Flow Cytometric Analysis of Asialoglycoprotein Receptor Expression Predicts Hepatic Functional Reserve after Hepatectomy

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1 ORIGINAL ARTICLE Flow Cytometric Analysis of Asialoglycoprotein Receptor Expression Predicts Hepatic Functional Reserve after Hepatectomy Zhang Ke, Jiang Li, Jia Zhe, Li Bao Liang, Lu Yan and Mu Yi ABSTRACT Objective: To validate a cheaper and more accessible flow cytometry-based method of assessing Asialoglycoprotein Receptor (ASGPR) expression for hepatic functional reserve. Study Design: A retrospective analysis. Place and Duration of Study: Beijing Ditan Hospital, Capital Medical University, Beijing, from January 2011 to October Methodology: Patients with Hepatocellular Carcinoma (HCC) undergoing major hepatectomy at Beijing Ditan Hospital, during the study period were retrospectively studied. The fraction of hepatocytes expressing ASGPR in liver tissues was assessed by flow cytometry. Patients were grouped according to the presence or absence of postoperative hepatic dysfunction. The correlation between ASGPR expression and pre-operative liver function parameters with the outcomes of hepatectomy were analyzed. Results: Fewer hepatocytes from patients with postoperative hepatic dysfunction expressed ASGPR [63.3 ( )] than from patients without postoperative hepatic dysfunction [72.4 ( ), p < 0.001]. Multiple logistic regression demonstrated ASGPR levels to be independently correlated with postoperative hepatic dysfunction (Odds ratio 3.34, 95% CI: , p < 0.001), and the Receiver Operating Characteristic (ROC) curve for prediction of postoperative liver dysfunction at 68.95% ASGPR + hepatocytes achieved a sensitivity of 100% and specificity of 90.6%. The ROC curve for prediction of postoperative liver failure related death at 58.53% ASGPR+ hepatocytes achieved a sensitivity of 100% and specificity of 99%. Conclusion: Flow cytometric assessment of ASGPR expression may be a useful predictor of liver dysfunction following major hepatectomy for HCC in Chinese patients. Key Words: Asialoglycoprotein receptor (ASGPR). Flow cytometry. Hepatocellular carcinoma. Liver failure. Hepatectomy. INTRODUCTION Assessment of hepatic functional reserve is critical in the pre-operative evaluation of patients undergoing hepatectomy. 1 The Child-Pugh classification is a simple system for grading liver function based on the easily measurable factors and has been considered a gold standard for more than four decades. 2 Regarding Child- Pugh criteria, not all variables may be assessed objectively such as the determination of the degree of ascites and hepatic encephalopathy. Additionally, some markers, such as albumin, can be altered by treatment. Therefore, the Child-Pugh score might not accurately reflect overall liver function. 3 Because this scoring system relies mainly on clinical manifestations and conventional tests for liver function, it likely reflects the Department of Hepatobiliary Surgery, Beijing Ditan Hospital, Capital Medical University, Beijing, China. Correspondence: Dr. Mu Yi, Department of Hepatobiliary Surgery, Beijing Ditan Hospital, Capital Medical University, Beijing , China. bjdtyywaike@126.com Received: April 14, 2014; Accepted: July 19, severity of liver damage and compensatory functions of liver cells, however, may not accurately predict the actual capacity of the hepatic reserve when encountering trauma, infection or surgery. 4 The Model for End-stage Liver Disease (MELD) score is based on a subset of variables which were shown to be significantly and independently correlated to the outcome by multivariate analysis. However, the MELD score also has limitations including the absence of clearly defined cut-off values for categorizing cirrhotic patients and the absence of validation in some clinical situations, 5 in addition, variables such as the creatinine and bilirubin can be adversely affected by renal injury and obstructive jaundice from tumor growth. 6 Several modifications of the MELD score have been proposed to include the contribution of hyponatremia, but currently the MELD score does not fully reflect the actual hepatic reserve of cirrhotic patients. 7 The Asialoglycoprotein receptor (ASGPR) is a receptor localized on hepatocytes that is involved in the clearance of glycoproteins containing terminal galactose residues from the circulation. 8 The level of ASGPR expression can serve as an objective biomarker of 820 Journal of the College of Physicians and Surgeons Pakistan 2014, Vol. 24 (11):

