Analysis of the Expression of Cathepsin S in Gastric Adenocarcinoma and in Helicobacter Pylori Infection

Background: recent experimental studies have shown a potential link between cathepsin S (CTTS) and gastric cancer progression. Herein, we aimed to evaluate the expression of CTTS in gastric adenocarcinoma. Methods: Cross-sectional study that included two groups, gastric adenocarcinoma (n=42) and gastritis (n=50). The gastritis group was then subdivided into H. pylori positive (n=25) x negative (n=25). Gastric tissue samples were analyzed in order to determine the CTTS expression through immunohistochemistry. Results: In patients with gastritis, the age ranged from 18 to 78 years. Among them, 34% were male, and 66% were female. In patients with gastric cancer, the age ranged from 37 to 85 years. Among them, 50% were male, and 50% were female. When comparing the expression of CTTS between the two groups, only 16% of the gastritis samples had an expression higher than 25%. On the other hand, among patients with gastric adenocarcinoma, 19% had expression between 25-50%, 14.3% between 51-75%, and 26.2% had expressions higher than 75% (p < 0.001). CTTS expression was signicantly higher in patients with positive test for H. pylori: 87.5% x 38.5% (p<0.001). There was no statistically signicant association between the positivity of CTTS and the clinical-pathological variables, including tumor staging, histological type, angiolymphatic invasion, recurrence, current status and death. Conclusion: CTTS has a higher expression in samples of gastric adenocarcinoma. Patients with gastritis by H. pylori also show a higher expression of CTTS compared with patients with negative results for this bacterium.


Introduction
Cathepsins are enzymes that comprise a family of 15 lysosomal proteases widely distributed in intracellular and extracellular spaces, among which ve have been implicated repeatedly in the progression of solid cancers (cathepsin B, H, K, L, and S) [1,2].
Cathepsins plays roles in a wide range of body activities based on their hydrolysis effect. In digestive cancers, the expression of cathepsin is positively regulated by tumor-promoting factors, such as C-myc, Kras, AGR2, MAPK, p38, and the Hedgehog (Hh) signaling pathways. Activated cathepsins hydrolyze growth factors, such as EGF, VEGF and TGFβ, promoting the proliferation of cancer cells, and they appear to play a role, although still uncertain, in the regulation of apoptosis [3,4]. In addition, analyses of expression of cathepsins in tumor microenvironments have sparked discussion about the role of these molecules in the response to anti-cancer therapy and in the phenomenon of therapeutic resistance [5].
Recently, studies have pointed to a supposed relation between gastric cancer and cathepsin expression, speci cally cathepsin S (CTTS). Data are however still incipient, suggesting a therapeutic, prognostic, and diagnostic potential of this enzyme in the evolution of this disease [6]. In vitro studies have shown that increased CTTS expression is related to increased tumor invasion and metastasis, and that its inhibition Page 3/16 is capable of preventing tumor cell invasion and migration in gastric cancer [7,8]. Parallel to this, studies have also shown that the serum expression of CTTS may be a great ally in the early diagnosis of gastric cancer, even presenting a sensitivity superior to the usual tumor markers, such as CEA, CA 19.9, and CA 72. 4 [8].
The present study aims to evaluate the expression of CTTS in gastric tissue samples of patients with gastric adenocarcinoma and compare it with the expression in gastric tissue samples of patients without cancer, only with gastritis. In addition, this study seeks to evaluate the impacts of H. pylori infection on CTTS expression in gastric tissue samples without cancer.

Study design
This is a cross-sectional study carried out at Hospital das Clínicas, Federal University of Pernambuco, Recife, Brazil, aiming to evaluate the expression of CTTS in gastric tissue samples of patients diagnosed with gastric adenocarcinoma (n = 42) and of patients diagnosed only with gastritis (n = 50). The group of patients with gastritis was subdivided into another two subgroups: one with a positive result for H. pylori (n = 25) and one with a negative result for H. pylori (n = 25). The primary result was to compare CTTS expression assessed by immunohistochemistry in gastric tissue samples from patients with adenocarcinoma and patients with gastritis (with and without H. pylori).
All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional research committee and the 1964 Helsinki declaration and its later amendments, or comparable ethical standards. This research's protocol was approved by the Ethics Committee of the Hospital das Clínicas da Universidade Federal de Pernambuco (HC/UFPE-EBSERH) under the protocol CAAE no. 38000620.9.0000.8807. Informed consent was obtained from all participants in the study.

Selection of patients
We included patients undergoing surgical treatment of gastric adenocarcinoma with curative intent in our center from 2017 to 2019. We excluded patients at stage IV and those undergoing neoadjuvant chemotherapy. After each surgical procedure for the treatment of gastric adenocarcinoma, we always selected the rst patient to present a con rmed histopathological result for gastritis in the Pathology sector of our institution, aiming to form the control group with the lowest possible risk of selection bias.
This group included patients who underwent esophagogastroduodenoscopy (EGD) with biopsy of lesions, con rming that it was gastritis. The search for H. pylori was performed in all patients using the urease test and con rmed by histopathology with Giemsa stain. We excluded patients previously submitted to gastroplasty and those with reports of previous treatment for H. pylori.

