the first universal method for direct real-time targeting of tumors based on biological guidance. Utilizing positron emission tomography (PET) emissions from the patient, this new technique allows for continuous and automatic dose conformation to multiple lesions, enabling dose escalation to the target(s) and toxicity reduction to sensitive tissues. The concept has been proven with the development of the first prototype machine. This proposal has four specific aims. Aims 1 and 2 are to validate the reproducibility and robustness of the treatment approach; Aims 3 and 4 are to develop a quality assurance (QA) device that can measure the performance of biology-guided radiotherapy (BgRT) and perform end-to-end tests to validate that planning goals can be accurately achieved in patient-like phantoms. Success of these aims will mitigate clinical risks and enable commercialization of the first biology-guided radiotherapy (BgRT) system.