Abstract 6235: Detection of γ-OHPdG in circulating liver cells and its potential use as a prognostic biomarker in patients with hepatocellular carcinoma recurrence
Authors: Aggarwal Monika , Zhu Zizhao , Gould Sophie , Johnson Peter , Beheshtian Samira , Fang Hongbin , Kallakury Bhaskar , Mueller Susette C. , He Aiwu Ruth , Chung Fung-Lung 
Published: June 2022

Abstract
Background: Primary liver cancer is the fourth most common cause of cancer-related death. Resection is the most widely used potentially curative treatment for patients with early-stage hepatocellular carcinoma (HCC). Recurrence within 2 years occurs in 30-50% of patients and is a major cause of mortality. A biomarker to predict the risk of HCC and its recurrence is urgently needed. Our studies showed that hepatic levels of γ-OHPdG, an endogenous adduct from lipid peroxidation, closely correlate with hepatocarcinogenesis in various independent animal models, including an obesity model. Importantly, we observed that higher levels of γ-OHPdG in liver specimens from HCC patients who underwent surgical resection were strongly associated with poor survival (p &lt; 0.0001) as well as with poor recurrence-free survival (RFS) (p = 0.007). These finding suggest that γ-OHPdG may serve as a prognostic biomarker of HCC and its recurrence. A non-invasive method to detect γ-OHPdG is highly desirable. There is a surge of interest in using circulating liver cells (CLCs) from patient blood to diagnose cancer stages and predict treatment outcomes, metastatic potential, and recurrence. We developed a non-invasive method for detecting and quantifying γ-OHPdG using CLCs from patient blood.
Method: The method was developed by following steps: (i) isolate CLCs from blood samples using a RosetteSep™ Human CD45 Depletion Cocktail and Ficoll Paque, (ii) detect γ-OHPdG by immunocytochemistry, (iii) quantify the images of γ-OHPdG-positive stained CLCs, and (iv) compare with that found in liver biopsies by immunohistochemistry.
Results: To determine the specificity of the anti-γ-OHPdG antibody developed previously in our laboratory, we showed that the proportion of γ-OHPdG-positively stained cells and staining intensity increased in HepG2 liver cancer cells upon acrolein treatment. Liver cells were co-stained with asialoglycoprotein receptor 1 (ASGPR1), a cell surface protein expressed solely on the surface of hepatic cells. CLC γ-OHPdG was quantified using Metamorph (BioVision)-based journal software, developed to quantify the percentage of ASGPR1-stained cells that also stain positively, and with what level of intensity, for γ-OHPdG. We then applied this method to 35 HCC patients’ blood samples. This method detects the number of ASGPR1 stained CLC and determines the cellular localization of γ-OHPdG, allowing the quantification of γ-OHPdG in these cells. While the nuclear γ-OHPdG reflects DNA damage, adducts in both the nucleus and the cytosol will be evaluated against IHC staining using biopsies and other clinical endpoints, such as disease stage and recurrence.
Conclusion: The CLC method descried above for detecting and quantifying γ-OHPdG can be potentially used to validate this adduct as a prognostic biomarker for predicting HCC risk and recurrence in clinical trials.
Citation Format: Monika Aggarwal, Zizhao Zhu, Sophie Gould, Peter Johnson, Samira Beheshtian, Hongbin Fang, Bhaskar Kallakury, Susette C. Mueller, Aiwu Ruth He, Fung-Lung Chung. Detection of γ-OHPdG in circulating liver cells and its potential use as a prognostic biomarker in patients with hepatocellular carcinoma recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6235.