The Immunohistochemically Estimation of CD63 in Iraqi Patients with Gastric Cancer

: CD63 is -one of the tetraspanin family proteins, which are regarded as: hallmark exosomal markers because it is absent from other types of vesicles. It is expressed in the cell membrane of cancer cells, and cytoplasm of stromal cells. Objective: To assess CD63 expression in gastric cancer (GC) patients, and detected if it could be used as a predictive marker. Furthermore, the current study aimed to find the correlation between CD63 expression and clinicopathological parameters as: gender, age, invasion depth, histopathological type, involvement of lymph nodes, grade and stages of GC (TNM). The current study is a retrospective study in the period time from (2018 to-2020); 50 randomly patients formalin-fixed paraffin embedded blocks (FFPE) of stomach tissue (10 cases normal tissue without GC as control, and 40 patients with GC) with its reports and diagnosis were collected from Pathology Department of the Gastroenterology and Hepatology Teaching Hospital and some private hospitals. The histological sections were stained by hematoxylin and eosin stain (H&E), and immunohistochemistry (IHC) stain for CD63. Statistical analysis accomplished by SPSS system at (P ≤ 0.05). This study indicated that there were significant differences between control group, and patients group in the expression of CD63, also there was a significant correlation between CD63 expression, and histopathological subtype, invasion depth, involvement of lymph node, and stages in patients, whereas there was a non- significant association between the age, grade, and gender of patients, and the expression of CD63.This result indicates that CD63 could be a good prospective marker in Iraqi cancer patients.


Introduction:
Gastric carcinoma (GC) comprises a universal health issue. It is a disease with high aggressive and heterogeneous nature. It is one of the most prevalent reasons of cancer related death and takes advantage of an important encumbrance on international health sponsor 1 . In Iraq malignant neoplasm's represented the second leading cause of death. Gastric cancer trend demonstrated an instant rise after 2007; it is the fifth of eighteenth cancers in Iraq 2 . GC is the third main reason of cancer-related mortality in the world, leading round about 783,000 deaths in 2018, and over 1,000,000 new cases of gastric carcinoma per year 3 . The Lauren classification is the most communal classification of GC. It includes three main subtypes: intestinal, diffuse, and mix 4 which they differ in many properties, such as: clinical characterize, genetics, morphology, epidemiology and development features 5 . Exosome is an extracellular vesicle excreted mostly by many eukaryotic cells. It can be utilized for tumor patients as a prognostic marker and/or grading basis. It is contributed in intercellular communication. It has an important function in controlling plentiful processes during cancer progression such as: tumor growth, metastasis, and angiogenesis, because of it contains proteins, DNA, mRNA, circular RNA, microRNA, long noncoding RNA, etc. 6 . Moreover, it is an intermediate signal transduction incident that has a main role in the cellular processes regulation such as adhesion motility and differentiation. Exosomes may have a major possibility to use as biomarkers for the premature diagnosis, the prediction of prognosis, and the valuation of the treatment impact in gastric cancer 7 . CD63 which is also called lysosome-associated membrane glycoprotein 3, melanoma-associated antigen ME491 or melanomaassociated antigen MLA1 is a glycoprotein cell surface 8 , and one of the tetraspanin family proteins (TM4SF), which is regarded as: hallmark exosome marker because it is absent from other types of vesicles 9 , and extremely reinforced on the membranes of intraluminal vesicle (ILV) 10,11 has been correlated with human tumor progression in non-small cell lung cancer, prostate cancer, breast cancer, astrocytomas, pancreatic cancer, and melanoma 12,13 . CD63 magnitude in gastric cancer patients has not been elucidated. Cancer cells and stromal cells exosomes derived could be a major role in the intracellular communications include in the development of cancers 14 . Objective of the current study: To estimate CD63 expression in Iraqi patients with gastric cancer, and detected if it could be used as a predictive marker. Furthermore, the current study aimed to find the correlation between CD63expression, and clinicopathological parameters as: gender, age, invasion depth, histopathological type, involvement of lymph nodes, grade, and stages of GC.

Samples collection
The current research is a retrospective study from January 2018 to December 2020. The total numbers of samples were 50 cases. Forty samples of gastric cancer patients formalin-fixed paraffin embedded blocks (FFPE) have been obtained randomly from surgically resected specimens in Gastroenterology and hepatology Teaching Hospital, Medical City/ Baghdad/Iraq with its reports (no chemotherapy received by these patients) after getting the official agreement from Iraqi Ministry of Health, and Department of Medical City/ Baghdad/ Iraq. Each report contains clinic-pathological parameters (age, gender, histopathological type of tumor, grade of tumor, depth invasion of tumor, lymph nodes involvement, tumor stage/ TNM), which were diagnosed by pathological doctors of hospital. Ten samples of normal stomach tissue have been selected randomly from patients undergo (Sleeve gastrectomy) by private hospitals. These patients' cases were classified depending on Lauren classification 4 .

