Annexin A2 Versus a-fetoprotein (AFP) as an Efficient Diagnostic Serum Marker for Hepatocellular Carcinoma

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1 Med. J. Cairo Univ., Vol. 85, No. 6, September: , Annexin A2 Versus a-fetoprotein (AFP) as an Efficient Diagnostic Serum Marker for Hepatocellular Carcinoma EHAB M. REYAD, M.D. 1 ; EHSAN H. ABD EL-BARI, M.D. 2 ; SELIM W. MORCOUS, M.D. 3 and SAHAR Z. MAKLAD, M.D. 4 The Departments of Clinical & Chemical Pathology 1, Pathology 2, Diagnostic Radiology 3 and Hepatology & Tropical Medicine 4, National Hepatology & Tropical Medicine Research Institute, Cairo, Egypt Abstract Background: The burden of hepatocellular carcinoma (HCC) has been increasing in Egypt due to high prevalence of HCV in Egypt. Early detection of HCC is difficult due to lack of reliable markers. Objective: This work was designated to assess the role of annexin A2 versus the widely used AFP for detection of HCC, especially in the early stage of the disease. Patients and Methods: This study included 24 patients with chronic hepatitis C, 26 patients with cirrhosis, 50 HCC patients and 20 healthy controls. All participants performed thorough assessment and laboratory investigations. Serum level of annexin A2 was detected using ELISA. Clinicopathological characteristics of circulating annexin A2 expression were analyzed, and its diagnostic efficiency in HCC was evaluated versus AFP. Results: We found that annexin A2 was significantly increased in the sera of HCC patients (median, 29.8ng/mL) compared with the cirrhotic (median, 13.8ng/mL, p=0.03), chronic hepatitis C (median, 10.8 ng/ml, p<0.001) and healthy controls (median, 11.2ng/mL, p<0.001). Importantly, annexin A2 levels in early stage HCC cases were not significantly different from that of cirrhotic patients (p=0.08) and late stage HCC cases (median, 30.7ng/mL, p=0.07). Area under the receiver-operating characteristic curve was 0.75 for annexin A2 and 0.83 for AFP. Combining detection of two markers could not improve the diagnostic efficiency for HCC. Serum annexin A2 expression in HCC patients was associated with AFP level (p<0.001), but was not correlated with patient sex, age, tumor size or tumor BCLC staging system. Conclusion: Annexin A2 may not be a good diagnostic biomarker for HCC. Key Words: AFP Annexin A2 Hepatocellular carcinoma Marke. Introduction PRIMARY liver cancer is the sixth most commonly occurring cancer in the world and the second Correspondence to: Dr. Ehab M. Reyad, The Department of Clinical & Chemical Pathology, National of Hepatology and Tropical Medicine Research Institute, Cairo, Egypt most deadly cancer worldwide [1]. Globally, the most common histology is hepatocellular carcinoma (HCC) [2]. Hepatitis C virus (HCV) is one of the major risk factors that contribute to HCC development by inducing an inflammatory, fibrogenic, and carcinogenic tissue microenvironment in the liver. Dissimilar to other solid tumors, the combination of inflammation and cirrhosis makes the early detection and prognostic assessment of HCC much more problematic [3]. In Egypt, the increased incidence of HCC, which is approximately 4.7% of chronic liver disease patients [4] is attributed to the high prevalence of HCV infection which was estimated nationally at 14.7% [5]. At the present time, the primary surveillance methods for patients with cirrhosis are combination of serum a-fetoprotein (AFP) level and hepatic ultrasonography with some limitations [6].However, the most commonly used marker AFP does not yield satisfactory results in the early diagnosis of HCC, particularly small hepatic tumors with low AFP expression. These false negative results limit its universality in HCC diagnosis [7]. The development of cellular and molecular techniques have led to discovery and utilization of several novel early detection biomarkers with potential diagnostic value, including glycoprotein antigens, cytokines, enzymes and isoenzymes, growth factors and their receptors as well as related genes [8]. By reasons of convenience, noninvasiveness and inexpensiveness, serum tumor markers are the most effective method for detecting malignant tumors. Annexin A2, a member of annexin family of cytosolic Ca2+-binding proteins localized at the cell surface of endothelial cells, macrophages and various types of tumor cells. It plays multiple roles in various biological processes, including phagocytosis, fibrinolysis and angiogenesis and apoptosis 2389

