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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 20  |  Issue : 3  |  Page : 239-244

The role of glypican 3, arginase 1, and CD34 in differentiation between benign and malignant primary hepatic lesions


Department of Pathology, Al-Azhar University, Faculty of medicine (Assuit), Egypt

Date of Submission05-Sep-2021
Date of Decision18-May-2022
Date of Acceptance07-Jun-2022
Date of Web Publication11-Oct-2022

Correspondence Address:
MD Said Abu-Alkhair Mohamed
Department of Pathology, Faculty of Medicine, Al-Azhar University, 44628 Assuit
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/azmj.azmj_105_21

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  Abstract 


Background and aim Benign hepatic nodular lesions mimicking hepatocellular carcinoma (HCC) may be categorized into hyperplastic nodular lesions, neoplastic nodular lesions, and miscellaneous nodular lesions. This study aimed to assess the role of glypican 3 (GPC3), arginase 1 (ARG-1), and CD34 in differentiation between benign and malignant primary hepatic lesions.
Materials and methods This study included 48 cases, where 25 (52.08%) cases were diagnosed as primary HCC in the liver and 23 (47.92%) cases were diagnosed as benign hepatic lesions. Of the 48 patients, five (10.42%) cases were diagnosed as focal nodular hyperplasia, four (8.33%) cases were diagnosed as hepatic adenoma, eight (16.67%) cases were diagnosed as dysplastic nodule, and six (12.50%) cases were diagnosed as regenerative nodule.
Results The sensitivity of GPC3 for differentiation between benign and malignant primary hepatic lesions was 80% and its specificity was 82.5%. The sensitivity of ARG-1 was 90%, and its specificity was 0.0%. The sensitivity of CD34 for HCC in the study group was 84% and its specificity was 91.3%.
Conclusion There was high sensitivity and specificity of both GPC3 and CD34 immunostaining for distinction of primary HCC from benign hepatic mimicker lesions, whereas there was high sensitivity of ARG-1 in both benign and malignant primary hepatic lesions but with no specificity for differentiating the benign from malignant primary hepatic lesions.

Keywords: arginase 1, CD34, glypican 3, immunohistochemistry, primary hepatic lesions


How to cite this article:
Mohamed SA, Eldowik YM. The role of glypican 3, arginase 1, and CD34 in differentiation between benign and malignant primary hepatic lesions. Al-Azhar Assiut Med J 2022;20:239-44

How to cite this URL:
Mohamed SA, Eldowik YM. The role of glypican 3, arginase 1, and CD34 in differentiation between benign and malignant primary hepatic lesions. Al-Azhar Assiut Med J [serial online] 2022 [cited 2023 Jan 27];20:239-44. Available from: http://www.azmj.eg.net/text.asp?2022/20/3/239/358030




  Introduction Top


Benign hepatic nodular lesions mimicking hepatocellular carcinoma (HCC) may be categorized into hyperplastic nodular lesions, neoplastic nodular lesions, and miscellaneous nodular lesions [1]. HCC is the fifth most common cancer in the world and the third cause of cancer-related mortality. HCC is responsible for more than a million deaths every year worldwide. The diagnosis of HCC may be difficult and requires the use of more than just microscopic techniques [2],[3].

Glypican (GPC) belongs to the family of heparan sulfate proteoglycans with similar structure, including 60–70-kD protein core. It is linked to the cell membrane surface by a glycosylphosphatidylinositol anchor, and the carboxy terminus is modified with a heparan sulfate side chain. Currently, six members of the GPC family have been identified in the genome of mammals. All GPC proteins are highly expressed during the process of embryogenesis [4]. Arginase (ARG) is a manganese-containing enzyme that catalyzes the last step of the urea cycle. ARG-1 is expressed in normal and neoplastic liver cells, especially in the periportal hepatocytes, but it is not expressed in the bile ducts and endothelial and Kupffer cells [5].

CD34 is not expressed in the normal sinusoidal endothelium. However, as capillarization of sinusoidal endothelium occurs during the progression of dysplastic nodules to HCC, immunostaining for the vascular marker CD34 has been found to be a useful marker of malignant transformation. However, although increased to diffuse vascular markings with CD34 is a suspicious finding in a liver tumor, no specific cutoff has yet been established in differentiation between dysplastic nodule and early HCC [6]. Our study aimed to assess the role of GPC3, ARG-1, and CD34 in differentiation between benign and malignant primary hepatic lesions.


