PROJECT SUMMARY Validating highly sensitive molecular analysis approaches that can fully exploit precious clinical specimens will transform cancer research and clinical applications. Tissue biopsy is the gold standard for cancer diagnosis and has been the primary source of clinical biospecimens in cancer research. However, one of the biggest challenges in basic and clinical cancer research is the frequent lack of sufficient amount of tumor materials from biopsy for multi-omics research (e.g., parallel RNA and DNA-based profiling) after diagnostic procedures, thus limiting progress in systems biology. As such, the ability to conveniently sample bodily fluids, including circulating cell-free DNA (cfDNA) from plasma, offers great promise for enabling highly-sensitive, minimally- invasive, cost-efficient cancer diagnostic methods, and facilitating screening of high-risk populations and patient monitoring. However, cfDNA typically exists in extremely low quantity (e.g., a few nanograms from several mL of blood). Cancer-derived cfDNA is expected to constitute an even smaller portion of the already scarce cfDNA. To accelerate and enhance cancer biology and clinical applications, this R33 aims to validate a novel technology for profiling DNA methylation, i.e., 5-methylcytosines (5mC), which contributes to cancer pathobiology, is reflected in patient-derived cfDNA, and has been integrated in several FDA-approved tests. Given the prevalence of 5mC in the human genome, its roles in gene regulation, and high chemical stability, validating a novel 5mC technology in nanogram or sub-nanogram-level DNA materials offers promising opportunities for various applications in cancer research that have been limited by technology that can utilize limited clinical samples. Specifically, we developed the T7-Linear Amplification based Bisulfite Sequencing (LABS-seq) that integrates a specially-designed bisulfite conversion procedure with the next-generation sequencing (NGS) for sensitively and unbiasedly detecting 5mC in nanogram or sub-nanogram-level DNA materials (e.g., 100 pg). Our preliminary results demonstrated the technical robustness of the LABS-seq and the feasibility of using this innovative technology to identify cancer-specific 5mC changes in cfDNA. In this R33, we will rigorously validate the LABS-seq technique using banked tumor tissues/cells and plasma cfDNA samples from 250 patients with diverse cancer types and 50 frequency-matched healthy controls as well as longitudinal samples from 200 prospectively recruited cancer patients. In Aim 1, we will validate the LABS-seq in genomic DNA (gDNA) from tumor tissues/cells to detect cancer type-specific epigenetic alterations. In Aim 2, we will validate the LABS-seq in cfDNA for the detection of cancer and longitudinal changes. Upon completion of this project, we will provide a highly sensitive, cost-efficient, transformative epigenetic approach applicable to both gDNA and cfDNA, opening up opportunities for research that have been limited by technology. Our established multidisciplinary team, well-annotated human clinical specimens, and excellent environment will ensure the success of this proposed project and future implementation in the clinic.

PROJECT NARRATIVE The proposed research is relevant to public health because it offers novel molecular analysis capabilities for detecting epigenetic alterations in various sources of DNA from limited tissue biopsies and liquid biopsies. The validation of this technique will provide opportunities to cancer research that have been limited by technology and the availability of limited clinical biospecimens, e.g., multi-omics studies and liquid biopsy-based early cancer detection. Therefore, the proposed research is relevant to the NIH's mission, in particular to the RFA- CA-21-004 that pertains to novel technologies and molecular analysis for clinical cancer research that will help reduce the morbidity and mortality of cancer.