Staining of immunohistochemistry (IHC) & hematoxylin and eosin (H&E)
Each FFPE were cut in 5 µm in thickness before staining, mounted in positive charged slides for IHC, and normal slides for H&E stained. Some of cutting sections for control, and patients groups were stained by routine staining sequent steps depending on Suvarna et al. 15 . CD63-antibody (Recombinant Rabbit Monoclonal antibody) (Code No. SY21-02; Dilution1/100; Thermo Fisher, USA) was used for IHC, and protocol of the manufacture company applied to accomplish this staining. Sections were incubated in serum blocking solution, and then slides were incubated with primary antibody (CD63), then with biotinylated link secondary antibody (Abcam, USA). After that slides were incubated with streptavidin-enzyme conjugate (Thermo Fisher, USA), then with substratechromogen (DAB) mixture (Abcam, USA). Finally, slides stained with hematoxylin, and mounted with aqueous mounting solution. After each of these steps slides were excess washed with PBS buffer.

Negative and positive control
The positive control of this antibody is lung tissue cancer as in Thermo Fisher Company, in which the cell membrane of these cells was visualized by chromogenic stain (brown color) ( Fig.1, B), whereas a negative control is a section from lung tissue cancer without adding primary antibody, so cell membrane was not stained with brown color (Fig.1A). The staining of lung tissue cancer slides was accomplished by using Thermo Fisher company protocol.

Scoring system
Colored cancer cells were calculated by selecting four areas, and total selected cancer cells were not less than 100 cells, at magnification power (10X and 40X). Colored cancer cells were calculated in hall slide, and cells with brown color in cell membrane consider being (positive), and cancer cells that were not colored in cell membrane being (negative). The percentage of colored cancer cells were divided as: Stained cells% (score 0: 0%, score +1: 10%, score +2: 20-30%, score +3: 40-100%) 14 .

Statistical analysis
Collected data analysis was accomplished by (statistical package for social science/SPSS version -24 software/ IBM): Chi square (X2) used to estimate the correlation between clinic-pathological correlations with expressions of the marker, and compered with control group. P-values valuable at the accepted level of significance in ≤ 0.05 was considered significant.

Results:
The results of 50 cases for this study referred to the fact that males' total numbers in control group were 2 (20%) statuses, while females were 8 (80%) statuses, with male: female (M: F) ratio 1:4 (the majority of statuses from females). In patients with gastric cancer group, the number of males was 24 (60%) cases, while females number was 16 (40%) cases, and 3:2 in (M: F) ratio (the majority cases from males). Age was divided into two age groups: equal or less than 50 years, and more than 50 years. Control group age ranged between  years for 10 (100%) statuses, which were within the age group equal or less than 50 years with mean age (33.9 ± 10.027), and the age of patients with gastric cancer which ranged from (19-83) years with mean (55.325 ± 15.423). Patients number of equal or less than 50 years was 16 (40%) cases with mean (39.875 ± 9.493), and the number of patients in more than 50 years was 24 (60%) cases with mean age (65.625 ± 8.234). Histopathological subtype distributed in: intestinal type: patients' numbers were recorded 23 (57.5%) cases. Diffuse type: 15 (37.5%) cases of the total number of patients, whereas mixed type: patients were registered 2 (5%) cases. The most cases for grade were moderately differentiated, which were registered 25 (62.5%) cases, while poorly differentiated were 15 (37.5%) cases of the total number of patients. Also, the current study showed that gastric cancer invasion (subserosa) found in about 15 (37.5%) cases. In invasion (serosa) total patients were 18 (45%) cases, while (muscularis propria) invasion were 7 (17.5%) cases in total patients. The total number of patients that diagnosed with lymph node involvement was about 32 (80%) cases, whereas patients without lymph node involvement total number was 8 (20%) cases. Invasive depth and lymph node metastasis (TNM) were used to distribute gastric cancer patients into advanced stages (III & IV), and early stage (II). Advanced stages (III & IV) total patient numbers were 27 (67.5%) cases. The total number of patients was about 12 (30%) cases in stage III, whereas IV stage total patient number was 15 (37.5%) cases. The total number of patients in early stage (II) was 13 (32.5%) Tab. 1

