Verify γ-OHPdG as a prognostic biomarker of HCC recurrence
Develop a new method for γ-OHPdG liquid biopsy
Prevent recurrence among HCC patients treated with Polyphenon E
Verify γ-OHPdG as a prognostic biomarker for HCC and its occurance
Prognosis following surgical resection differs substantially between patients with HCC, and its reasons are unknown. Biomarkers such as AFP and AFP-L3 have shown to be predictive but lack acceptance in clinical practice because of a wide variety of cutoff values, low sensitivity, and high false positivity from hepatitis and cirrhosis. Our lab showed that γ-OHPdG levels in HCC tissue coincided with adjacent non-tumorous tissue 50% of the time in a small sample. We hypothesize γ-OHPdG may predict HCC aggressiveness in a larger sample size. Our lab aims to recruit 200 patients with hepatocellular carcinoma (HCC) to verify the relationship of γ-OHPdG with recurrence. We will address additional questions on its relationships with its underlying etiology, clinical prediction for aggressiveness, and tumor mutation load. We will also compare its levels with those found in adjacent non-tumorous tissues.
γ-OHPdG is a ubiquitous endogenously formed DNA adduct derived from acrolein as a product of polyunsaturated fatty acid peroxidation. Our lab found γ-OHPdG IHC immunoscores in liver tumor tissue can predict the risk of HCC recurrence after surgical resection. Subsequently, an important goal for the lab is to develop a non-invasive method for detecting γ-OHPdG in blood or urine. Based on reported levels of εdA DNA adduct in liver and urine, we believe our quantified γ-OHPdG levels will be a proxy for its levels in the liver. This is a prerequisite for applying γ-OHPdG as a clinical prognostic biomarker. A liquid-chromatography mass-spectrometry method will be developed for urine samples.
Curative HCC resection or liver transplantation remains the only curative treatment modality for HCC. However, hepatic decompensation, neoplastic progression, and lifelong immunosuppression before and after transplantation increases morbidity and mortality in patients. Polyphenon E (Poly E) is the only green tea extract formula approved by the FDA (IND No. 58367), and our research represents the first attempt to study the effects of Poly E on HCC recurrence following curative resection. Previous studies show that consumption of green tea correlates with lower liver cancer risk in humans. HCC patients shown to have high γ-OHPdG levels will be treated with Poly E. Their γ-OHPdG levels will be determined before and after treatment while they are monitored for recurrence.
Detection and Quantification of an Endogenous DNA Adduct Through MASLD Progression in Circulating Epithelial Cells
There is a need for a more comprehensive group of biomarkers that can not only identify liver injury, but also provide accurate insight into the degree of injury and predisposition to progression to cirrhosis, end-stage liver disease, or HCC. Understanding the molecular changes throughout disease development can help to determine these targets for prediction and prevention. The goal of this study is to detect and quantify γ-OHPdG in circulating epithelial cells (CEC) throughout the progression of Metabolic Dysfunction Associated Liver Disease (MASLD) to understand its correlation to disease progression and HCC development. Further we will quantify oxidative stress through the GSH/GSSG ratio alongside γ-OHPdG to identify a MASLD stage-dependent correlation between oxidative stress and cellular lipid peroxidation (LPO) leading to endogenous DNA adducts. With this information we hope to gain insight into the mechanisms of hepatocarcinogenesis as well as identify clinically relevant biomarkers for liver injury and disease progression.
