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Abstract
Background: A disintegrin and metalloproteinases (ADAMs) have emerged as therapeutic targets in many cancers. ADAM10 was particularly studied in hepatocellular carcinoma (HCC) for its potential role in hepatocarcinogenesis and HCC progression. Objective: To investigate the immunohistochemical (IHC) expression of ADAM10 in HCCs and the adjacent noncancerous tissues from 70 HCC patients, attempting to elucidate any association between ADAM10 and HCC development and/or progression. Materials and Methods: IHC staining for anti-ADAM10 was performed using horseradish peroxidase technique. An extent and intensity-dependent scoring was applied dividing samples into high- and low-expression groups. HCCs were statistically compared in relation with gender, age, cirrhosis, hepatitis C virus (HCV) status, alpha-fetoprotein (AFP) serum level, tumor size, multiplicity, encapsulation/invasion, grade, histological pattern and variant, mitosis, necrosis, vascular emboli, portal thrombosis, stage, recurrence, and mortality. Kaplan–Meier's method was used to analyze disease-free and overall survival (DFS and OS). Results: ADAM10 was expressed in 77.1% of HCCs compared with 42.9% of noncancerous tissues. Differential expression showed significant statistical difference (P = 0.02), as 38.6% of HCCs showed high expression, whereas 92.8% of noncancerous samples showed low expression. No significant differences were observed when high- and low-ADAM10 expression HCCs were compared with respect to all tested prognostic parameters except the HCV status. Patients whose tumors showed high-ADAM10 expression had relatively longer DFS and OS times, but with insignificant log-rank differences. Conclusions: ADAM10 is frequently expressed in HCCs compared with noncancerous hepatic tissues suggesting its role in hepatocarcinogenesis, especially in association with HCV. It has no association with HCC progression or survival. Further studies should be sought to investigate its validity as a therapeutic target.
Keywords: ADAM10, hepatocellular carcinoma, immunohistochemistry, prognostic, survival
How to cite this article: Introduction 
According to the Global Cancer Observatory estimates in 2018, hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide (4.7% of cancers with 841,080 new cases in 2018), and the third most lethal cancer being responsible for 8.2% of cancer-related mortalities.[1] It is the most frequent histological type of primary liver malignancies accounting for 85%–90% of primary liver cancers. Geographical variations in overall HCC incidence, age-specific incidence, and mortality rates are ascribed to different levels of exposure to HCC-associated risk factors such as chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infections, liver cirrhosis, and exposure to aflatoxin in developing countries, smoking and alcohol abuse in developed countries.[2]
Despite increasing advances in the diagnostic and therapeutic surgical or nonsurgical techniques, HCC patients' have unsatisfactory survival rates mainly due to the high rate of recurrence, metastasis, and the emergence of postresection tumors.[3] Long-term survival is likely only in patients with early stage, small, asymptomatic HCCs that can be treated by resection, and liver transplantation in patients fulfilling Milan criteria, or by nonsurgical methods including percutaneous ethanol or acetic acid injection and percutaneous radiofrequency ablation in patients not fulfilling the abovementioned criteria. Meanwhile, transarterial chemoembolization is recommended for patients who do not have vascular invasion or extrahepatic spread but have unresectable large or multifocal tumors. Radioembolization with 90Y-labeled glass beads has shown effectiveness to treat patients with intermediate-stage disease not responding to transarterial chemoembolization.[4] Yet, most studies report a 5-year survival rate of less than 5% in patients with symptomatic or advanced HCCs who are basically resistant to radiotherapy and chemotherapy. Thus far, sorafenib, a tyrosine kinase inhibitor, is the only recommended therapeutic agent in advanced-stage disease refractory to other therapeutic options. For this reason, better knowledge of the molecular mechanisms responsible for HCC development and progression may serve to discover novel efficacious therapeutic agents for HCC, especially in the field of tumor regression therapies.[3],[4],[5]
In the past few years, A disintegrin and metalloproteinases (ADAMs) have emerged as new therapeutic targets in many cancers, especially of gastrointestinal origin.[6],[7] Lately, ADAM10 was investigated in HCC and was claimed to be a promising therapeutic target; however, most of these studies have been performed on HCC cell lines,[8],[6],[7],[8],[9],[10],[11],[12] and only a few studies applied immunostaining on HCC tissues obtained from patients relied on clinical analysis to serve for this target.[13]
In the present study, we investigated the differential immunohistochemical (IHC) expression of ADAM10 in HCC tissues and the adjacent noncancerous tissues obtained from 70 HCC patients. Moreover, we attempted to elucidate if there is an association between ADAM10 expression and HCC development and/or progression through analyzing its expression level in conjunction with several clinicopathological prognostic parameters and with patients' survival.