The IHC expression of CD63
This study shows that CD63 expressed in high level in patients with gastric cancer only reached 40 (100%), whereas control groups were not expressed in any status for this marker. In patients with gastric cancer (score 0) was not expressed CD63 in any case, while number of patients expressed CD63 at (score +1) were 16 (40%) cases, 10 (25%) cases in (score +2), and 14 (35%) cases at (score +3). This difference in expression level between two groups leads to statistically significant association at P ≤ 0.05, P= 0.0001 as shown in Tab.2, and (Figs.2, 3, 4, 5).    Tab.3 shows the expression of CD63 correlated with clinic-pathological parameters. This study donates that 60% of total numbers in gastric cancer patients were positively expressed CD63 in males, which was recorded in 24 cases, and (40%) 16 cases for female, statistically, no significant association between gender and the expression of CD63, P-value were larger than 0.05 (P > 0.05). Patients' age ranged from 19 to 83 years, patients from less or equal 50 years were recorded (40%) in expression of CD63, and 60% in patients more than 50 years. P-value = 0.579 larger than 0.05 (P > 0.05) so there was not a significant association between age and the expression of this marker. The current study showed that the total number of patients with intestinal type that positively expressed CD63 were 57.5%, diffuse were 37.5%, and 5% in mixed type. These differences made a significant association statistically at (P ≤ 0.05) P= 0.041. There was an expression of CD63 in patients with gastric cancer at moderately differentiated grade 62.5%, more than in poorly differentiated grade, which recorded 37.5% but there was not a significant correlation (p=0.164). The positive expression of CD63of invasion depth recorded 15% in pT2, and 85% in pT (3 &4), a significant association statistically at P ≤ 0.05, p=0.025. Also, the study indicated that there was 80% of CD63 positive expression in patients involving lymph nodes (N1, N2&N3), whereas in patients without involvement of lymph nodes (N0) patients were 20%. Statistically, these differences made a significant association between involvement of lymph nodes and the expression of this marker, p=0.0001. The highest records of marker positive expression were in advanced stage (III&IV) of gastric cancer in 67.5%, while it was 32.5% in early stage. Statistically, these differences made a significant association between stage of gastric cancer, and the expression of marker at p ≤ 0.05, p= 0.0005.

