Establishment of an evidence-based biospecimen practice for urine liquid biopsy on the detection of HCC DNA in urine.

Hepatocellular carcinoma (HCC) is the world’s 2nd leading cause of cancer-related death and one of the fastest-growing cancers in the US; 85% of patients die within 5 years, mainly due to late detection, limited treatment options, and high recurrence. Currently, HCC recurrence and treatment responses are monitored by serum AFP and serial imaging. Unfortunately, serum AFP can only be used for ~50% of HCC cases due to low sensitivity. While MRI/CT imaging is the gold standard for diagnosis, it is expensive and less accessible. It also has limited utility in the detection of small tumors (< 2 cm) and is challenging in the presence of dysplastic nodules. Early detection of recurrent HCC is ineffective with the current available methods. Genetic liquid biopsies have been routinely used in clinics for almost all cancer types as part of the National Comprehensive Cancer Network (NCCN) guideline. However, its usage in HCC is limited even though its promises have been well documented in the literature, mostly for blood and some for urine. Our hypothesis is that by establishing an evidence-based biospecimen practice for urine collection, storage, and transrenal DNA (trDNA) isolation, urine HCC DNA analysis with tailored sensitive assays can provide HCC genetics with more frequent tumor surveillance and treatment response assessment compared to current standard of care for prognosis. As a pioneer in developing urine cancer liquid biopsies, we have established the standard operating procedure (SOP) for urine collection for the Early Detection Research Network colon cancer validation study (protocol ID: 320) and other non-urinary tract cancers. We investigate pre-analytic variables that influence urine HCC DNA assay performance in the process of developing an HCC urine DNA test for HCC liquid biopsies. In this study, we use a liver-specific trDNA marker, hepatitis B virus (HBV) DNA, which is found in urine, from patients with chronic HBV infection to validate and mitigate these pre-analytic variables. As a result, an evidence-based urine biospecimen practice can be established to help support urine HCC DNA assay development, validation for HCC genetic liquid biopsy and assessment of treatment response. Developing a robust evidence-based urine biospecimen practice for sample collection, processing, and long-term storage will lead to the validation of urine tests for assessing HCC treatment in clinical trials.

Molecular mechanism of HBV cccDNA and integrated DNA in HBV mono-infection and HIV-coinfection

Hepatitis B virus infection affects more than 296 million people globally and is a major cause of cirrhosis and hepatocellular carcinoma (HCC); these complications are particularly high among patients with HBV/HIV coinfection. Complete HBV cure with the elimination of cccDNA and integrated DNA can definitively arrest the disease progression and HCC risk, but these are unattainable with current therapy. We aim to understand the molecular mechanisms of cccDNA and integrated DNA transcriptional activities in both HBV/HIV coinfection and HBV mono-infection that can facilitate the development of novel HBV therapies for a complete HBV cure.

Chronic hepatitis B (CHB) remains a major public health problem despite the availability of effective vaccines and approved therapies. Functional cure with HBsAg clearance is the current HBV treatment endpoint. Despite prolonged therapy with nucleos(t)ide analogs to suppress HBV DNA replication, the rate of functional cure remains low. With functional cure, it is expected that the intrahepatic cccDNA would be either eliminated or in a transcriptional inactive state and regardless the status of integrated DNA. We and others provided evidence that integrated DNA provides the major source of HBsAg especially in HBeAg-negative CHB. The current serum assay cannot distinguish HBsAg generated from intrahepatic cccDNA versus integrated DNA. In this proposal, we focus to 1) evaluate both the concentrations and the transcriptional activities of cccDNA and integrated DNA in CHB with and without HIV; 2) understand the epigenetic mechanisms on cccDNA transcription and treatment response; 3) to correlate integrated DNA in plasma and in liver biopsy and with HBsAg levels and treatment outcome. This is a unique translational research study that utilize well-characterized liver and blood samples as well as advanced molecular technologies and methods to understand the virological mechanisms of HBV persistence, inactivity, and clearance. The success of our study will have significant impacts on the development of novel therapies leading to complete HBV cure.