Materials and Methods Patients and tissue samples
This retrospective cohort study included a total of 70 HCC patients who underwent partial hepatic surgical resection at the Operations Department, Gastrointestinal Surgery Center (GISC), Faculty of Medicine, Mansoura University, Egypt, during the period from July 2014 to June 2017 and whose resection specimens were submitted for diagnosis at the pathology laboratory of the same center. For all the 70 HCCs, adjacent noncancerous hepatic tissues (at least 1.5 cm. from the tumor), were included as a comparison group.
Cases were selected provided that none of the patients had received preoperative percutaneous or transarterial chemotherapy, or radiotherapy, the formalin-fixed, paraffin-embedded (FFPE) tissue blocks are available with adequate tissue (for both the HCC tumor tissue and the adjacent noncancerous tissue) for further IHC study, and the clinical and follow-up data (for 36 months) are readily accessible from our institute database for medical records.
Data collection
Demographic, clinical, and serology testing parameters of prognostic significance including gender, age, tumor size, multiplicity, metastasis, portal vein thrombosis, HCV virology test status, and alpha-fetoprotein (AFP) serum levels were collected. For analysis of disease-free and overall survival (DFS and OS, respectively), the date of surgery was used as the beginning of the follow-up period, disease recurrence/relapse (either a radiologically or a histopathologically confirmed local reappearance of the tumor at the same site/or at a metastatic site during the follow-up) or HCC-related death were recorded by time. Patients who died from HCC nonrelated events or lost for follow-up were excluded from the survival analysis.
Histopathological evaluation and staging
The FFPE tissue blocks and the hematoxylin and eosin (H&E)-stained microscopic slides were retrieved from the pathology laboratory archives. H and E slides were reviewed by two pathologists for histopathological diagnosis, HCC histological pattern identification, determination of any specific HCC variants, and grading according to the World Health Organization (WHO) criteria.[2] Hep-par-1 and/or glypican-3 IHC staining was reviewed/requested to confirm diagnosis whenever indicated. Tumor encapsulation/capsular invasion, necrosis, vascular emboli, and the number of mitotic cells in 10 nonoverlapping high-power fields (HPFs) were evaluated and recorded.[14] The adjacent noncancerous hepatic tissue was evaluated for the presence or absence of cirrhosis. Finally, the tumor stage was determined based on the American Joint Committee of Cancer (AJCC) Cancer Staging Manual, 8th edition.[15]
Immunohistochemical (IHC) staining
About 3- to 4-μm-cut paraffin sections on positively charged silanized glass slides (VitoGnost SIL adhesive microscope slides) were stained using the standard horseradish peroxidase (HRP) technique. Antigen was retrieved in 0.01 M citrate buffer (pH 6.0) for 10 min in microwave followed by 3% hydrogen peroxide incubation for 10 min. Slides were incubated with a rabbit IgG polyclonal anti-human-ADAM10/MADM antibody corresponding to the extracellular region of the human ADAM10 (Chongqing Biospes Co., Ltd China; Cat. No. YPA2221, concentrated, 100 μL, 1:400 dilution, pH 7.4) at room temperature for an hour, followed by a poly HRP conjugate for 30 min. Diaminobenzidine (DAB; Sakura USA, Poly HRP DAB kit; Cat. No. 54-0117) was applied as a chromogen followed by hematoxylin counterstaining, dehydration, and mounting. Negative controls were prepared under identical conditions but omitted the primary antibody. Invasive ductal breast carcinoma with confirmed positivity was used as positive control tissue.