  Materials and methods Top


The material of this study included 48 cases as formalin-fixed paraffin-embedded tissue blocks of primary hepatic lesions that were collected from the medical research institute, Alexandria University, as well as from private pathology laboratories over a period of 2 years. The available clinicopathological data of the cases were obtained from the pathology reports of the cases. A total of 25 (52.08%) cases were diagnosed as primary HCC in the liver and 23 (47.92%) cases were diagnosed as benign hepatic lesions. Five (10.42%) cases were diagnosed as focal nodular hyperplasia (FNH), four (8.33%) cases were diagnosed as hepatic adenoma, eight (16.67%) cases were diagnosed as dysplastic nodule, and six (12.50%) cases were diagnosed as regenerative nodule. Tumor classification was performed according to the WHO criteria [7]. Four sections from each block (4 μm thickness) were cut. One section was stained by ordinary stain to confirm the diagnosis, whereas the other three sections were mounted on poly-L-lysine-coated slides for immunohistochemical study using the avidin-biotin-peroxidase complex method. Three primary antibodies were used: GPC3 incubation with primary GPC3 rabbit monoclonal antibody for 30 min at room temperature [monoclone Anti-GPC3 antibody (SP86) (ab95363), 1 : 100 dilution; ABCAM Cambridge, Cambridge, UK); ARG-1 incubation with anti-liver ARG monoclonal antibody (SP156) ab183333 for 30 min at room temperature (Clone number SP156, 1 : 100 dilution, ABCAM); and CD34 incubation with anti-CD34 for 30 min at room temperature (diluted 1 : 50, QB End, Dako, Glostrup, Denmark). The resulting immune complex was detected by the ultravision detection system (Catalog # TP-015-HD, ready-to-use, Thermo Scientific, Fremont, CA, USA). Sections were deparaffinized, rehydrated, incubated in 3% H2O2, rinsed in PBS, and placed in a pressure cooker for antigen retrieval. The slides were incubated in jars containing 10% normal goat serum (Vector Laboratories, Burlingame, California, USA) and with serum-free protein block (Dako, Carpentaria, California, USA). The primary antibodies (two drops) were placed on the sections for 30 min and then washed with PBS. Then, the secondary antibody and streptavidin peroxidase complex were applied. The sections were visualized and were counterstained in hematoxylin and rinsed gently in tap water. Finally, the slides were dehydrated and cleared.

Scoring system

GPC3 was expressed in a cytoplasmic and/or membranous pattern, and the tumor was considered positive if more than 5% of the tumor cells were stained with this antibody [8]. The ARG-1 was expressed in cytoplasmic or cytoplasmic plus nuclear staining, and the tumor is considered positive if more than 5% of tumor cells were stained [9]. CD34 was expressed by counting the microvessels using a light microscope at 200 magnification. The immunostained slides for CD34 were carefully scanned at a low power field, and the three areas considered to have the highest vascularity (hotspots) in the section were selected. Microvessels were counted manually in a single microscopic field in each hotspot at a high power field as the MVD score [10].

Statistical analysis

Data collected were analyzed using IBM SPSS software, version 20 (IBM Corp., Armonk, New York, USA). Qualitative data were described using the number and percent. To verify the normality of distribution, quantitative data were described using range (minimum and maximum), mean, SD, and median using the Kolmogorov–Smirnov test. The diagnostic value of each immunoprofile was analyzed according to its sensitivity, which is the proportion of tumor cases that were targeted by a certain antibody and showed positive results for this antibody [true positive cases/true positive cases+false negative cases)×100]; specificity, which is the proportion of tumor type cases that were not targeted by a certain antibody and showed negative results for this antibody [(true negative cases/true negative cases+false positive cases)×100]; and positive predictive value [(true positive cases/true positive cases+false positive cases)×100]. The diagnostic accuracy of a certain antibody was calculated as the number of correctly assessed cases in relation to the number of all cases [(true positive cases+true negative cases)/true positive cases+false positive cases+true negative cases+false negative cases)×100].


  Results Top


This study included 48 cases; 25 (52.08%) cases were diagnosed as primary HCC and 23 (47.92%) cases were diagnosed as benign hepatic lesions. Five (10.42%) cases were diagnosed as FNH, four (8.33%) cases were diagnosed as hepatic adenoma, eight (16.67%) cases were diagnosed as dysplastic nodule, and six (12.50%) cases were diagnosed as regenerative nodule ([Table 1]).
Table 1 Relationship between the studied cases and the diagnosis

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Regarding GPC3, 24 (50%) of 48 cases were positive, and 24 (50%) cases were negative. Benign hepatic lesions showed negative reaction in 19 (82.6%) of 23 cases and focal positive reaction in four (17.4%) cases. HCC showed positivity in 20 (80%) of 25 cases and negativity in five (20%) cases. The sensitivity of GPC3 antibody for the diagnosis of HCC from benign component in the study group was 80% and its specificity was 82.5%, positive predictive value was 83.3%, negative predictive value was 79.2%, and the diagnostic accuracy of GPC3 was 81.3% ([Table 2] and [Figure 1] and [Figure 2]).
Table 2 Agreement (sensitivity, specificity, and accuracy) for glypican 3 to differentiate hepatocellular carcinoma from benign lesions

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Figure 1 HCC GIII showing Strong diffuse positive cytoplasmic GPC3 expression in (GPC3, X200). GPC, glypican; HCC, hepatocellular carcinoma.

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Figure 2 HCC GII with pseudoglandular differentiation showing strong diffuse positive cytoplasmic GPC3 expression in GPC3 (×100). GPC, glypican; HCC, hepatocellular carcinoma.