Discussion:
In Iraq malignant neoplasm's represented the second leading cause of death. GC trend demonstrated an instant rise after 2007; it is a fifth of eighteenth cancers in Iraq 2 . GC comprises a universal health issue. It is a malignancy disease with high aggressive nature. It is one of the most prevalent reasons of cancer related death, and takes advantage of an important encumbrance on international health sponsor 1 . Gastric cancer treatment is restricted because of its heterogeneity and genetic complicated 16 so finding of special biomarkers is important for management of the development of gastric cancer, and identified the effective treatments for patients 17 . The current study indicated that the total number of male more than female in about (1.5:1) in (M: F) ratio (the majority cases from males) and the number of older patients (more than 50 years) more than younger (equal or less than 50 years), The result of the current study is agrees with several previous Iraqi studies which indicated that the males are the most gastric cancer incidence than females in about (2.3:1) as in Hermize et al., 18 , while the study of Razak et al., 19 declared that the males' higher percentage than females in about 58%, 42% for males, and females respectively. Also, the study of Saeed, 20 which found that males' ratio is more than females, and in another review to the same researcher found that males recorded double in ration (2:1) 21 . Furthermore, the result of this study is corresponded with the different Arab countries studies: as in Bahrain study, which found that gastric cancer in males recorded a high incidence compared with female about doubles (2:1) 22 . In study from Yamen there was a difference in the incidence in gastric cancer between genders in about (2.5:1) (M: F) 23 . Globally, the results of this study matched with the study of Lou et al. 24 , which is showed that men are higher incidence than women, and the study of Li et al., 25 that founded that males recorded a significant higher incidence than female in (number of patients, stages, and grade) of gastric cancer. Moreover, as in the Globocan 2012 report, standardized rates of age of gastric cancer was twice more in men than in women 26 . The study of Radkiewicz et al. 27 demonstrated that rise of risk for gastric cancer is associated with male sex, the portion of cancer explicated by factors correlated to male sex is huge, and males suffer poorer survival in most cancer sites. A probable exposition is either the preventative impact of estrogen in women, or other effects as diversity in diet and occupational exposure may participate to raise gastric cancer incidence in males 28,29 . This study indicated that the higher ratio of gastric cancer incidence at ages more than 50 years in ratio 60% with mean 65.625, which matched with several Iraqi studies as the study of Hermiz et al., 18 , which mentioned that the ratio of gastric cancer incidence increases with age, and the most diagnosis cases were up to 50 years in age ranged from 30-80 years. In study of Lafta and Al-faisal, 30 the two researchers mentioned that the elderly people are the most incidence exposure to gastric cancer than younger. In Saeed's et al., 21 , they mentioned that the gastric cancer incidence raised with age in age 50 years and more, compared with patients less than 50 years, and in mean age about 59 years. Furthermore, this study is corresponded to many Arab countries, which found that gastric cancer incidence increases with age.  37,38 . Also, in 39 author reminded that (54%) of cases were intestinal subtype, which are located in distal stomach (non-cardiac), and it is the highest ratio compared with diffuse, and mix type. This study corresponded with the most Arabic studies, as in Saudi Arabia study by Alahmadi et al., 32 , which indicated that 91% of the cases were intestinal subtype, and with Egyptian study by Badary et al., 40 , which mentioned that cases distributed as follows: intestinal subtype was the common subtype in ration 59.5%, diffuse 21.4%, and mixed 19%. Also, with the study from Yamen by Kassim et al., 23 , the total number of cases was 130 cases; about 82.5% of these cases were intestinal subtype. The results of this study disagree with some Iraqi studies as Al-Kaptan, 41 , which indicated that the most diagnosis cases were diffuse subtype in about 63% among other subtypes. Also, the current study does not match with some Arabic studies, such as the study of Awad et al. 42 in Jordin, which refers to that about 52% of cases were diffuse subtype. Globally, this study does not corresponded to a study from Vietnam by Phan et al. 43 study, which mentioned diffuse subtype occurs in 52% of study cases, and with a study in Brazil by Braga-Neto et al., 44 , which declared that diffuse is appearing in younger patients in about 70%, comparing with elderly intestinal subtype that recorded about 33.7%. The construction of these differences in results is that these two subtypes of gastric cancer develop out of various mechanisms, the intestinal subtype is more correlated to environmental factors as dietary factors, lifestyle, and H. pylori infection; the diffuse subtype is more related to genetic factors 19 . Also, these differences in results may be due to environment variation, occupational, and geographical differences, ethics, differences in the size, and type of the samples: some of researchers depends on biopsies, and other on surgical excision samples. The current study referred that the moderately differentiated was the common differentiated grade among diagnostic cases in about 62.5%. This result matches with several previous studies in Iraq. In study of Abdulla et al., 45 , which indicated that the moderately differentiated is the general differentiated grade among other grades. Also, with the study of Abdul Jabar and Al-Faisal, 46 , they mentioned that about 44.4% of diagnosis cases were moderately differentiated. In study of Ashour et al., 47 , this indicated that about 63.3% of cases were moderately differentiated. Moreover, this study matches with Mohammed and Raziq, 36 , which declared that about 52.38% of diagnosis cases were moderately differentiated grade. Furthermore, the current study also agrees with some Arabic, and global studies, as in some Egyptian studies, which refers that about 61.8% was the moderately differentiated grade ratio of diagnosis cases, while poorly differentiated grade ratio about 27.6% (Abdel-Salam et al. 48 , and Harras and Mowafy 49 study, which indicated that moderately differentiated grade was the common grade among other differentiated grades in about 52%, and 36% for poorly differentiated. In a study from Mexican found that moderately differentiated grades are the common grade comparing with other differentiated grades in ration 55.5%, and 27.7% for poorly differentiated grades 50 . In Indian