2 2390 Annexin A2 Versus a-fetoprotein (AFP) [9]. Moreover, it has been reported that interactions between annexin A2 and its binding proteins play an important role in the tumor microenvironment and act together to enhance cancer metastasis [10]. The main issue raised by this work is to assess annexin A2 versus AFP as a diagnostic serological marker for detection of HCV-related HCC, especially in the early stage of the disease. Patients and Methods A case control study was performed from December, 2014 till December, 2015 in National Hepatology and Tropical Medicine Research Institute (NHTMRI) and included 100 patients who presented to the outpatient clinic of the NHTMRI, Cairo, Egypt. Their ages ranged from 20 to 74 years old for both sexes. Signed informed consent was obtained from each patient. The present study was approved by the corresponding Ethical Committee and conducted in accordance with the ethics principles of Declaration of Helsinki. The diagnosis of HCV infection was confirmed by the presence of ant-hcv antibodies and HCV RNA by polymerase chain reaction (PCR). Patients were classified into 3 groups: Group (l) included patients with chronic viral hepatitis C (n=24), group (II) patients with HCV-related cirrhosis without evidences of HCC (n=26). The presence of cirrhosis was diagnosed by clinical, laboratory and ultrasonographic features cirrhosis. Group (III) included 50 treatment-naive patients who fulfilled the diagnostic criteria of HCC by the American Association for the Study of Liver Disease (AASLD) guidelines [11]. The diagnosis of focal lesions was originally detected by ultrasonography. We used dynamic multiphase contrast-enhanced CT-scan to confirm the presence of arterial enhancement of a nodule with subsequent washout in the portal venous or delayed phases. If lesions showed atypical findings, confirmatory dynamic contrast-enhanced MRI was the following step because MR imaging is more sensitive than CT in the diagnosis of HCC (81% vs 68%) [12]. No patients needed to be biopsied. Blood samples were collected prior to initiation of any HCC treatment. HCC patients were subclassified according to the previously described criteria of Barcelona Clinic Liver Cancer staging (BCLC) into early and late substages of HCC [13]. Of the 50 HCC cases, 20 (40%) was categorized as early stage (BCLC 0 and A) and 30 (60%) as late stage (BCLC B and C) [14]. In addition, 20 apparently healthy persons (group IV) were included as a control group. They had normal liver biochemical tests, negative viral hepatitis markers and normal ultrasonic examination. Serum samples of control group were tested for annexin A2 to set up the normal reference range. The clinically acceptable normal serum AFP was defined as <20ng/mL. Any patient with history of cancers other than HCC, diabetes mellitus, cardiac or renal diseases was excluded from the study [15]. Five ml of venous blood were withdrawn from all participants, then the sera were separated and divided into two aliquots, one for the blood biochemical tests which was assayed on the same day of sample collection, the second for AFP and annexin A2 were stored at 80 C. Methods: All subjects underwent full history taking, thorough clinical examination including general and local abdominal examination, and Child-Pugh score evaluation [16]. Laboratory investigations included aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin, serum albumin. All were performed on Olympus AU400 Chemistry Analyzer. Serum AFP level was measured by a commercially LIAISON analyzer based on the Chemiluminescence Immunoassay (CLIA) technology (DiaSorin, USA). Serum annexin A2 were detected using a human double-antibody sandwich ELISA kit (Uscan Life Science Inc., USA) according to the manufacturer s instructions. Wells were precoated with a monoclonal antibody to annexin A2. To each well was added 100µL of serum sample or standard separately, and soon after, 100µL of detection reagent A working solution was added for 1h at 37 C, followed by aspiration and washing. Then, 1 00µL of detection reagent B working solution was added for 30min at 37 C followed by 5 wash processes. Subsequently, 90µL of substrate solution was added for 25min at 37 C. Ultimately, 50µL of stop solution was added, and absorbance was read at 450nm on STATFAX 303 Plus Microstrip, Awareness Technology, Inc.USA. The results were calculated from standard curve (recombinant human ANXA2; range, ng/mL). Each sample was measured in duplicate and the mean values were taken. Statistical analysis: Statistical Package for Social Science (SPSS, Chicago, IL , USA) program version 17.0 was used for data analysis. Mean and standard deviation (SD) or median and inter-quartile range

3 Ehab M. Reyad et al (IQR) were estimates of quantitative data while frequency percentage were estimates of qualitative data. Differences in clinical and biochemical characteristics were tested by Student s t-test or Mann- Whitney test for quantitative data and by chi-square test for qualitative data. p-value <0.05 was considered statistically significant. Kruskal-Wallis H Test for non-parametric data was used to compare the difference between serum level of annexin A2 and AFP between different studied groups. A Spearman's rank correlation was performed to analyze linear association between serum annexin A2 and serum AFP values in cases with HCC. Receiver operator characteristic (ROC) curve was drawn for prediction of cutoff values for annexin A2 level, AFP level and suggested values for both markers. Results The studied subjects comprised 120 individuals. The patient characteristics are shown in Table (1). Annexin A2 were significantly increased in the sera of HCC patients compared to cirrhosis patients, chronic hepatitis and healthy controls (median, 29.8 vs. 13.8; p=0.03, 10.8; p<0.001, and 11.2ng/ ml; p<0.001, respectively). However, there was no significant difference between serum annexin A2 levels of normal controls and those with hepatitis and cirrhosis backgrounds ( p=0.5 and p=0.06, respectively). Similarly, there was no significant difference between hepatitis and cirrhotic patients (p=0.2). Similar to the annexin A2 results, serum AFP levels were significantly higher in the HCC group compared to cirrhosis, hepatitis, and healthy control groups (median, 49.8 vs. 12.9; p<0.001, 9.6; p<0.001, and 5.3ng/mL; p<0.001, respectively) (Table 2). Compared with control group, AFP levels in hepatitis and cirrhosis groups also were significantly higher (p=0.02 and p<0.001, respectively). However, there was no difference between serum AFP levels of cirrhotic patients and those of hepatitis group (p=0.5). Table (1): Clinical characteristic of individuals enrolled in the study. Characteristic Chronic viral hepatitis (I) (n=24) Cirrhosis (II) (n=26) HCC (III) (n=50) Controls (IV) (n=20) Age (years) 38.7± ± ± ± 14.1 Male/female, n 13/11 12/14 32/18 11/9 Size of focal lesion, n: <2 cm cm 21 >5 cm 17 BCLC stage, n: Early 20 Late 30 PVT, n 11 AFP ( 20 ng/ml), n Age is presented as mean ± SD. HCC: Hepatocellular carcinpma, BCLC: Barcelona Clinic Liver Cancer staging, PVT: Portal vein tumor thrombus, AFP: α-fetoprotein. Table (2): Serum annexin A2 and α-fetoprotein levels in studied groups. Group n Median (IQR) p-value AFP ng/ml: Chronic hepatitis a (25.6) <0.001 Liver cirrhosis a (34.3) HCC (161) Healthy controlsb (3.95) Annexin A2 ng/ml: Chronic hepatitis a (16.4) <0.001 Liver cirrhosis a (33.95) HCC (34.4) Healthy controls a (7.4) Results presented as median (inter-quartile range). The difference between groups having the same letter is not statistically significant.