IHC evaluation and scoring
Staining (cytoplasmic and/or membranous) was visualized by observing slides under light microscope using 20× objective independently by two blinded pathologists depending on two parameters: the extent of staining and the staining intensity. Extent of staining was categorized as: 0 = no staining or “ADAM10 negative;” 1 = focal positive; and 2 = diffuse positive staining. For “ADAM10 positive” samples, the staining intensity was categorized as: 1 = faint, 2 = modest; and 3 = dense. The sum of both parameters provided the final scores for ADAM10 in each sample, in which a final score of 2 or 3 was defined as weak positive, score 4 as moderate positive, and score 5 as strong positive. For statistical analysis, negative and weak positive samples were described as ADAM10 low-expression group, whereas moderate and strong positive were considered as ADAM10 high-expression group.[16]
Ethics
The study protocol was approved by the local Institutional Research Board (Code 20.12.1118 R1). Informed consent was obtained from patients. No additional surgical or nonsurgical interventions were applied to the patients as a part of the study whose anonymity and confidentiality was secured throughout and after the study. All procedures were done in accordance with the current revision of the Helsinki Declaration of medical research involving human subjects.[17]
Statistical analysis
Statistical Package of Social Science (SPSS) program (standard version 23) was used for data entry and analysis. Data normality was tested with a one-sample Kolmogorov–Smirnov test. Data were presented as numbers, percentages, ranges (minimum and maximum), and/or mean ± standard deviation (SD) whenever appropriate. Comparison of ADAM10 expression levels in HCC and the noncancerous tissue samples and the association between ADAM10 protein expression level and the clinicopathological variables were analyzed using Chi-square test. Monte Carlo test was used when the expected cell count was less than 5. Kaplan–Meier survival analysis was applied to evaluate the DFS and OS. A statistically significant difference was accepted at a P value of ≤0.05 and the level of significance was assumed to be higher whenever P value was lower.
Results Demographic and clinicopathological criteria
The study encompassed 70 HCC patients including 52 men and 18 women, with a mean age of 61.2 years (27–85 years), of which 90% were cirrhotic and 85.7% had an HCV-positive serologic test. At the initial diagnosis, their AFP levels ranged from 2–2,000 ng/mL with a mean of 499.9 ng/mL, and 8.6% presented with portal vein thrombosis. The mean tumor size was 6.5 cm and 15.7% of patients had multiple masses (considering the size of the main mass in case of multiple masses). About 54.3% of tumors were encapsulated and 24.3%, 44.3%, and 61.4% of tumors showed capsular invasion, necrosis, and vascular emboli, respectively. Concerning the tumor's histological pattern, the most frequent was the trabecular pattern (64.3%), followed by the pseudoglandular (27.1%) then the solid (8.6%) patterns. Grade 1; well-differentiated tumors were the most frequent (52.9%) followed by grade 2 and 3 (moderate and poor differentiated; 25.7 and 18.6%, respectively), whereas grade 4; undifferentiated tumors comprised a minority of cases (2.9%). Sixty-four cases (91.4%) were classified as classic HCC, whereas 6 cases (8.6%) were of the clear cell variant. No other HCC variants were detected [Figure 1]. The mean mitotic count was 8.1/10 HPF (range: 1–30). Based on the standard AJCC criteria for HCC staging, 85.7% of patients were classified at early stage disease (I and II) and the remainder (14.3%) were classified at advanced stage disease (III and IV). During the 3-year follow-up period, 32.9% of patients developed recurrence, 34.3% of them died due to HCC-related factors, and five patients (7.1%) were lost for follow-up. The mean DFS and OS times for the 65 patients who completed follow-up were 24.6 and 26.9 months, respectively [Table 1].
Figure 1: Different histological patterns, variants, and grades of hepatocellular carcinoma (Hematoxylin and Eosin, ×200)
Table 1: The clinicopathological criteria of the 70 hepatocellular carcinoma patients included in the studyDifferential ADAM10 IHC expression in HCC and noncancerous tissue
ADAM10 was expressed in 77.1% of HCCs compared with 42.9% of the adjacent noncancerous hepatic tissues. Positivity was categorized as weak (38.5 and 35.7%), moderate (34.3 and 7.2%), and strong (4.3 and 0%) for HCC and the noncancerous tissues, respectively [Figure 2] and [Figure 3]. Comparing the high and low-ADAM10 expression levels, there was a significant statistical difference between HCC and the noncancerous samples (P = 0.02), as 38.6% of HCCs showed high-expression level while most noncancerous samples (92.8%) showed low expression [Table 2].
Figure 2: Diagrammatic presentation of differential ADAM10 expression in hepatocellular carcinoma (HCC) and the noncancerous hepatic tissues. A case of HCC showing high-ADAM10 expression compared with the low expression in the adjacent noncancerous tissue (Immunohistochemistry; Diaminobenzidine, ×200)
Figure 3: High- and low-ADAM10 expression levels demonstrated in different histological patterns, variants, and grades of hepatocellular carcinoma (Immunohistochemistry; Diaminobenzidine, ×200)
Table 2: High- and low-ADAM10 expression levels as compared with hepatocellular carcinoma and noncancerous hepatic tissuesADAM10 associations with the prognostic parameters
In HCC [Table 3], there were no observed significant statistical differences when the high- and low-ADAM10 expression groups were compared with respect to almost all tested prognostic parameters with exception of the positive HCV status that was significantly associated with a high-ADAM10 expression level (P = 0.04). Concerning the DFS and OS, the ADM10 high-expression group had somewhat longer DFS and OS times compared with those with low expression but the difference did not reach the level of significance.