study by Raj et al., 51 demonstrated that in ratio 46% the moderately differentiated were the common differentiated grades among all patients cases. This study does not correspond with some previous Iraqi studies such as: Lafta and Al-Faisal, 30 study mentioned that about 80% of cases were poorly differentiated. Also, the study does not match with some Arabic, and global studies, in Saudi study by Alahmadi et al.,32 , which refers to that about 50% of cases were poorly differentiated grade, and in study from Tunis by Gharsall et al., 52 , which mentioned that about 47.83% of cases were poorly differentiated. Also, the study of Zhang et al. 53 , which found that poorly differentiated was the domain differentiated. The result of this study refers that advanced gastric cancer (third and fourth) were higher than early stage (second) in about 67.5%, which match with several previous Iraqi studies. In Al-Kaptan, 41 study found that about 75% of diagnosis cases were in advanced stage. In Hermize et al., 18 study, which indicated that third advanced stage was the higher than early stage in about 93%. Also, in Ashour et al., 47 , which declared that the advanced stage (third and fourth) were the highest comparing with early stage (second) in ratio 53.3%. Furthermore, the study of Mohammed and Raziq,37 , which found that 66.15% cases were in advanced stage. The current study is in agreement with some Arabic studies. A study by Alahmadi et al., 32 from Saudi Arabia, which refers to the fact that advanced stage, was higher than early stage in about 59.1%, 40.9% respectively. Globally, the result of this study is corresponded with several previous studies. The study of Braga-Neto et al., 44 , which mentioned that advanced gastric cancer was in high ratio in about 60.9%, comparing with early stage in about 39.1%. The study of Feng et al., 54 , declared that advanced stage was raised in ratio 70.1%. Also, the Li et al., 55 study that indicated about 50.3% of diagnosis cases were in advanced stage of gastric cancer.
The current study is not in agreement with some previous Arabic studies, which demonstrated that early stage of gastric cancer was the general stage comparing with advanced. In Abdel-Salam et al., 48 study from Egypt refers to that the ratio of early stage was about 56.6%, and advanced in 43.4%.
Furthermore, when comparing some global studies, the result of this study is not corresponded with it, as a study by Katai et al., 56 , which mentioned that early stage was the common stage comparing with advanced in ratio 58.7%, while about 41.3% for advanced. Also, a study by Silva et al., 57 which refers to that early stage recorded in about 54.2%, while advanced 45.8%. Gastric cancer is a disease that is often times revealed delayed, at a stage when medical treatment is difficult to achieve. The identification and demarcation of early precancer harms more defy than in the esophagus, and colon because of the numerous number of folds, known as rugal folds, that increase surface area of the stomach 58 . GC starts without any apparent signs of symptoms until advanced stages; therefore, it is not easy to diagnose at early stage without routine screening by endoscopy. It is a heterogeneous disease from histological, and molecular estimation, also the diversity, and complexity of this disease is not enough evident understand of molecular mechanisms including in tumorigenesis, tumor progression, and metastasis of its 59 . This can explain the high ratio of advanced stage comparing with early stage of gastric cancer. Furthermore, the causes of differences in results may be due to partly result by screening, mechanisms, plans that depending on diagnosis patients. Also, using advanced techniques in development countries such as: computed tomography (CT), which can detect early small lesions. Moreover, the differences in the type of samples, and the number of studying samples. Tab.3 showed the expression of CD63 correlated with clinicopathological parameters, which indicated that there was not significant association in positive expression of the marker, and age, gender, and grade, but significant in invasion depth of tumor, and lymph node involvement; these foundlings resemble the results study of Miki et al. 14 . CD63 have a major role in niche formation metastasis 60 ; this study mentioned that this marker is a tissue inhibitor of metalloproteinase-1(TIMP1) receptor, which generates a tumor niche in a small environment of the liver, causing in metastasis of pancreas cancer cells in liver. Knockout of TIMP1 or CD63 in mice was not consistent liver metastasis by cancer cells of pancreas injected. This leads to infer that TIMP1 (pancreatic cancer cells) use CD63 receptor to activate stellate cells (liver), which intermediate the formation of niche to create metastasis. Furthermore, CD63, and TIMP1 coexpression have been recorded in glioblastoma, and astrocytoma patients 61 . Moreover, CD63 has also been involved in coordination with the roles of the tumor development-associated protein, membrane-associated type-1 MMP in extracellular matrix transformation, which leads to rise cell invasion, and metastasis 62 ; this can explain the significant correlation between positive expression of CD63, and invasion depth of GC, involvement of lymph node, and stage of GC (TNM) of the current study. The interaction between CD63, and numerous other proteins as CD9, CD81, and β1 integrins participating to the downstream of the signaling pathway of the cell 63 . Seubert et al 64 remind that this marker rise the intrinsic metastatic predisposition of tumor cell by initiating β-catenindependent in the epithelial-mesenchymal transition, that influence on the plasticity of the cell in ovary cancer, gastric cancer in human, and mouse melanoma cells. Furthermore, as in study of Mohammed et al., 65 , which mention that Fumonisin B1 (FB1) (a mycotoxin produced in some grains specially in corn) by Fusarium species has a significant effect on TGF-β1 and p16 protein expression, so its role in cancer development is proposed. Moreover, a decrease in E-cadherin together with raised Vimentin, MMP-2 and MMP-9 are significant markers that association with poor prognosis of transitional cell carcinoma TCC, also increase in men comparing to women in age up 50 years 66 .

Conclusions:
The results of the currents study indicated that CD63 could be a good prospective marker for Iraqi patients with gastric cancer. Cancer cells exosomes include many contents as: (proteins, DNA, mRNA, miRNA, lncRNA, and circRNA). Some of them act as biomarkers; we can take these features for cancer: early detection, early diagnosis, prognosis prediction, and therapeutic efficacy evaluation, and develop new strategies relying on engineered exosomes carrying with tumorsuppressing proteins, nucleic acid components, or targeted drugs function as precision medicine.