2 Flow cytometric measurement of ASGPR expression hepatic functional reserve. 9 Technetium- 99m -diethylenetriaminepentaacetic acid-galactosyl human serum albumin ( 99m Tc-GSA), an analog of asialoglycoproteins, has been employed as an estimate of liver function pre or post surgery. 10 However, only a few tertiary centers are able to perform 99m Tc-GSA scintigraphy in China, and the cost of this procedure limits widespread application in clinical practice. In this study, flow cytometric analysis of ASGPR level on the surface of hepatocytes, obtained from liver specimens of patients undergoing hepatectomy was evaluated as a marker of hepatic functional reserve. The objective of this study was to assess hepatic functional reserve and the risks of hepatectomy by analyzing the correlation ASGPR expression and preoperative liver function parameters with the outcomes of hepatectomy. METHODOLOGY One hundred and thirty-four patients with primary hepatocellular carcinoma (HCC) underwent partial hepatectomy in the surgical department of Beijing Ditan Hospital between January 1, 2011 and October 31, Patients undergoing major hepatectomy were included. Liver function before surgery was classified as Child-Pugh A in all patients. The study protocol was approved by the Institutional Review Board at the Beijing Ditan Hospital Affiliated to Capital Medical University, and written informed consent was obtained from each subject. All patients were diagnosed with HCC before treatment according to European Association for the Study of the Liver guidelines: 11 (a) demonstration of typical features of HCC with two imaging techniques or positive findings on one imaging study together with an alpha-fetoprotein level of more than 400 ng/ml (n=93) or (b) cytologic and/or histologic diagnosis of HCC (n=16). Liver biopsies were systematically performed when noninvasive criteria were not satisfied. Technical feasibility was established if residual liver volume with optimal blood inflow, blood outflow, and biliary drainage was expected to be sufficient. Surgical resection was carried out through a bilateral subcostal incision with the patient under general anesthesia. Intraoperative ultrasound was performed to evaluate the tumor burden, the liver remnant, and the possibility of a negative resection margin. Anatomic resection, in the form of segmentectomy as described by Hasegawa et al., was the preferred surgical method for liver resection. 12 The Pringle maneuver was routinely used, with clamping and unclamping times of 10 and 5 minutes, respectively, repeatedly throughout the entire procedure. Hemostasis of the surface of the raw liver was achieved with suturing. Postoperative hepatic dysfunction was defined as hyperbilirubinemia, or serum total bilirubin level over 5.0 mg/dl, and persistent ascites or pleural effusion. 13 All patients were followed for 3 months after surgery. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB), cholinesterases (CHE), serum albumin (ALB), prothrombin activity (PTA), international normalized ratio (INR) and blood platelet count (PLT), and MELD score were examined using standard procedures before surgery. Postoperative hepatic dysfunction was defined as hyperbilirubinemia, or serum total bilirubin level over 5.0 mg/dl, and persistent ascites or pleural effusion. 13 To measure indocyanine green retention rate at 15 minutes (ICGR15) before surgery, indocyanine green (ICG) was injected intravenously at a dose of 0.5 mg/kg body weight with a 15 minutes retention rate, measured by a photopiece applied to the fingertip (DDG-3300K; Nihon Kohden Corp., Tokyo, Japan) without blood sampling. 14 The histology of background liver disease was reviewed using surgical specimens after operation and the histological hepatic fibrosis was graded using the Ishak score. 15 Two or more liver segments containing the tumors were resected during hepatectomy. Because pathological changes of cirrhosis are not the same in different liver lobes, two liver tissue specimens were drawn from each liver segment for flow cytometric assessment of ASGPR expression, and the distance between two liver tissue specimens from the same liver segment was no less than 5 mm. In order to ensure the liver tissue specimens did not contain the tumor, the liver tissue specimens was extracted more than 5 mm away from the leading edge of tumor. Erythrocytes were removed from the liver tissue specimens by washing with Hank's Balanced Salt Solution (HBSS). The sampled liver tissue was then reduced into 1-2 mm 3 pieces and digested in 0.05% collagenase type-ii at 37 C for 10 minutes. The liver tissue specimens were filtered through a 200 µm mesh cell strainer. The digestion was stopped by addition of 2% FBS/DMEM. (ZuRui Biotechnology