Table 3: Associations between ADAM10 expression level and the prognostic parameters of hepatocellular carcinomaSurvival analysis
As demonstrated in Kaplan–Meier survival curves [Figure 4], patients whose tumors showed high-ADAM10 expression levels had slightly longer DFS and OS times (27.1 and 28 months, respectively) compared with those with low expression (23.8 and 26.2 months, respectively) [Table 3], however, the log-rank test showed no significant difference between the high- and low-expression groups for either DFS (P = 0.26) or OS (P = 0.45).
Figure 4: Kaplan–Meier survival curves for disease-free survival (DFS; left) and overall survival (OS; right) times in high- and low-ADAM10 expression levels. The log-rank test showed no significant difference between high- and low-expression groups (P = 0.255 and 0.447, respectively) Discussion The ADAMs are a family of zinc-dependent cell surface and secreted proteins that possess both potential adhesion and protease domains. They play important roles in regulating cell phenotype via their effects on cell adhesion, migration, proteolysis, and signaling. Sheddase, a generic name for the ADAMS, that functions to cleave various substrates such as amyloid precursor protein (APP), N-cadherin, E-cadherin Proto-cadherin-c, VE-cadherin, cluster designation (CD) 23, Delta-like ligand-1 (Dll1), Pro-EGF, HER2/neu, CD30, CD44, and L1-CAM at the cell surface, thus releasing soluble ectodomains with an altered location and function. A key ADAMs family member is the ADAM10 also known as CDw156 or CD 156c encoded by the human ADAM10 gene at 15q21.3. It is normally expressed in human mesenchymal stem cells, placenta, bladder, blood, and bone marrow myeloid cells. ADAM10 is a principal player in signaling via the Notch and Eph/ephrin pathways and is a sheddase for APP, MHC class I polypeptide-related sequence A (MICA), HER2/neu; E-cadherin, N-cadherin, CD44, and Notch, most of which play a significant role in proliferation, migration, invasion or stemness of cancer cells, and drug resistance as well.[3],[9],[18],[19],[20] Previous studies have shown that ADAM10 is overexpressed in a variety of cancers such as breast, oral squamous cell, gastric, and nasopharyngeal carcinomas.[18],[21],[22],[23] Moreover, ADAM10 overexpression in nasopharyngeal carcinoma cell lines was accompanied by the downregulation of E-cadherin and upregulation of N-cadherin and vimentin confirming the role of ADAM10 in epithelial-mesenchymal transition (EMT),[23] and therefore, its oncogenetic capabilities.[24]
In 2010, Mazzocca et al.,[25] raised a question concerning the involvement of ADAMs in tumorigenesis and progression of HCC: Is it merely fortuitous or a real pathogenic link? Their recommendation was to develop more detailed large-scale clinical analyses to allow better knowledge of the role of ADAMs, and hopefully lead to more translational applications of the obtained results in patients with HCC.
As ADAM10 silencing was described to suppress HCC cell proliferation, migration, invasion, and metastasis both in vitro and in vivo,[3],[26] some studies have linked ADAM10 with worse prognosis,[13] and shorter patients' survival in HCC,[13],[16] and hence attempts have been made to develop novel therapeutic agents against HCC cells with inhibitory effects on ADAM10 enzyme activity.[9],[12] However, this issue may merit further investigation from a clinicopathological context.
We retrospectively investigated the IHC expression of ADAM10 in HCCs and the adjacent noncancerous tissues among cohort of 70 HCC patients of whom 63 patients were cirrhotic and 60 patients were HCV positive. An unequivocal cytoplasmic and/or cell membrane staining was observed in the majority of HCCs. In differential expression, 38.6% of HCCs showed high-expression level while most noncancerous samples (92.8%) showed a low-expression level rendering a significant difference between groups (P = 0.02). Likewise, the IHC positivity rates of ADAM10 were 73.3% and 75.6% in two earlier comparable studies,[13],[16] and the frequency of ADAM10 in HCC tissues was significantly higher than that in the noncancerous tissues.[16] Our findings indicate that ADAM10 could be involved in the development of HCC as an early event in hepatocarcinogenesis, possibly via tumor-stromal interactions.[25]
In our locality, HCC represents the fourth most common cancer and is the most common cause of mortality-related and morbidity-related cancer.[27] Many hospital-based studies reported increasing HCC incidence that could be attributed to improvement in screening programs and diagnostic tools and the increased survival rate of cirrhotic patients. The high prevalence of HCV infection in our locality (especially genotype 4) contributes to the high incidence of HCC as viral hepatitis markers were reported to be negative in just 3.5%–14.5% of HCC patients.[27],[28],[29] Thus, a better understanding of the underlying pathological mechanisms in the development of HCC in this population may uncover more efficient ways to limit its burden.
