检测肝细胞癌患者循环肿瘤细胞中的γ-羟基脱氧鸟苷作为复发的潜在预后生物标志物

Detection of γ-OHPdG in Circulating Tumor Cells of Patients With Hepatocellular Carcinoma as a Potential Prognostic Biomarker of Recurrence.

作者信息

Aggarwal Monika, Kuo Mark, Zhu Zizhao, Gould Sophie, Zhang Kevin, Johnson Peter, Beheshtian Samira, Kuhlman Laura, Zhao Zijun, Fang Hongbin, Kallakury Bhaskar, Creswell Karen, Mueller Susette, Kroemer Alexander, He Aiwu Ruth, Chung Fung-Lung

机构信息

Department of Oncology, Lombardi Comprehensive Cancer Centre, Georgetown University, Washington, District of Columbia.

MedStar Georgetown Transplant Institute, MedStar Georgetown University Hospital and the Center for Translational Transplant Medicine, Georgetown University Medical Center, Washington, District of Columbia.

出版信息

Gastro Hep Adv. 2024 Apr 24;3(6):809-820. doi: 10.1016/j.gastha.2024.04.006. eCollection 2024.

DOI:10.1016/j.gastha.2024.04.006

PMID:39280920

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11401592/

Abstract

BACKGROUND AND AIMS

Blood-based biomarkers for hepatocellular carcinoma (HCC) and its recurrence are lacking. We previously showed that hepatic γ-hydroxy-1, -propano-2'-deoxyguanosine (γ-OHPdG), an endogenous DNA adduct derived from acrolein by lipid peroxidation, increased during hepatocarcinogenesis. Additionally, higher hepatic γ-OHPdG from HCC patients after surgery were strongly associated with poor survival ( < .0001) and recurrence-free survival ( = .007) (Fu et al, Hepatology, 2018). These findings suggest that γ-OHPdG is a potential prognostic biomarker for HCC and its recurrence. To attain the goal of using γ-OHPdG as a biomarker in future preventive and therapeutic trials, we developed a blood-based method to detect γ-OHPdG in circulating liver tumor cells from HCC patient blood.

METHODS

We first established the specificity of anti-γ-OHPdG antibody by determining its dose-response in HepG2 cells treated with acrolein. Then, HepG2 cells in spiked blood of healthy volunteers and circulating tumor cells (CTCs) from 32 HCC patients were isolated using a RosetteSep CD45 Depletion Cocktail and Ficoll Paque. The HCC CTCs identified with anti-asialoglycoprotein receptor 1, a surface protein expressed solely in hepatocytes, were stained with an anti-γ-OHPdG antibody. The number of total HCC CTCs and γ-OHPdG-positive CTCs, as well as the staining intensity, were quantified using MetaMorph software. As an initial effort toward its clinical application, we also evaluated γ-OHPdG in CTCs from these patients along with certain clinical features.

RESULTS

The γ-OHPdG antibody specificity was demonstrated by an acrolein concentration-dependent increase of γ-OHPdG-positive HepG2 cells and the intensity of γ-OHPdG staining. The recovery of HepG2 cells from spiked blood was ∼50-60%, and the positivity rate of CTCs in blood from 32 patients with advanced HCC was 97%. The MetaMorph analysis showed a wide variation among patients in total number of CTCs, γ-OHPdG positivity, and staining intensity. Statistical analysis revealed that γ-OHPdG in CTCs of these patients appears to be associated with multifocality and poor differentiation.

CONCLUSION

A blood-based method was developed and applied to HCC patients to evaluate the potential of γ-OHPdG in CTCs as a prognostic biomarker.

摘要

背景与目的

肝细胞癌（HCC）及其复发的血液生物标志物尚为空白。我们之前发现，肝脏γ-羟基-1, -丙基-2'-脱氧鸟苷（γ-OHPdG），一种由脂质过氧化产生的内源性DNA加合物，在肝癌发生过程中增加。此外，肝癌患者术后肝脏中较高水平的γ-OHPdG与较差的生存率（<0.0001）和无复发生存率（=0.007）密切相关（Fu等人，《肝脏病学》，2018年）。这些发现表明γ-OHPdG是HCC及其复发的潜在预后生物标志物。为了在未来的预防和治疗试验中实现将γ-OHPdG用作生物标志物的目标，我们开发了一种基于血液的方法来检测HCC患者血液中循环肝肿瘤细胞中的γ-OHPdG。

方法

我们首先通过测定抗γ-OHPdG抗体在用丙烯醛处理的HepG2细胞中的剂量反应来确定其特异性。然后，使用红细胞吸附分离CD45去除鸡尾酒和Ficoll Paque从健康志愿者的加标血液和32例HCC患者的循环肿瘤细胞（CTC）中分离出HepG2细胞。用抗去唾液酸糖蛋白受体1（一种仅在肝细胞中表达的表面蛋白）鉴定的HCC CTC用抗γ-OHPdG抗体染色。使用MetaMorph软件对总的HCC CTC和γ-OHPdG阳性CTC的数量以及染色强度进行定量。作为其临床应用的初步尝试，我们还评估了这些患者CTC中的γ-OHPdG以及某些临床特征。

结果

γ-OHPdG阳性HepG2细胞的数量和γ-OHPdG染色强度随丙烯醛浓度的增加而增加，证明了γ-OHPdG抗体的特异性。从加标血液中回收的HepG2细胞约为50 - 60%，32例晚期HCC患者血液中CTC的阳性率为97%。MetaMorph分析显示，患者之间的CTC总数、γ-OHPdG阳性率和染色强度存在很大差异。统计分析表明，这些患者CTC中的γ-OHPdG似乎与多灶性和低分化有关。