4 2392 Annexin A2 Versus a-fetoprotein (AFP) 250 (A) 4000 (B) Annexin (A2) ng/ml AFP ng/ml Chrenic viral Cirrhosis HCC Controls Chrenic viral Cirrhosis HCC Controls hepatitis hepatitis Fig. (1): Simple boxplot displaying serum annexin A2 level (A) and serum AFP level (B) in different studied groups. 0 Importantly, serum annexin A2 levels were significantly increased in the early stage HCC patients (graded BCLC 0-A) compared with hepatitis and control groups (median, 28.1vs. 10.8; p=0.003 and vs. 11.2; p<0.001, respectively) (Table 3). It is noteworthy that annexin A2 levels in early stage HCC cases were statistically similar to that of cirrhosis group (median, 13.8ng/mL, p=0.08) and late stage HCC cases (graded BCLC B-C) (median, 30.7ng/mL, p=0.7) (Table 4). In contrast to the annexin A2 results, AFP levels for early stage HCC cases were significantly higher compared to hepatitis, cirrhosis and control groups (median, 25.2 vs. 9.6; p=0.02, 12.9; p=0.04, and 5.3ng/mL; p<0.001, respectively) (Table 3). Furthermore, patients with early stage HCC had significantly lower AFP values than those with late stage HCC (median, 91.6ng/mL, p=0.002) (Table 4). Table (3): Comparison of serum annexin A2 and AFP concentration in early HCC patients and other groups of patients. Number Annexin A2 (ng/ml) AFP (ng/ml) Median (IQR) p Median (IQR) p Early HCC (25.3) (31.6) 0.04 Cirrhosis (33.95) 12.9 (34.3) Early HCC (25.3) (31.6) 0.02 Chronic hepatitis (16.4) 9.6 (25.6) Early HCC (25.3) (31.6) Controls (7.4) 5.3 (3.95) The association between the biomarkers and various clinicopathologicl features of HCC were shown in (Table 4). Both annexin A2 and AFP abundance were not age, gender, serum ALT level and serum albumin concentration dependent in HCC patients (p>0.05 for all). In contrary, the higher levels of serum annexin A2 and AFP expression were affiliated with the occurrence of portal vein tumor thrombus (p=0.02 and p=0.006, respectively). Assessment of AFP levels according to the tumor size, revealed that, there was significant elevation of AFP among patients with HCC lesions >5cm compared to those with smaller ones (2 to 5cm ones and <2cm ones) (median, 307 vs and 21.7ng/mL; p=0.01 for both, respectively). In addition, AFP values were correlated with serum bilirubin levels in HCC patients (p=0.03). In con- trast, no significant association between serum annexin A2 levels and tumor size (p=0.4) as well as serum bilirubin concentrations (p=0.7). Taken together, elevations of annexin A2 were affiliated only with the occurrence of portal vein tumor thrombus, whereas, AFP values were elevated along with the increase of serum bilirubin and tumor size and with the occurrence of portal vein tumor thrombus. Using 20ng/mL as a clinically acceptable threshold for AFP, we found that 26% (13/50) of the studied HCC cases were AFP-negative and 74% (37/50) AFP-positive. Moreover, AFP-negative HCC patients had significantly lower annexin A2 levels compared to the AFP-positive HCC cases (median, 20 vs. 37.8ng/mL; p=0.00 1).