Immunohistochemistry
We performed immunohistochemical staining (IHC) to study the expression of CTTS in 42 samples of human gastric cancer tissue (Fig. 1) and in 50 samples of gastric tissue with gastritis (Fig. 2). We made 3-µm sections in series for immunohistochemical analysis and placed them on Superfrost Plus glass slides. We performed immunostaining using the Ventana BenchMark ULTRA System automated staining system using rabbit polyclonal antibody directed against CTTS (Clone No. A13482; ABclonal, Massachusetts, United States). We used a 1:100 dilution and incubated it for 30 min to 37 ºC. We used the DAB IHC Detection Kit as the chromogen substrate. All specimens were counterstained with hematoxylin. We interpreted immunohistochemical reactions using a standard optical microscope and analyzed them according to the speci c pattern of the investigated antibody. We assessed marking intensity using the following grading: For analysis purposes, the CTTS expression intensity was categorically assessed: high expression or low expression. We de ned a high expression as a color index score > 4, while low expression was a score ≤ 4. An index = 0 corresponds to a missing expression.  When comparing the expression of CTTS between the two groups, in gastritis samples 38% did not express CTTS, 46% had low expression (1-25%), and only 16% had an expression higher than 25%. On the other hand, among patients with gastric adenocarcinoma, 19% had expression between 25-50%, 14.3% between 51-75%, and 26.2% had expressions higher than 75%, with statistically signi cant results (p < 0.001). Analyses involving the CTTS staining index in IHC and the intensity of expression also showed a statistical signi cance, being higher in the group of patients with gastric adenocarcinoma ( Table 2). In the evaluation of CTTS expression in the group of patients with gastritis, CTTS expression was signi cantly higher in patients with positive test for H. pylori: 87.5% x 38.5% (p < 0.001) ( Table 3). In the evaluation of CTTS expression in the group of patients with gastric adenocarcinoma, there was no statistically signi cant association between the positivity of the expression and the clinical-pathological variables presented in Table 4.

Discussion
The cathepsins that have already shown an increased expression in the presence of gastric cancer are B, E, K, L, S, X, and Z. To date, there are only a few studies that sought to assess the relation between CTTS and gastric cancer [7,8]. This enzyme appears to play an important role in the tumor invasion process through the degradation of the extracellular matrix, modulation of the immune response, and regulation of several cell signaling pathways, including the activation of tyrosine kinase receptors, especially c-Met, matrix metalloproteinases, IL-11, CXCL16, and Integrin alpha-6-beta-4 [2,7,9]. Still, speci cally for gastric adenocarcinoma, CTTS appears to have an activating effect on the MKN7 and MKN45 cancer cell lines [7]. Yang et al. [7] studied the expression of cathepsins through a proteomic analysis of cultures of normal cells and gastric cancer. We observed a higher protein expression and a positive regulation of cathepsin S in gastric cancer cell secretome. There were no statistically signi cant differences in CTSS expression between the intestinal, diffuse, and mixed subtypes.
Researchers have shown a correlation between CTTS and disease characteristics, such as tumor size, lymph node invasion, distant metastases, and overall survival, noting that higher CTTS expressions were related to more advanced TNM stages and worse survival rates [8]. In the present study, there was no statistically signi cant association between CTTS expression and tumor staging or survival rates. A possible explanation for such a difference between the studies is the number of patients included, which was noticeably lower in our analysis.
Infection of the gastric mucosa by H. pylori is known to be an important risk factor for the development of gastric adenocarcinoma. However, the exact mechanisms of activation of carcinogenesis are not yet fully elucidated [10]. One of the possible mechanisms pointed out in this process is the pro-in ammatory response orchestrated by Th17 cells in the infected gastric mucosa [11,12]. Previous studies have shown an association between infection by H. pylori and increased levels of expression of cathepsins D and X. However, there are no studies determining the behavior of CTTS in the presence of an infection by H. pylori [13,14]. In the present study, we evaluated the expression of CTTS in samples of gastric mucosa infected by H. pylori. We observed that 87.5% of the samples in the gastritis group with H. pylori showed positive expression for CTTS, contrasting with only 12.5% of the gastritis group without H. pylori. These results reinforce the hypothesis that CTTS is involved in the process of carcinogenesis of gastric adenocarcinoma, since it also has a higher expression.
This study has some limitations that deserve attention. First is the sample size, which, as a result of the single-center character of this study, was limited, with a sample power of 72.7%. As the sample was nonprobabilistic, and selected by convenience, we did not calculate the sample size since we included in the analysis all patients operated on during the study period. Another limitation that is worth mentioning is in relation to the observational and cross-sectional nature of this study. A longitudinal analysis could provide more accurate information about the relationship between CTTS expression and patient survival.
However, for our primary result, the methodology applied was adequate.
In contrast to the limitations discussed above, the present study reports important data that provide robustness and authenticity to the analysis. It is one of the few studies to study the expression of CTTS in samples of gastric adenocarcinoma in humans and the rst to attest a possible relationship between the expression of this enzyme and infection by H. pylori, an important risk factor for the development of gastric adenocarcinoma.

Conclusion
Considering the results of the present study, the authors conclude that CTTS has a higher expression in samples of gastric adenocarcinoma compared to samples of non-tumor tissue. In addition, as a secondary nding, we report that patients with gastritis by H. pylori also show a higher expression of CTTS compared with patients with gastritis with negative results for this bacterium.