Summary The analysis of liquid biopsy (eg, cell-free DNA [cfDNA]; circulating tumor cells [CTC]) in blood is increasingly integrated in clinical contexts including diagnosis, disease monitoring, understanding resistance, and early detection of relapse. The key challenge of detecting these analytes is that they are present at a very low proportion of the biospecimens, and therefore are heavily influenced by pre-analytical factors associated with acquisition and processing. Understanding effects of these pre-analytical variables on the quality of data generated in downstream molecular CTC and cfDNA assays is critical for robust clinical implementation of liquid biopsy tests. To date, research efforts have focused on effects of preservation methods, processing time, storage temp, and shipment conditions on quality of CTC and cfDNA in blood plasma. There are no studies reported on effects of patient-specific context such as fasting, administration of anti-emetics, or biospecimen acquisition procedures (eg, order of blood collection aliquots, time of day when blood is drawn, etc.) There is a lack of data on this type of pre-analytical variable that impacts design of clinical trials such as optimal timing for blood draw and interpretation of data to distinguish technical variables introduced by these pre-analytical factors from the biological signals being evaluated. We propose to address this gap by extending the work done by our team members on evaluating effects of sample processing protocols on cfDNA and CTC analysis, to further investigate effect of patient-specific context. Hypothesis: Pre-analytic variables may affect signal-to- noise ratio in cfDNA and CTC analysis and thus have a higher impact on quantification at levels close to the assay limit of detection. Aim 1: Determine the effect of patient-specific context on the quality of cell-free DNA (cfDNA) and circulating tumor cells (CTC) in prostate cancer patients. Aim 2: Evaluate the impact of these variables on the performance of downstream cfDNA and CTC molecular profiling assays. We will apply an adaptive design in which we perform initial analysis with 20 patients per cohort, then adjust as needed. In a foundation-funded pilot study, we focused on one cohort to study effect of draw order. Results confirm the variability of biomarkers quantification as a result of pre-analytical variables. Significance: Results will elucidate effects of multiple pre-analytical variables specific to individual patient context on performance of blood-based biomarker analysis in cfDNA and CTC. These data will inform the design of liquid biopsy-incorporated clinical trials by identifying optimal timing of blood collection to minimize effects of pre-analytical variables. Innovation: This will be the first study to examine the effect of patient- specific context on quality of liquid biopsy data. We will collaborate closely with commercial liquid biopsy test developers as part of the Blood Profiling Atlas in Cancer (BloodPAC) project, with the goal of sharing knowledge across different sectors and working toward harmonization of pre-analytical procedures for liquid biopsy testing.

Narrative Analytes such as cell-free DNA (cfDNA) and circulating tumor cells (CTC) exist in very low proportion in liquid biopsy specimens, and therefore analyses of these samples are potentially heavily influenced by pre-analytical factors associated with acquisition and processing. We propose to expand our foundation-funded pilot study to evaluate effects of sample processing protocols on cfDNA and CTC analysis to further investigate effect of the patient-specific context. Results will inform the design of liquid biopsy-incorporated clinical trials by identifying optimal timing of blood collection to minimize effects of pre-analytical variables, and will be used to facilitate implementation of liquid biopsy testing in a clinical setting.