5 Ehab M. Reyad et al Table (4): Serum annexin A2 and AFP levels and their clinical significance in HCC patients. Parameter n Annexin A2 levels (ng/ml) AFP levels (ng/ml) Median (IQR) p-value Median (IQR) p-value Age (years): <_ (44.6) (314.1) 0.7 > (24.8) 38.8 (101.9) Gender: Male (30.7) (225.4) 0.9 Female (52.9) 57.6 (166.9) S.bilirubin (mg/dl): < (27.7) (49.3) 0.03 * > (43.2) 73.2 (314) Serum ALT (U/L): <_ (31.5) (91) 0.4 > (41.1) 57.4 (332.6) Serum Albumin (g/dl): < (48.5) (266.7) 0.2 > (29) 34.5 (103.4) Size of focal lesion: <2cm (15) (34.6) 0.01 * 2-5 cm (33.1) 68.2 (85.8) >5cm (49.2) 307 (747.4) BCLC stage: Early (25.3) (31.6) 0.002* Late (45.8) 91.6 (341.1) PVT: No (29.2) 0.02* 37.5 (73) 0.006* Yes (63.6) 350 (869.2) Results presented as median (interquartile range). p<0.05 is considered significant. AFP p=0.000 r= Anexin A2 Fig. (2): Spearman correlation for annexin A2 and AFP in cases with HCC. Evaluation of serum annexin A2 level for HCC diagnosis: Receiver Operating Characteristics (ROC) curve was used for evaluating the clinical performance characteristics of annexin A2 in relation to AFP in HCC patients. The diagnostic indices and corresponding ROC curves using these cuto-ff values are shown in Table (5), Fig. (3). The optimum annexin A2 cutoff value was calculated as 19.6ng/ ml (AUC=0.75; 95% confidence interval [CI]: ; sensitivity, 72%; specificity, 74.3%), AFP as 15.0ng/mL (AUC=0.83; 95% CI: ; sensitivity, 84%; specificity, 74.3%). In the differential diagnostic accuracy evaluation, annexin A2 had a lower AUC for identifying HCC from healthy controls and non-hcc groups than AFP (AUC=0.75 vs. 0.83). Because annexin A2 and AFP had a significant positive correlation ( r=0.6; p<0.001 by Spearman's rank correlation (Fig. 2), combined use of annexin A2 and AFP assays for HCC diagnosis resulted in modest increase only in specificity (77%) but the sensitivity (76%) could not be improved. The accuracy of annexin A2 plus AFP was higher than that of annexin A2 alone and statistically similar to that of AFP alone (AUC=0.84 vs and 0.83, respectively) (Fig. 3A). Interestingly, by using 19.6 ng/ml as a cutoff value for annexin A2, we found that 53.9% (7/13) of the studied AFP-negative HCC cases displayed annexin A2 levels > 19.6ng/mL. We next explored the diagnostic efficacy of the biomarkers in various HCC subgroups using ROC. In discriminating

6 2394 Annexin A2 Versus a-fetoprotein (AFP) patients with early HCC (BCLC 0-A) from those without HCC, the optimal cut-off value was 18.7ng/ ml for annexin A2 and of 15.1ng/mL for AFP. The area under ROC curve (AUC) was 0.76 for either marker alone and for the combination of the two markers (Fig. 3B). The sensitivity and specificity of annexin A2 were 80% and 71.4%, respectively, and those of AFP were 80% and 74.3%. Combining annexin A2 with AFP mildly increased only the specificity (77.1 %) for early HCC diagnosis, whereas, sensitivity, PPV and NPV could not be improved (Table 5). Table (5): Efficiency evaluation of serum annexin A2 and/or AFP levels for diagnosis of hepatocellular carcinoma, particularly early stage HCC. HCC vs. all groups: AFPa Annexin A2b Combined Early HCC vs all groups: AFP c Annexin A2d Combine d AUC Sensitivity (%) Specificity (%) PPV (%) NPV (%) Accuracy (%) AUC: Area under ROC. acutoff value of AFP at 15.0 ng/ml. ccutoff value of AFP at 15.1 ng/ml. PPV: Positive predictive value. bcutoff value of annexin A2 at 19.6 ng/ml. dcutoff value of annexin A2 at 18.7 ng/ml. NPV: negative predictive value. Sensitivity Combined AFP Annexin A2 Sensitivity Combined AFP Annexin A Specificity (A) Healthy controls, hepatitis & cirrhosis groups vs. HCC Specificity (B) Healthy controls, hepatitis & cirrhosis groups vs. early stage HCC. Fig. (3): ROC curves for comparison between annexin A2 and AFP and both markers