Co., Ltd., Shanghai, China) Following centrifugation at 1, 200 rpm for 5 minutes, the supernatant was discarded and the pellet was resuspended in 3-5 ml HBSS. Cell morphology and number were examined under a phase contrast microscope (Olympus, Tokyo, Japan) and cells were kept in x 10 6 / ml at 4 C µl of cell suspension was incubated with 5 µl FITC-labeled anti-asgr1 antibody (HyCult Biotechnology, Uden, Netherlands) at 4 C for 30 minutes. The cells were then washed twice, by centrifugation at 1,000 rpm for 5 minutes, using 1 ml HBSS. The final cell number was maintained at x 10 6 / ml. A mouse isotype IgG1, labeled with FITC, served as a negative control. The level of ASGPR expression on the surface of hepatocytes was detected on a flow cytometer Journal of the College of Physicians and Surgeons Pakistan 2014, Vol. 24 (11):

3 Zhang Ke, Jiang Li, Jia Zhe, Li Bao Liang, Lu Yan and Mu Yi (Beckman Coulter, Los Angeles, USA), and the average percentage of ASGPR + hepatocytes from each patient was used to calculate the final ASGPR value. All statistical analyses were performed using IBM SPSS software (IBM, Armonk, New York City, USA). ASGPR values are expressed as median (interquartile range). Dichotomous variables were compared using Fisher's exact test, and continuous variables were compared using the Mann-Whitney U-test. Multiple logistic regression using the stepwise method was used to evaluate independent predictors of postoperative hepatic dysfunction. Receiver Operating Characteristic (ROC) curve analysis was performed for predictive variables. The Area Under the Curve (AUC) and the corresponding values of sensitivity and specificity were used to extrapolate a cut-off value. All significance tests were two-tailed. Differences with a p-value < 0.05 were regarded as statistically significant. RESULTS One hundred and nine patients underwent surgical liver resection of two or more liver segments (major hepatectomy), 67 males and 42 females, with an average age of 49.4 years (range years). Eighty five patients (78%) were assigned to group A (absence of postoperative hepatic dysfunction), and 24 patients (22%) were assigned to group B (presence of postoperative hepatic dysfunction). All patients were followed for 3 months after surgery, during which period no cases were lost. Of the 24 patients in group B, 7 died of hepatic failure and associated complications within 40 days of hepatectomy. There were no statistically significant differences in age, sex, surgical duration, blood loss, hepatic hilum blocking time, types of hepatic resection, MELD score, ALT, AST, ALB, PTA, PLT, ICGR15 and background liver disease between the two groups (all p > 0.05), but TB, INR and CHE differed significantly between the two groups (all p < 0.05, Table I), and the percentage of ASGPR + hepatocytes detected by flow cytometry was significantly lower in patients with postoperative hepatic dysfunction (63.3 ( )), than patients without postoperative hepatic dysfunction [72.4 ( ), p < 0.001, Figure 1 and Table I]. Since TB, INR, CHE and ASGPR value differed significantly between the two groups, we assessed the ability of these factors to predict postoperative hepatic dysfunction, however, multiple logistic regression revealed that the ASGPR value was the only independent parameter correlated with postoperative hepatic dysfunction [OR 3.34 (95% CI: )] (p < 0.001, Table II). The optimal cut-off value of ASGPR for the prediction of postoperative hepatic dysfunction was 68.59%. The area under the curve was (95% CI: ), accompanied by a sensitivity of 100% and a specificity of 90.6% (Figure 2). There were no postoperative deaths in group A but 7 in group B. Among these 7 patients (6 men and one woman), 3 had right lobectomy, 2 right trisegmentectomy, one central bisegmentectomy and one left Table I: Demographic and clinical characteristics of patients. Group A Group B p-value (n=85) (n=24) Age (years) 49.0 ( ) 50.0 ( ) Gender Male 48 (56.5%) 19 (79.2%) Female 37 (43.5%) 5 (20.8%) Surgical duration (minutes) ( ) ( ) Blood loss (ml) ( ) ( ) The hepatic hilum blocking time (minutes) 24.0 ( ) 23.5 ( ) Background liver disease Ishak 5 59 (69.4%) 11 (45.8%) Ishak 6 26 (30.6%) 13 (54.2%) Operation Left lateral lobectomy 20 (23.5%) 3 (12.5%) Left lobectomy 13 (15.3) 5 (20.8%) Right bisegmentectomy 22 (25.9%) 4 (16.7%) Right trisegmentectomy 11 (12.9%) 3 (12.5%) Right lobectomy 10 (11.8%) 5 (20.8%) Central bisegmentectomy 9 (10.6%) 4 (16.7%) MELD score 5.0 ( ) 5.0 ( ) ALT (IU/L) 40.2 ( ) 45.1 ( ) AST (IU/L) 55.5 ( ) 46.9 ( ) TB (umol/l) 14.8 ( ) 21.6 ( ) <0.001 CHE (IU/L) ( ) ( ) <0.001 ALB (g/l) 38.9 ( ) 39.0 ( ) PTA (%) 78.9 ( ) 74.4 ( ) INR 1.42 ( ) 1.48 ( ) PLT (x10 9 /L) 89.0 ( ) 83.0 ( ) ICGR15 (%) 17.6 ( ) 20.4 ( ) ASGPR (%) 72.4 ( ) 63.3 ( ) <0.001 Table II: Multiple logistic regression analysis for predicting postoperative hepatic dysfunction. Odds ratio 95% CI p-value ASGPR to 6.02 <0.001 CHE to TB to INR to Figure 1: Histograms of hepatocyte ASGPR levels analyzed by flow cytometry. (A) A typical patient without postoperative hepatic dysfunction. (B) A typical patient with postoperative hepatic dysfunction. 822 Journal of the College of Physicians and Surgeons Pakistan 2014, Vol. 24 (11):