A strong correlation between ADAMs and chronic liver diseases of different etiology, including HCV infection has been confirmed.[25],[30] Since HCV is an important risk factor for developing HCC,[2] it is reasonable to assume a contribution of ADAMs during the initial phases of hepatocarcinogenesis via controlling important oncogenic signaling pathways in HCC cells of HCV-infected patients.[25],[30] In this context, this study verified a significant association between the high ADAM10 expression level. This finding proposes the contribution of ADAM10 in the HCV-promoted liver cell injury and hence, hepatocarcinogenesis in the setting of continuing inflammation and fibrosis. Likely, a recent study demonstrated that ADAM10 expression markedly increases in chemically induced liver tissue injury where its overexpression aggravates inflammation and hepatic lesions.[31] Moreover, ADAM10 was documented to be increased in plasma of untreated human immunodeficiency virus (HIV)-infected patients and was found to correlate with HIV progression via its sheddase activity on soluble T cell immunoglobulins,[32] suggesting its pathogenetic linkage to viral infections.
In this study, we investigated the potential association between ADAM10 expression and a myriad of demographic and clinicopathological prognostic parameters of HCC as well as DFS and OS. Except for the HCV positive status, we did not report any significant difference when the prognostic parameters were compared between the ADAM10 high- and low-expression groups. Although Yuan et al.,[16] reported a significant association between ADAM10 IHC expression and the clinical outcome in HCC patients with a shorter OS in the ADAM10 high-expression group, they were unable to detect any difference between the high- and low-expression HCCs in relation to age, gender, risk factors (alcoholism, smoking history, and HBV infection), clinical stage of disease, histological grade, or microvascular invasion. On the contrary, ADAM10 IHC expression level correlated with the tumor size, differentiation, Tumor-Node-Metastasis (TNM) stage, and the Kaplan–Meier survival analysis in another study.[13] Though ADAM10 was hypothesized to be a poor prognostic HCC marker, previous reports that may relate to this hypothesis using IHC are scarce to resolve this dispute. So far, we were unable to demonstrate any evidence of ADAM10 involvement in HCC progression. In agreement with our findings, a recent study on gastric adenocarcinoma demonstrated no statistically significant association between ADAM-10 expression and clinicopathological parameters or patients' survival in multivariate analysis.[33]
Support to our findings comes through two observations. First, ADAM10 is synthesized as an inactive proform, which is activated within Golgi apparatus via proteolytic removal of the ADAM10 inhibitory prodomain. This active form of ADAM10 is responsible for the shedding of membrane-spanning proteins.[20] Therefore, the IHC detection of ADAM10 may not necessarily equate that it is in the active sheddase form. Second, ADAM10 was found to exhibit different prognostic associations in HCC depending on genetic polymorphisms. In a recent study that investigated ADAM10's single-nucleotide polymorphisms (SNPs) and their relation to clinical stage; tumor size; lymph node metastasis; distant metastasis; vascular invasion; grade; HBsAg; anti-HCV; and liver cirrhosis in HCC patients, the results of five SNPs indicated that one variant was correlated with a higher risk of developing lymph node metastasis, a second variant was correlated with a higher risk of developing distant metastasis and higher levels of AFP, and the remaining three variants showed no significant association with the studied clinicopathological parameters.[10]
Conclusions Using the immunohistochemical approach, ADAM10 was frequently expressed in HCC tissues as compared with the adjacent noncancerous hepatic tissues. Despite its potential role in early hepatocarcinogenesis, especially in association with HCV, it seems that ADAM10 seems to have no role in HCC progression or patient survival. Further studies concerning the regulatory mechanisms, functions, and the potential prognostic role of ADAM10 in HCC patients should be sought to investigate its validity as a therapeutic target in HCC.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient (s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Acknowledgments
The authors acknowledge the support of Taif University Researchers Supporting Project number (TURSP-2020/127), Taif University, Taif, Saudi Arabia.
Financial support and sponsorship
Taif University Researchers Supporting Project number: TURSP-2020/127, Taif University, Saudi Arabia.
Conflicts of interest
There are no conflicts of interest.
References 
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ijpm.ijpm_608_21
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