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Expression of GOLM1 Correlates with Prognosis in Human Hepatocellular Carcinoma

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Expression of GOLM1 Correlates with Prognosis in Human Hepatocellular Carcinoma Ann Surg Oncol DOI 10.1245/s10434-013-3101-8 ORIGINAL ARTICLE – TRANSLATIONAL RESEARCH AND BIOMARKERS Expression of GOLM1 Correlates with Prognosis in Human Hepatocellular Carcinoma Ming-Huang Chen, MD, PhD1,2, Yi-Hua Jan, PhD3,4, Peter Mu-Hsin Chang, MD, PhD1,2, Yung-Jen Chuang, PhD4, Yi-Chen Yeh, MD5, Hao-Jan Lei, PhD6, Michael Hsiao, PhD3, Shiu-Feng Huang, PhD7, Chi-Ying F. Huang, PhD2,8, and Gar-Yang Chau, MD, PhD6 1Division of Hematology and Oncology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan; 2Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan; 3Genomics Research Center, Academia Sinica, Taipei, Taiwan; 4Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan; 5Department of Pathology and Laboratory Medicine, Taipei Veterans General Hospital, Taipei, Taiwan; 6Division of General Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan; 7Institute of Molecular and Genomic Medicine, National Health Research Institute, Zhunan, Taiwan; 8Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei, Taiwan ABSTRACT liver tissues (p \ 0.01). After a median follow-up of 51 Background. Serum Golgi membrane protein 1 (GOLM1) months, multivariate analysis showed that portal vein is a novel biomarker for hepatocellular carcinoma (HCC). invasion (hazard ratio [HR], 1.515; 95 % confidence However, few studies have investigated the relationship interval [95 % CI], 1.008–2.277; p = 0.046) and high between GOLM1 protein expression and clinicopathologic GOLM1 protein expression (HR, 1.696; 95 % CI, features in HCC patients. The aim of this study was to 1.160–2.479; p = 0.006) were independent prognostic investigate the expression of GOLM1 in human HCC and factors for poor overall survival. High GOLM1 protein its correlation with clinicopathologic parameters. expression still significantly correlates with worse overall Methods. Clinicopathologic data were obtained through a survival as well as disease-free survival in the validation detailed retrospective review of the medical records of 193 cohort (p \ 0.001 and p = 0.002). patients with HCC who had undergone surgical resection Conclusions. Overexpression of GOLM1 is associated between 1990 and 2006 at the Taipei Veterans General with poor prognosis in human HCC. Hospital. Another 120 HCC tissue samples provided by the Taiwan Liver Cancer Network were used as validation cohort. Immunohistochemical staining was used to deter- Hepatocellular carcinoma (HCC) is the most common mine the expression of GOLM1 in archived formalin-fixed, liver malignancy and one of the leading causes of cancer paraffin-embedded tissue specimens. death worldwide. HCC is an aggressive cancer with a 5-year Results. GOLM1 expression was significantly higher in survival rate below 12 %.1 Surgical resection is the main resected HCC tumor tissues than in corresponding normal form of therapy for HCC; however, this is not a viable option for many patients because of the difficulty in diagnosing early-stage HCC.2,3 Moreover, high recurrence rates of up to 70 % are observed after curative resection of HCC.4 Ming-Huang Chen and Yi-Hua Jan contributed equally to this work. Diagnostic imaging is currently the best approach for the early detection of relapsed HCC and future localized Ó Society of Surgical Oncology 2013 treatment plans.2,5 Alpha fetoprotein (AFP), a serum bio- First Received: 5 February 2013 marker, has been widely used as a surveillance tool for HCC recurrence.6,7 However, serum AFP is not an ideal C.-Y. F. Huang, PhD screening method because of the low sensitivity and e-mail: [email protected] specificity.8 Recently, several studies confirmed that serum G.-Y. Chau, MD, PhD Golgi membrane protein 1 (GOLM1) is a novel and e-mail: [email protected] M.-H. Chen et al. promising biomarker for early HCC detection and follow- panel of carcinogenesis progression from normal liver, cir- up after resection.9–12 GOLM1, a type II transmembrane rhosis, dysplasia to HCC in background of HCV infection. protein of the Golgi cisternae, is typically expressed in the The raw intensities in *.CEL files were normalized by robust epithelial cells of normal human tissues.13,14 Serum multichip analysis, and fold-change analysis was performed GOLM1 has been reported to be a more reliable biomarker using GeneSpring GX11 (Agilent Technologies). Differen- for the early diagnosis of HCC than current biomarkers tially expressed genes were identified by applying a twofold such as AFP.11 Although overexpression of GOLM1 pro- change cutoff with p \ 0.01 as threshold for significance. tein should first be observed in the cytoplasm before protein secretion by