4 Flow cytometric measurement of ASGPR expression lobectomy. Their median age was 49.0 ( ) years. The background liver diseases diagnosed by histological analysis were 2 patients with Ishak 5 and 5 patients with Ishak 6. The mean time to death after hepatectomy was 26.5 ( ) days. The optimal cut-off value of ASGPR for the prediction of postoperative liver failure related death was 58.53%. The area under the curve was (95% CI: ), with a sensitivity of 100% and a specificity of 99% (Figure 3). Figure 2: Receiver Operating Characteristic curve of ASGPR for the prediction of postoperative liver dysfunction. Figure 3: Receiver Operating Characteristic curve of ASGPR for the prediction of postoperative liver failure related death. DISCUSSION The level of ASGPR expression on hepatocytes has been previously established as objective biomarker of hepatic functional reserve, and decreased ASGPR expression has been observed in patients with chronic liver disease. 16,17 In this study, the researchers sought to find a reliable alternative to the 99m Tc-GSA test for assessment of hepatic functional reserve as the cost and complexity of this procedure limits widespread clinical application in China. The authors utilized flow cytometry to measure ASGPR expression on the surface of hepatocytes in tumoradjacent liver tissue specimens from the liver segments excised during major hepatectomy. It was found that specimens from patients that suffered postoperative liver dysfunction and liver failure related death contained a lower fraction of ASGPR + hepatocytes. In this sample, conventional laboratory tests were also applied for pre-operative assessment of postoperative liver function and calculated the MELD score, a system for assessment of the severity of chronic liver disease, for each patient. The MELD scores of patients that suffered postoperative liver dysfunction did not differ significantly from the MELD scores of patients that did not suffer postoperative liver dysfunction, suggesting that the MELD scores were not a reliable predictor of hepatic functional reserve in this population, as has been observed elsewhere in multiple clinical settings. 6,7 The level of TB, INR and CHE did differ significantly between the two patient groups, but multiple logistic regression revealed only ASGPR levels to be independently correlated with postoperative hepatic dysfunction, and ROC curves indicated that ASGPR levels below 68.59% were a reliable predictor of postoperative liver dysfunction and ASGPR levels below 58.53% were a reliable predictor of postoperative liver failure related death. This validates the prior results as previously established by alternative methods of assessing ASGPR. 10 The conclusions are somewhat limited by the size and nature of the sample. We studied a small sample from a single academic center. These findings will need to be validated in a larger multicenter cohort. Furthermore, patients older than 65 years were not studied, nor were the predictive capacity of other parameters measured, such as the volume of the remaining liver, and the severity of the portal hypertension. 18 CONCLUSION Flow cytometric measurement of ASGPR may be a useful method for predicting postoperative liver dysfunction and hepatic functional reserve in this population than conventional laboratory tests of functional reserve. Flow cytometric measurement of ASGPR levels could be conducted with liver biopsies before surgery or intraoperative assessment, which may aid in the selection of appropriate treatments and management strategies in patients with liver tumors following resection. Acknowledgement: This work was supported by the Beijing Municipal Science and Technology Commission Capital Characteristic Clinical Application Research (No. Z ). REFERENCES 1. Golse N, Bucur PO, Adam R, Castaing D, Sa Cunha A, Vibert E. New paradigms in post-hepatectomy liver failure. J Gastrointest Surg 2013; 17: Journal of the College of Physicians and Surgeons Pakistan 2014, Vol. 24 (11):

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