in sensitivity and specificity. Annexin A2 is green, AFP is blue and combination of annexin A2 and AFP is grey. Discussion Early detection of HCC is critical for the application of potentially curative therapies and improving patient outcome [17]. Although AFP is still the golden standard and most commonly used biomarkers for patients at risk for HCC, its diagnostic value has several shortcomings. For example, the low sensitivity (39-64%) and poor diagnostic accuracy at the early stage of HCC [18], therefore, finding new markers for screening and diagnosing HCC at an early stage is of the utmost importance. Conventionally, annexin A2 is known as a calcium-binding cytoskeletal protein and is docu- mented as one of key players in tumorogenesis. It is significantly elevated in cancers of different sites such as breast [19], prostate [20], pancreas [21] and gastric cancer [22]. Previous reports implied that annexin A2 expression was up-regulated in HCC both on the mrna and the protein levels; and it could be a useful biomarker for early diagnosis of HCC patients [23]. In the present study, a typical multistage hepatocarinogenesis model (healthy, chronic viral hepatitis C, HCV-related cirrhosis and HCC) was used to explore the role of annexin A2 as a serum biomarker that could distinguish HCC from non-malignant HCV-related liver diseases, particularly in early stage of the disease.

7 Ehab M. Reyad et al In accordance with the previous studies [23-25], we found that high serum concentration of annexin A2 differentiated HCC patients from those with non-malignant HCV-related liver diseases and control individuals. Moreover, we found no significant difference of the annexin A2 levels among the three non-hcc studied groups. These results indicated that the development of HCC in patients with chronic hepatitis C and/or cirrhosis might be predicted by an increasing level of annexin A2.The ROC curve comparing HCC versus non-hcc revealed sensitivity of 72% and specificity of 74.3% at the optimal cut-off of 19.6ng/mL. Our finding came in agreement with previous studies about the diagnostic value of annexin A2 in HCC which showed a range of sensitivity from 80.4 to 100% and a range of specificity from to 91% [23,24,25,27]. In addition, annexin A2 in the present study recorded an AUC of 0.75 indicating its usefulness as a serological marker of HCC. This finding was similar to the previous studies which documented a range of AUC from 0.73 to 0.91 for annexin A2 [23,24,25,27]. However, by performing a head-to-head comparison between annexin A2 and AFP as serological markers of HCC, our findings revealed that AFP remained superior to diagnose HCC. At first, the diagnostic sensitivity, PPV, NPV, and overall accuracy of annexin A2 were inferior to that of AFP in detecting patients with HCC. By contrast, Zhang et al. [23] reported that annexin A2 had higher sensitivity (86.96% vs % for AFP), NPV (87.39% vs % for AFP), and diagnostic accuracy (75.28% vs % for AFP). Second, the current study showed that the AUC for annexin A2 was inferior to that of AFP in detecting patients with HCC. The same finding was also reported by El-Abd et al. [24] (0.91; 95% CI: for annexin A2 vs. 0.93; 95% CI: for AFP). In the contrary, different previous studies reported that annexin A2 had a superior AUC in comparison with AFP in detecting patients with HCC [23,25, 27,28]. Moreover, the present work revealed that the combined assay of annexin A2 and AFP did not increase diagnostic yield for HCC (76%) whereas, other studies showed that the combined assay of two markers can increase the rate of HCC diagnosis [23,25,27]. We stated that the significant positive correlation between annexin A2 and AFP (r=0.6, p<0.001) in cases with HCC may be explained unfavorable outcome of the combined assay of two markers. Meanwhile, this