Development of cell-free DNA assays for HCC screening and liquid biopsy

Development of cell-free DNA assays for HCC screening and liquid biopsy This proposal is for the development of a panel of non-invasive, cell-free (cf) DNA markers, found in the blood or urine, for clinical usage as biomarkers of hepatocellular carcinoma (HCC). This panel will be used in a Certificate for Laboratory Improvement Act (CLIA) lab setting for the early detection and liquid biopsy. The development of this panel will be accomplished by the partnership with two industrial partners, Medical Diagnostic Laboratories (MDL), a leading diagnostic company for over 130 CLIA-certified PCR diagnostic tests in-house, and JBS Science Inc., a company specialized in detecting fragmented cell-free HCC DNA modifications, and two clinical sites, Thomas Jefferson University and Johns Hopkins University. Like other cancers, HCC is a disease of the genome; identification of the DNA modifications underlying the development of HCC should provide unambiguous detection of HCC and personalized care, if HCC is detected. Although there have been numerous attempts to develop cfDNA blood-based, liquid biopsy tests for cancers, most have failed. Of the few attempts designed for HCC, there have not been any used clinically for that capacity. This proposal is for the development of assays that will overcome the obstacles of bringing biomarker discovery to clinical use in the form of liquid biopsies. We will achieve this through detection of a panel of DNA modifications that will enable screening of HCC, identifying cancer subtypes, optimizing drug treatment plans, and monitoring residual diseases to meet the unmet need in early detection and personalized treatment of the cancer. Briefly, in preliminary studies, we have shown that most cfDNA that is present in the blood or urine, and is derived from tumor cells, is low molecular weight (LMW), <300 nts, and thus requires methods specifically designed for small DNA detection and amplification. In a study of 74 HCC urine samples, we detected 77% of the HCC cases and distinguished these cases from liver cirrhosis (n=45) and hepatitis (n=42) with 95% specificity. This was accomplished by detection and quantification of DNA fragments corresponding to DNA modifications (mutations or methylation) within three cfDNA markers: TP53 mutations and methylated GSTP1 and RASSF1a. We believe that the addition of just two HCC-associated DNA modifications, Tert and CTNNB1 mutations, covering the other major HCC cancer pathways will greatly improve the performance of this panel, since almost all (98%, 60/61) HCC tissue tested contained at least one of these five DNA modifications (mutations or methylation). The assays for detecting these five DNA markers in the circulation must be standardized, and the studies must be confirmed. This proposal will therefore develop and optimize the five cfDNA assays for reduction to practice and to determine their clinical utilit in the early detection and precision management of HCC. The deliverable components will be CLIA-certified assays for which their clinical usefulness will be determined, and these assays will be ready for use in commercial CLIA labs, with which we have partnered.