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Regarding ARG-1, 45 (93.7%) of 48 cases were positive cases, and three (6.3%) cases were negative. ARG-1 was positive in all benign hepatic cases [23/23 (100%)] (FNH, HA, NRN, and DN). HCC showed positivity in 22 (88%) of 25 HCC cases and three (12%) cases were negative. The sensitivity of ARG-1 for HCC from benign component in the study cases was 90% and its specificity was 0.0%. The positive predictive value was 60%, negative predictive value was 0.0%, and the diagnostic accuracy was 56% ([Table 3] and [Figure 3] and [Figure 4]).
Table 3 Agreement (sensitivity, specificity, and accuracy) for arginase 1 to differentiate hepatocellular carcinoma from benign lesions

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Figure 3 A case of HA showing diffuse positive cytoplasmic and nuclear staining of ARG-1 expression (ARG-1, ×100). ARG, arginase.

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Figure 4 A case of FNH showing diffuse positive cytoplasmic and focal nuclear staining of ARG-1 expression (ARG-1, ×100). ARG, arginase; FNH, focal nodular hyperplasia.

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Regarding CD34, 23 (47.9%) of 48 cases were positive, whereas the remaining 25 (52.1%) cases were negative. Benign hepatic lesions showed a negative reaction in 21 (91.3 %) of 23 cases, and only two (8.7%) cases showed diffuse positive staining for CD34. HCC showed positivity in 21 (84%) of 25 cases and negativity in four (16%) cases. The sensitivity and specificity of CD34 staining for differentiating HCC and benign component was 84 and 91.3%, respectively. The positive predictive value was 91.3%, negative predictive value was 84 %, and the diagnostic accuracy was 87% for CD34 ([Table 4] and [Figure 5],[Figure 6],[Figure 7]).
Table 4 Agreement (sensitivity, specificity, and accuracy) for CD34 to differentiate hepatocellular carcinoma from benign lesions

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Figure 5 Immunostaining for CD34 in normal hepatic tissue showing negative staining in all endothelial cells surrounding the normal hepatocytic cells (×200).

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Figure 6 Case of hepatocellular carcinoma showing strong diffuse positive CD34 immunostaining (×200).

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Figure 7 A case of hepatocellular carcinoma showing trabecular pattern surrounded by strong positive CD34 immunostaining (×400).

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  Discussion Top


The GPC3 expression in HCC can be explained by fetal phenotype reactivation, which is common in malignant tumors [11]. The current study revealed that benign hepatic lesions show negative reaction in 82.6%, and only 17.4% of benign hepatic lesions showed focal positivity for GPC3 immunostaining. Multiple previous studies [12],[13],[14],[15] confirmed either low frequency of focal GPC3 staining or negative GPC3 staining in benign liver lesions in the examined benign hepatic lesions including hepatocellular adenomas, FNH, and large regenerative nodules. HCA and FNH, which mimic HCC, were always reported as negative for GPC3 expression [16]. In our study, GPC3 was expressed in 80% of HCC cases. This was in agreement with several studies [8],[14],[17], which have demonstrated that GPC3 was positive in most HCC, and positivity varied from 52.5 to 100% of HCC cases. The sensitivity and specificity of GPC3 for HCC detection in our study were 80 and 83.3%, respectively. These findings were consistent with other previous study [18], which revealed high specificity and sensitivity of 86 and 94%, respectively. The study by Xu et al. [15] also agreed with our study. They founded that the sensitivity and specificity of GPC3 for HCC detection were 82 and 100%, respectively.ARG-1 has been described in the literature as a new potential immunostaining marker of hepatocytes differentiation [19]. In this study, ARG-1 was positive in all benign hepatic cases (100%) (FNH, HA, NRN, and DN) as well as positive in 90% of HCC cases. Several studies [15],[19],[20],[21] have agreed with our results, which approved diffuse and strong reactivity of ARG-1 in most HCC cases and all benign liver masses. Our results were in agreement with other studies [22],[23],[24] that approved high sensitivity of ARG-1 in both benign and malignant primary hepatic lesions but with no specificity for primary hepatic lesions.

CD34 immunoreactivity is in fact highly sensitive for the diagnosis of HCC, as it outlines clearly the abnormal growth pattern of HCC [25]. Regarding CD34 immunohistochemical staining, 91.3% of cases of benign hepatic lesions showed negative staining, whereas only two (8.7%) cases showed diffuse positive staining. HCC showed positivity in 84% of cases and negativity in 16% of the cases. This finding was in agreement with other previous studies [16],[25],[26],[27]. In this study, sensitivity and specificity of CD34 in HCC were 84 and 91.3%, respectively. These findings were consistent with many previous studies [25],[26],[27], which revealed the value of CD34 in making a discrimination between benign hepatic lesions and HCC.


  Conclusion Top


We concluded that the high sensitivity and specificity of both GPC3 and CD34 immunostaining can be used for distinction between primary HCC from benign hepatic mimickers, whereas there was a high sensitivity of ARG-1 in both benign and malignant primary hepatic mimicker lesions but with no specificity for differentiating the benign from malignant primary hepatic lesions.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
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  [Table 1], [Table 2], [Table 3], [Table 4]



 

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