result indicates that annexin A2 assay could not complement measurement of AFP in the diagnosis of HCC, or in other words, the former could not be used as an independent and significant serological marker for the detection of HCC in patients with chronic hepatitis C and/or cirrhosis. Conversely, Sun et al. [27] reported that the correlation coefficient between serum annexin A2 and AFP values was not significant (p=0.1984), indicating that annexin A2 could be used as an independent biomarker for the diagnosis of HCC. In clinical practice, both the early detection and monitoring of small size tumors have been always challenging due to the lack of tumorsensitive and specific biomarkers that can characterize the formation and progression of HCC development [29]. Herein, we found no significant difference of annexin A2 among early stage HCC, cirrhotics, and late stage HCC groups as well as no statistical association between annexin A2 and tumor size. The efficiency evaluation of serum annexin A2 or/and AFP levels for early HCC diagnosis (Table 5, Fig. 3B) indicated that the diagnostic performance of annexin A2 was found to be statistically similar to that of AFP in ROC analysis, whereas the combined use of the two markers mildly improved AUC and specificity for early HCC diagnosis. However, previous report revealed that serum annexin A2 had better diagnostic performance than AFP in detecting early stage HCC, and combination of the two markers may increase the diagnostic power for early HCC [27]. Additionally, in discordance with the earlier studies [26,27], our results demonstrated that annexin A2 levels in early HCC patients were statistically similar to those of cirrhotic cases (p=0.08), whereas AFP levels were higher in early HCC cases as compared with those with chronic hepatitis or cirrhosis. The increased AFP level in early HCC patients may be due to the initial rise of AFP in early stages of the disease followed by drop before rising again as disease progression occurs [30]. Despite of our disencouraging findings regarding the efficiency of annexin A2 as discriminative biomarker for HCC, we documented a bright light represented by the increased annexin A2 levels above 19.6ng/ ml among 53.9% of AFP-negative HCC cases. Similarly, Sun et al. [27] found that serum annexin A2 levels were greater than a cutoff value of 17.3 ng/ml in 78.4% of AFP-negative HCC cases. Therefore, they documented that annexin A2 was helpful to AFP especially for AFP- negative cases. However, the low number of AFP-negative HCC cases in the current study might limit the importance of this finding. Regarding the clinicopathologic features of circulating annexin A2 expression in HCC patients, we found significant correlations between serum annexin A2 and presence of vascular invasion

8 2396 Annexin A2 Versus a-fetoprotein (AFP) indicating that serum annexin A2 may reflect vascular invasive potential of HCC. Likewise, Zhang et al. [23] reported a significant correlation between serum annexin A2 level and portal vein invasion in HCC patients ( t=2.859, p=0.005). Consistent with our findings, preceding reports found that there were no significant correlations between serum annexin A2 level and age, gender, [27] tumor size [23,27,30] or tumor stage [27] in HCC patients. Besides, this study found no significant correlation between annexin A2 and serum ALT level (p=0.6), and this finding seems to be line with that of Sun et al. (Spearman's rank correlation test, p=0.3096) who suggested that liver injury was not the major cause to release annexin A2 in HCC [27]. The limitation of our study is the small number of patients. Furthermore, chronic hepatitis C was the underlying etiology for HCC in all of the participating patients which may limit the generalization of our findings to other chronic liver