Detection of DNA adduct γ-OHPdG in circulating tumor cells (CTCs) and its use as a prognostic biomarker in patients with hepatocellular carcinoma (HCC).

Abstract

594 Background: A blood-based biomarker to predict the risk of hepatocellular carcinoma (HCC), and its recurrence is needed. Previously, we showed that γ-OHPdG, a mutagenic DNA adduct formed by lipid peroxidation, in liver biopsies from HCC patients, as detected by IHC, is inversely associated with overall survival (p<0.0001) and recurrence-free survival (p<0.007) after surgical resection. This finding suggests that γ-OHPdG may serve as a prognostic biomarker of HCC and its recurrence. A non-invasive method to detect γ-OHPdG is needed. Liquid biopsy is preferred over tissue biopsy because it is non-invasive, allows repeated sampling, lower risk to patient, and lower medical care cost. We developed a non-invasive method for detecting and quantifying γ-OHPdG using CTCs from HCC patient. Methods: The method involved following steps. First, CTCs from blood samples were isolated using a RosetteSep CD45 Depletion Cocktail and Ficoll Paque-based method. Isolated cells were identified as liver CTCs using asialoglycoprotein receptor 1 (ASGPR1), a cell surface protein expressed solely on the surface of hepatic cells, and γ-OHPdG by immunocytochemistry. The percentage of ASGPR1 and γ-OHPdG-positive stained CTCs and γ-OHPdG staining intensity was quantified using Metamorph software. The staining intensities in livers CTCs will be compared with that found in liver biopsies by IHC. Results: To determine the sensitivity and specificity of testing -γ-OHPdG level in circulating tumor cells using an-anti γ-OHPdG antibody, we showed that the proportion of γ-OHPdG-positively stained cells and staining intensity increased in HepG2 liver cancer cells upon acrolein treatment at increasing concentration. Furthermore, when the HepG2 was used to spike blood of healthy volunteers’, the recovery rate of γ-OHPdG positivity was > 50-60%. CTCs from 32 HCC patients, detected at a positivity rate of ~97%, were tested for γ-OHPdG levels using the method developed in this project. The clinical factors including demographics, risk factors, alpha-fetoprotein (AFP), HCC size, multifocality, vascular invasion, extrahepatic metastasis have been correlated with the levels of γ-OHPdG in CTCs. Conclusions: The CTC method developed for detecting and quantifying γ-OHPdG warrant validations as a prognostic biomarker for predicting HCC risk and recurrence in clinical trials.