Leveraging canine spontaneous cancer to optimize the power of blood biopsy

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ABSTRACT Blood biopsy is a rapidly advancing technology in which circulating elements (DNA, RNA, proteins, exosomes) released from tumor cells are collected and assessed. In most cases, the circulating free DNA (cfDNA) is collected, and the circulating tumor DNA (ctDNA) within the cfDNA is quantified and analyzed. Blood biopsy has the potential to transform cancer treatment, but several challenges remain that prevent widespread application including the need to standardize collection and processing procedures, optimize sequencing/analysis platforms, and correlate data generated with long-term patient outcomes. Our current NCI R01 award (CA255319) leverages pet dogs with spontaneous cancer as a tool for improving blood biopsy technologies and as a tool for advancing effective implementation in human patients. The aims of this work award are to 1) assess ctDNA/tumor mutation concordance and optimize the parameters for diagnostic sampling; 2) utilize cancer in pet dogs to improve ctDNA deep duplex sequencing diagnostics; and 3) determine the accuracy of blood biopsy for monitoring disease status and predicting outcome in affected dogs. Over the past 2 years we have made significant progress on these goals, including sequencing more than 260 samples from over 110 dogs. Our data demonstrate that location of blood draw (central versus peripheral vein) influences ctDNA yield, and that ctDNA becomes detectable an average of 90 days prior to clinical relapse in dogs with lymphoma. We further identified clonal hematopoiesis of indeterminate potential (CHIP) mutations in 10.8% of samples, supporting the routine incorporation of CHIP detection in blood biopsy diagnostics. However, up to 20% of canine samples had no detectable copy number changes and a low tumor fraction, despite a very high clinical tumor burden. These data are concordant with recent findings in human cancer patients, creating a diagnostic challenge, particularly in tumors with high structural complexity and low incidence of recurrent point mutations. To address these issues, there is broad interest in evaluating other factors affecting cfDNA, such as methylation. Indeed, unique tissue- specific methylation signatures can determine the cell of origin of DNA in circulation, and in combination with tumor fraction and copy number analysis, have the potential to increase sensitivity and accuracy of liquid biopsy detection. As such, the purpose of this administrative supplement is to analyze the methylome of cfDNA from dogs with histologically confirmed cancers and incorporate these results into our existing ctDNA copy number pipeline. To accomplish this, we propose to first validate the methylation signatures of cfDNA in canine cancers, then characterize the accuracy of multi-modal cfDNA analysis for detection and monitoring of these cancers. All necessary samples have already been collected, along with associated patient outcome data, permitting completion of planned studies within 12 months. Data generated from this administrative supplement will support prospective incorporation of tumor methylome analytics into a multi-parameter blood biopsy diagnostic platform designed to enhance clinical impact in human cancer patients.