Epigenomic analysis of cell-free nucleosomes for cancer research

PROJECT SUMMARY Cell-free DNA (cfDNA) levels are elevated in cancer patients, driving a rising interest in liquid biopsy (LB)- based assays for cancer research. However, current cfDNA assays (which assess the abundance, sequence, and/or methylation status of cfDNA) have limited ability to differentiate cell types and disease states for diagnostics and disease monitoring. Of note, cfDNA is primarily found as nucleosomes (cfNucs). Chromatin features including histone post-translational modifications (PTMs) on nucleosomes play key roles in gene regulation, and analysis of these features can inform tissue of origin and disease states. Thus, analysis of cfNucs can provide important information that is lacking from current cfDNA assays. Early applications of cell-free ChIP- seq (cfChIP-seq) have demonstrated the potential of this approach to reveal disease-associated transcriptional programs and drug targets. However, commercial ChIP-seq assays have not yet been optimized for cfNuc analysis, and the ChIP-seq field is fraught with issues including poor sensitivity, cross-reactive antibodies, and lack of standardized controls. To fully leverage LB for clinical applications, there is a need for development of new epigenomic assays that have the sensitivity and reliability to resolve cancer biomarkers from human plasma. EpiCypher is developing cfNuc-seq™, the first commercial platform for epigenomic profiling from human plasma samples. cfNuc-seq assays will be quantitative (enabling reliable cross-sample comparisons), sensitive (able to detect disease signatures from limited inputs), and specific (on-target signal, ability to distinguish distinct disease states). A key innovation of this proposal is the application of ultra-affinity detection reagents to develop a highly sensitive and specific cfChIP-seq workflow to quantitatively compare cfNuc profiles across disease states. A second key innovation is the incorporation of DNA-barcoded designer Nuc (dNuc) spike-in controls to enable in-assay assessment of antibody specificity as well as technical monitoring, normalization, and quantitative cross-sample comparison. Notably, the development of highly sensitive and standardized assays for LB sample analysis is essential due to the low and variable abundance of cfNucs across patients and disease states. Overall, cfNuc-seq assays will be the first commercial epigenomic profiling assays specifically optimized for human plasma, providing ready-to-use assays for easy adoption by clinical researchers. In Phase I, we established strong proof-of-concept by identifying high-performance antibodies and applying them to distinguish cancer-specific signatures in human plasma-based cfNuc-seq assays (with a limit of detection of <0.1%). In Phase II, we will perform essential development to enable commercialization of cfNuc-seq assay kits and services for cancer (pre)clinical research. We will first develop and optimize cfNuc-seq assays for multiple high- value targets (Aim 1). Next, we will perform robust assay validation and develop a bioinformatic pipeline to facilitate deconvolution of cancer signatures from heterogenous samples (Aim 2). Finally, we will prepare for commercialization by assembling beta-kits and performing validation using real-world clinical samples (Aim 3).