disease etiologies. Conclusion: On the strength of these results, we can conclude that the diagnostic performance of annexin A2 alone or in combination with AFP was not better than AFP alone in discriminating HCV-related HCC patients, especially those at early stages of the disease, from those with HCV-related chronic hepatitis and/or cirrhosis. Therefore, it is not a good biomarker for HCC diagnosis and did not add benefits to the diagnosis when used with AFP. Consistent with our conclusion, Liu et al. [31] provided evidences that annexin A2 was not a good biomarker for HCC in cirrhosis. Acknowledgments: This study was supported by National Hepatology and Tropical Medicine Research Institute (NHTMRI) grant ITH References 1- PARKIN D.M., BRAY F., FERLAY J. and PISANI P.: Global cancer statistics, CA Cancer J. Clin., 55: , JEMAL A., BRAY F., CENTER M.M., FERLAY J., WARD E. and FORMAN D.: Global cancer statistics. CA Cancer J. Clin., 61: 69-90, GAO J., XIE L., YANG W.S., ZHANG W., GAO S., WANG J. and XIANG Y.B.: Risk Factors of Hepatocellular Carcinoma-Current Status and Perspectives. Asian Pacific J. Cancer Prev., 13: , LEHMAN E.M. and WILSON M.L.: Epidemiology of hepatitis viruses among hepatocellular carcinoma cases and healthy people in Egypt: A systematic review and meta-analysis. Int. J. Cancer, 124 (3): , EL-ZANATY F. and WAY A.: Egypt Demographic and Health Survey Cairo, Egypt: Ministry of Health, El-Zanaty and Associates, and Macro International, TRINCHET J.C., ALPEROVITCH A., BEDOSSA P., DEGOS F., HAINAUT P. and BEERS B.V.: Epidemiology, prevention, screening and diagnosis of hepatocellular carcinoma. Bulletin du Cancer, 96: (In French), ZHAO Y.J. and QIANG J.U.: Tumor markers for hepatocellular carcinoma (Review). Mol. and Clin. Oncology. 1: , ZHU K., DAI Z. and ZHOU J.: Biomarkers for hepatocellular carcinoma: Progression in early diagnosis, prognosis, and personalized therapy. Biomarker Research, 1: 10, GERKE V. and MOSS S.E.: Annexins: from structure to function. Physiol. Rev., 82: , LOKMAN N.A., WEEN M.P., OEHLER M.K. and RIC- CIARDELLI C.: The role of annexin A2 in tumorigenesis and cancer progression. Cancer Microenviron, 4 (2): , BRUIX J. and SHERMAN M.: Management of hepatocellular carcinoma. An update. Hepatology, 53 (3): , COLLI A., FRAQUELLI M., CASAZZA G., MASSIRONI S., COLUCCI A., CONTE D., et al.: Accuracy of ultrasonography, spiral CT, magnetic resonance, and alphafetoprotein in diagnosing hepatocellular carcinoma: A systematic review. Am. J. Gastroenterol., 101 (3): , MARRERO J.A., FONTANA R.J., BARRAT A., ASKARI F., CONJEEVARAM H.S., SU G.L. and LOK A.S.: Prognosis of hepatocellular carcinoma: Comparison of 7staging systems in an American cohort. Hepatology, 41: , VERSLYPE C., ROSMORDUC O., ROUGIER P., on behalf of the ESMO Guidelines Working Group. Hepatocellular carcinoma: ESMO-ESDO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann. Oncol., 23 (7): vii41-vii48, XU X.H., PAN W., KANG L.H., FENG H. and SONG Y.Q.: Association of annexin A2 with cancer development (Review). Oncology Report, 33: , LEVY I. and SHERMAN M.: Staging of hepatocellular carcinoma: Assessment of the CLIP, Okuda and Chil- Pugh staging systems in a cohort of 257 patients in Toronto. Gut., 50: , DAVILA J.A., MORGAN R.O., RICHARDSON P.A., DU X.L., McGLYNN K.A. and EL-SERAG H.B.: Use of surveillance for hepatocellular carcinoma among patients with cirrhosis in the United States. Hepatology, 52 (1): , ZHAO Y.J., JU Q. and CHENG G.L.: Tumor markers fo hepatocellular carcinoma. Mol. Clin. Oncol., 1 (4): , SOBRAL-LEITE M., WESSELING J., SMIT V.T.H.B.M., NEVANLINNA H., VAN MILTENBURG M.H., SAND- ERS J., HOFLAND I., et al.: Annexin A1 expression in breast cancer: Tumor subtypes and prognosis. BMC Medicine, 13 (156), 2015.

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