exocytosis, few studies have investi- Tissue Microarray Construction gated the value of GOLM1 protein expression as a and Immunohistochemical Staining prognostic biomarker in HCC.15 In the present study, we analyzed GOLM1 expression in resected human HCC Hematoxylin and eosin staining of all 193 samples was tumors by immunohistochemical staining and investigated reviewed by a pathologist. The HCC tissue microarrays the relationship of GOLM1 expression with clinicopatho- were constructed by obtaining 3 1-mm-diameter cores from logic parameters. In our study, a relatively homogenous each tumor. Archived formalin-fixed, paraffin-embedded population from a single institute with standardized treat- tumor specimens were used for immunohistochemical ment and medical care and a long-term follow-up were staining. Briefly, specimens were cut into 5-lm sections, used to further clarify the relationship between GOLM1 dewaxed in a 60 °C oven, deparaffinized in xylene, rehy- expression and HCC prognosis. In addition, the prognostic drated through serial dilutions of alcohol, and washed in role of GOLM1 expression was further validated in another phosphate-buffered saline (pH 7.2). A polyclonal anti- validation cohort. GOLM1 antibody (dilution 1:200; ABNOVA) was used for immunohistochemical staining. Immunohistochemical staining was performed on the fully automated Bond-Max MATERIALS AND METHODS autostainer (Leica Microsystems) using onboard, heat- induced antigen retrieval in citrate buffer (ER1 protocol) Patient Clinicopathologic Data for 20 min and VBS Refine polymer detection system (Leica Microsystems). Immunohistochemical staining was This study has been approved by the Ethics Committee of developed using diaminobenzidine (Leica Microsystems) the Taipei Veterans General Hospital (201010021IC). Clini- as a chromogen and hematoxylin as a counterstain. copathologic data were obtained through a detailed retrospective review of the medical records of 193 patients with HCC who had undergone surgical resection between 1990 and Scoring of GOLM1 Expression 2006 at the Taipei Veterans General Hospital. Patients who suffered from tumor recurrence could receive surgery, tran- The intensity of staining was scored using a 4-tier scale sarterial chemoembolization, and percutaneous ethanol and defined as: 0, no staining; 1?, weak staining; 2?, injection before 2002. After 2002, patients had other thera- moderate staining; 3?, strong staining. The extent of peutic choices, such as radiofrequency tumor ablation and liver staining was scored by the percentage of positive cells transplantation. No patients in this study received sorafenib (0–100 %). The final IHC scores (0–300) were obtained by because the drug was unavailable from 1990 to 2006. staining intensity score multiplied by the percentage of The HCC tissue samples of validation cohort were positive cells. All cases were divided into two groups provided by the Taiwan Liver Cancer Network (TLCN). according to the final IHC scores. High GOLM1 expression The TLCN is funded by the National Science Council to level was defined as a score more than and including 150 provide primary liver cancer tissue and associated clinical itself and a score less than 150 was defined as low GOLM1 information. The use of the 120 HCC tissues in this study expression. All of the immunohistochemical staining was approved by our Institutional Review Board and the results were reviewed and scored independently by two TLCN User Committee. pathologists. In the case of discrepant results from the same slide, a final consensus was obtained after a discussion HCC Microarray Dataset Analysis between the two pathologists. In order to further validate our data, we used an automated image analysis system GSE6764 microarray datasets were collected from Gene (Aperio Technology, Vista, CA), which uses a color Expression Omnibus (GEO) database (http://www.ncbi. deconvolution algorithm to visualize GOLM1 protein nlm.nih.gov/geo/query/acc.cgi?acc=GSE6764).16 The series expression in HCC.17,18 Quantification of immunohisto- contain gene expression profiles of 75 samples that represent a chemical staining was performed with color translation and GOLM1 Expression Predicts Prognosis in HCC an automated thresholding algorithm from Aperio Tech- GOLM1 protein expression and the clinicopathologic features nology (Fig. 1). of HCC are summarized in Table 1. Statistical Analysis Identification of GOLM as 1 of the Potential Biomarker from HCC Microarray Analysis Statistical analysis was performed using SPSS (version 15.0; SPSS Inc., Chicago, IL). Fisher exact and chi-squared To identify potential early HCC markers, we analyzed HCC tests were used to assess the association between GOLM1 microarray datasets from GSE6764 that contained 10 normal expression and the various clinicopathologic
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