Single-molecule Liquid Biopsy Systems for Multiplexed Detection of Epigenetic Modifications and Oncoproteins for Cancer Diagnostics

Analysis of cell-free DNA (cfDNA) and proteins from liquid biopsy samples holds great potential for non-invasive diagnostics and monitoring of various pathologies including cancer. In cancer patients, the death of tumor cells increases significantly the amount of cfDNA and tumor-biomarkers in the plasma. Yet, at early stages of the disease, when the tumor burden is low, confident detection of tumor-originating markers is extremely challenging, requiring highly sensitive methods. Furthermore, adapting a multi-parametric approach can increase specificity and accuracy of detection. We recently established a single-molecule multi-parametric assay to comprehensively profile the epigenetics of plasma-isolated nucleosomes (EPINUC). The power of our approach, relies on the fact that blood cfDNA is kept predominantly in the form of nucleosomes, maintaining their tissue- and cancer-specific epigenetic state. EPINUC harness single-molecule imaging for high-resolution detection of six active and repressive histone modifications, their ratios and combinatorial patterns on millions of individual nucleosomes. This facilitated accurate detection of colorectal cancer, even at early stages. Furthermore, direct single-molecule DNA sequencing revealed the tissue of origin of colorectal, pancreatic, lung and breast tumors. Finally, the highly sensitive and quantitative nature of our imaging platform provides means for detection of non-secreted, oncoproteins, such as mutant H3 and p53, even when present in very low levels in the plasma, as in the case of liquid biopsies from pediatric glioma patients. Overall, we provide a proof-of-concept for the utility of multi-layered clinically relevant information collected from limited (<1 ml) liquid biopsy material.