Quantification of γ-OHPdG from Formalin-Fixed Paraffin-Embedded Hepatocellular Carcinoma Tissue
Formalin-fixed paraffin-embedded tissues are among the most abundant biological tissue resources due to their easy and cost-effective storage at room temperature. As such, there is an abundance of available tissue samples for analysis. It has previously been hypothesized that further tissue oxidation occurs in FFPE tissues. This presents a barrier to utilizing FFPE tissues for the quantification of oxidative DNA adducts. DNA adducts are important markers of oxidative stress resulting from α,β-unsaturated aldehydes. DNA adduct quantification can be used as a predictor of metastasis and relapse. To examine the relationship between the duration of FFPE embedding and tissue oxidation of tissue, a series of DNA isolations from paraffin-embedded hepatocellular carcinoma tissue (HCC) were performed at intervals of four weeks. The quantity of adduct assessed from FFPE tissue was compared against a sample isolated from fresh-frozen tissue. Oxidation will be quantified by measuring the amount of y- hydroxy-1,N2- propane deoxyguanosine (y-OHPdG), an endogenously formed DNA adduct that results from lipid peroxidation, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Preliminary results suggest that DNA purity decreases following isolation from paraffin as compared to isolation from fresh tissue.
2,3-Epoxy-4-hydroxynonenal reduces DNA repair activity through proteasome-mediated degradation
Oxidative DNA damage by lipid peroxidation products of polyunsaturated fatty acids (PUFAs) contributes to hepatocellular carcinoma (HCC) risk in individuals with metabolic dysfunction-associated steatotic liver disease (MASLD). 4-hydroxy-2-nonenal (HNE) and acrolein are two α,β-unsaturated aldehydes implicated in dual detriments of mutagenesis: bulky DNA adduct formation and DNA repair protein degradation. Ames tests of an oxidation product of HNE, 2,3-epoxy-4-hydroxynonenal (EH), suggest EH has higher mutagenicity than HNE. EH may also reduce DNA repair activity due to high epoxide and aldehyde electrophilicity. This study seeks to quantify DNA repair proteins XPA, hOOG1, and MLH1 in HepG2 exposed to EH in vitro. DNA repair protein adducts will then be identified in vitro with mass spectrometry7. These results may verify that acrolein, HNE, and EH may be mechanistic biomarkers of HCC risk.
Hepatocellular carcinoma (HCC) is the third leading cause of cancer–related deaths worldwide, mainly because of its poor prognosis. A valid and reliable mechanism-based prognostic biomarker is urgently needed. gamma-Hydroxy-1,N2-propanodeoxyguanosine (γ-OHPdG) is an endogenous mutagenic DNA adduct derived from lipid peroxidation (LPO). We studied the relationship between hepatic γ-OHPdG and hepatocarcinogenesis in three animal models and the potential of γ-OHPdG to be used as a prognostic biomarker for recurrence in HCC patients after surgical resection, and we found that the increased γ-OHPdG levels in liver DNA correlated with HCC development in all three animal models. Furthermore, Theaphenon E treatment significantly decreased γ-OHPdG levels in the liver DNA of Xpa-/- mice, and this decrease was closely associated with its remarkable effect to reduce HCC incidence (from 100% to 14%). Theaphenon E also effectively inhibited HCC development in DEN-injected mice.
Using two independent sets of clinical samples from 90 and 45 HCC patients, our studies demonstrated that higher levels of γ-OHPdG in HCC biopsy specimens are strongly associated with low survival and low recurrence-free survival, respectively. These results support γ-OHPdG as a promising biologically relevant biomarker for predicting the risk of HCC and its recurrence. In this project, we propose to extend these exciting findings to include a larger patient population to further address questions on γ-OHPdG’s relationships with the underlying etiology of HCC, and clinical predictors for aggressiveness. We also want to compare its levels with those of the adjacent non-tumorous tissues and examine the tumor mutation load. We will develop a non-invasive method for detecting γ-OHPdG in human urine. Our hypothesis is that the adduct levels in urine reflect those in liver DNA. Furthermore, we will conduct an intervention trial with Polyphenon E, an FDA-approved equivalent of Theaphenon E, to prevent HCC recurrence in high-risk patients identified with high liver γ-OHPdG. Our lonγ-term goal is to develop γ-OHPdG as a prognostic biomarker for HCC and its recurrence for intervention trials.
Please see ‘Recent Publications’ for more information about these projects.
Read more about Dr. Chung, his current work, and research interests.