Overexpression of Clusterin in Human Hepatocellular Carcinoma YUN KYUNG KANG, MD, SEONG WOO HONG, MD, HYUCKSANG LEE, MD, AND WOO HO KIM, MD Clusterin has been reported to play a signiﬁcant role in tumori- genesis, and its overexpression occurs in various human malignan- cies. We examine the clusterin overexpression in human hepatocel- lular carcinoma (HCC) and verify its clinical usefulness as a candidate biomarker by clinicopathologic and survival analysis. We examined clusterin overexpression immunohistochemically in 100 surgically resected HCCs using the tissue microarray method. A total of 89 HCCs exhibited clusterin overexpression, in 2 distinct staining patterns, cytoplasmic (n  35) and canalicular (n  54). Clusterin positivity demonstrated an inverse correlation with tumor cell apo- ptosis evaluated by the terminal deoxynucleotidyl transferase–medi- ated deoxyuridine triphosphate nick-end labeling assay (P  0.024). Within the clusterin-positive group, cytoplasmic overexpression had a positive correlation with tumor cell proliferative activity measured by the Ki-67 labeling index (P  0.003). HCCs demonstrating cytoplas- mic clusterin overexpression were associated with poor Edmondson’s histological grade and high TNM stage (P < 0.05). In the survival analysis, the cytoplasmic-positive group demonstrated an overall poorer prognosis than the canalicular-positive group, according to univariate and multivariate analysis (P < 0.05). In HCC, clusterin may play an important role in tumorigenesis and progression, correspond- ing to its subcellular localization. Cytoplasmic clusterin overexpres- sion could be a potential new prognostic marker for the aggressive- ness of HCC. HUM PATHOL 35:1340-1346. © 2004 Elsevier Inc. All rights reserved. Key words: hepatocellular carcinoma, apolipoprotein, tissue mi- croarray, immunohistochemistry, survival analysis. Abbreviations: ABC, avidin-biotin streptavidin-peroxidase com- plex; HCC, hepatocellular carcinoma; LI, labeling index; TAE, transarterial chemoembolization; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling. Clusterin, also known as apolipoprotein J (ApoJ), sulfated glycoprotein 2 (SGP-2), testosterone-repressed prostate message 2 (TRPM-2), glycoprotein III, se- creted protein 40,40 (SP-40,40), and complement lysis inhibitor (CLI), is a heterodimeric glycoprotein present in most animal tissues and body ﬂuids. 1,2 Clus- terin is implicated in various physiological processes, including lipid transport, reproduction, complement regulation, tissue remodeling, senescence, and cell– cell interaction. 2,3 It has also been reported to play a signiﬁcant role in stress response, 4 apoptosis, 5,6 and tumorigenesis. 2 Clusterin overexpression occurs in various human malignancies, including cancers of the prostate, 7 breast, 8 kidney, 9 urinary bladder, 10 pancreas, 11 and co- lon 12 and lymphoma. 13 The role of clusterin in neo- plastic transformation and progression has not yet been clearly elucidated; there are a number of controversial results, probably due to the diverse isoforms of clus- terin derived from posttranslational modiﬁcation. 2,14 Nevertheless, it is apparent that this protein plays a signiﬁcant role in the tumorigenesis of several human cancers. Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the third most common cancer in the Korean population. 15,16 The molecular mechanisms of hepatocarcinogenesis are still incompletely deﬁned, and the clinicopathologic and prognostic signiﬁcance of the genetic changes has not been schematically described. As part of a partici- pant study group in the 21st Century Frontier Program of the Functional Human Genome Project of Korea, we prepared a high-throughput tissue array set of livers, including surgically resected HCCs, nonneoplastic liv- ers, and fetal livers. We identiﬁed clusterin during the process of rapid screening for an expression analysis using the array set. Because clusterin expression has not been described in HCC before, we further exam- ined the relationships of clusterin overexpression with proliferation and apoptosis. We also tried to precisely examine the role of clusterin overexpression in hepa- tocarcinogenesis and progression by means of clinico- pathologic and survival analysis. MATERIALS AND METHODS Tissue Samples A total of 116 HCCs resected at Inje University Seoul Paik Hospital between 1991 and 2001 were included in the tissue array blocks. All tissues were routinely ﬁxed in 10% buffered formalin and embedded in parafﬁn blocks. Screening Liver Tissue Array Core tissue biopsy specimens (2 mm in diameter) were taken from individual donor blocks and arranged in a new recipient parafﬁn block (tissue array block) using a trephine apparatus (Superbiochips Laboratories, Seoul, Korea). The resulting tissue array block (TA79) contained 30 HCCs (se- lected from the 116 HCCs), 10 cirrhotic liver tissue speci- From the Departments of Pathology and Surgery, Inje University, Seoul Paik Hospital, Seoul, Korea and the Department of Pathology, Seoul National University College of Medicine, Seoul, Korea. Ac- cepted for publication July 29, 2004. Supported by 21st Century Frontier Functional Human Genome Project grant FG03-11-02 from the Ministry of Science and Technol- ogy of Korea. Address correspondence to Woo Ho Kim, Department of Pathol- ogy, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-799, Korea. 0046-8177/$—see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2004.07.021 1340