PROJECT SUMMARY/ABSTRACT Neoadjuvant chemoradiation therapy (nCRT) followed by surgery is the standard of care for patients with locally advanced rectal cancer (LARC), which accounts for ~60% of newly diagnosed rectal cancer cases. Although nCRT benefits many patients, it is currently not known who will or will not respond favorably. About 20% patients that receive nCRT will experience a pathologic complete response (pCR), while ~25% patients will exhibit no response. Recently, several clinical trials have assessed new treatment paradigms, and their results present new treatment strategies which were previously not available for LARC patients. With the availability of these new strategies, it is crucial to be able to a priori identify LARC patients, via a predictive biomarker, who have a higher likelihood of responding or not responding to nCRT. Our goal is to optimize and validate an efficient assay that is accurate, precise, and minimally invasive to assess biomarker(s) that will predict response to nCRT in the setting of LARC. The assay that will be validated in this proposal is a Luminex multiplexed assay that uses the xMAP technology. We have defined six candidate biomarkers that will be validated in this study. Here, using the optimized and validated assay we will then achieve the clinical validation of the candidate biomarker(s) in a real-world clinical laboratory setting using LARC patient specimens. At the end of this study, we will achieve a minimally invasive, blood-based assay for patient treatment stratification which will be of tremendous benefit to LARC patient(s) and the healthcare system. Using the xMAP assay, we are detecting candidate biomarkers of DNA damage response (DDR) signaling in cell lysates from cryopreserved peripheral blood monocytes (PBMCs) of LARC patients. In our preliminary work, the change in the expression of these biomarkers significantly associated with response to nCRT in LARC patients. These candidate biomarkers were identified through our recent hypothesis-driven translational study funded by the DOD. This proposal was developed through a collaboration between the two primary investigators bringing together complementary expertise. The assembled multi-disciplinary team brings together expertise in clinical and basic science, assay development/validation, and their clinical deployment. Here, we will analytically validate our assay (UH2 phase) and then establish the clinical validity by using biospecimens from LARC patients (UH3 phase). The biospecimens used are from LARC patients and healthy controls from the Fox Chase BioSample Repository Facility, and from LARC clinical studies/trials evaluating therapeutic response. In the UH2 phase we will perform pre-analytical (biospecimen variability) and analytical (e.g., precision, accuracy) validation. In the UH3 phase, we will establish clinical validity by evaluating clinical utility parameters (e.g., sensitivity/specificity, optimal cut-offs) and compare with nCRT response status. Successful completion will yield validated biomarkers/assays for use as investigational assays in clinical trials/studies. Over the long-term, this work may improve patient stratification for treatment in LARC patients.

PROJECT NARRATIVE/RELEVANCE TO PUBLIC HEALTH We have developed a minimally invasive approach to determine the expression of DNA damage response (DDR) biomarkers in human PBMCs (from whole blood sample). In this proposal, we will analytically validate the Luminex multiplexed assay which uses the multi-analyte platform (xMAP) technology (UH2 phase) and then establish clinical validity by using biospecimens from the Fox Chase Cancer Center Biosample Repository Facility and from two clinical studies in locally advanced rectal cancer (UH3 phase). The ultimate goal of this research is to utilize the blood-based DDR biomarkers to guide patient stratification for treatment in the setting of locally advanced rectal cancer and potentially other cancers in the future.