Mast cell density in gastric cancer and its relation to aggressive behavior

Introduction: Stomach cancer is a serious health problem worldwide, as it is one of the most common cancers and the fourth leading cause of cancer-related death. Mast cells perform functions in the immunity system and are a type of discriminated myeloid cell. In this study, we evaluated the correlation between tryptase-positive mast cell density with prognostic histopathological findings in gastric cancer. Materials and Methods: The study was conducted as a cross-sectional study, using tissue samples from 40 patients who underwent radical gastrectomy at Sina Hospital between 2022 and 2023. After histopathological examination and determination of tumor histopathological characteristics, the samples were subjected to immunohistochemical staining using a monoclonal antibody against mast cell tryptase. Results: In this study, the median density of mast cells in tumor tissue was 8/10 high power fields. There was no significant relationship between mast cell density and the number of lymph nodes involved, as well as tumor type, grade, location, and size. Furthermore, there was no significant relationship between mast cell density and tumor vascular invasion or neural invasion. Conclusion: Mast cells have vital roles in normal immune systems and pathological situations. Mast cell density in tumor tissue might be considered for the prognosis of patients before treatment but the function of mast cells has not been completely explained in gastric cancer and needs confirmation to introduce new target therapy. Mast cell density in gastric cancer and its relation to aggressive behavior Elham Nazar¹, Elahe Farmani¹, Aysan Nozheh¹* 1. Department of Pathology, Sina Hospital, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Corresponding author: Aysan Nozheh Department of Pathology, Tehran University of Medical Sciences, Tehran, Iran https://orcid.org/0009-0007-8862-3008

Introduction: Stomach cancer is a serious health problem worldwide, as it is one of the most common cancers and the fourth leading cause of cancer-related death. Mast cells perform functions in the immunity system and are a type of discriminated myeloid cell. In this study, we evaluated the correlation between tryptase-positive mast cell density with prognostic histopathological findings in gastric cancer.

The study was conducted as a cross-sectional study, using tissue samples from 40 patients who underwent radical gastrectomy at Sina Hospital between 2022 and 2023. After histopathological examination and determination of tumor histopathological characteristics, the samples were subjected to immunohistochemical staining using a monoclonal antibody against mast cell tryptase.

In this study, the median density of mast cells in tumor tissue was 8/10 high power fields. There was no significant relationship between mast cell density and the number of lymph nodes involved, as well as tumor type, grade, location, and size. Furthermore, there was no significant relationship between mast cell density and tumor vascular invasion or neural invasion.

Mast cells have vital roles in normal immune systems and pathological situations. Mast cell density in tumor tissue might be considered for the prognosis of patients before treatment but the function of mast cells has not been completely explained in gastric cancer and needs confirmation to introduce new target therapy.

Stomach cancer, mast cell density, prognosis

Stomach cancer is the fourth most common cancer in the world and the second cause of cancer death [1-3]. Due to poor awareness, gastric malignancies are often diagnosed at a late stage, and because conventional treatments are ineffective, most patients with surgery have a very low survival rate, about five years, and die [4]. The mainly of solid cancers are related to chronic inflammation and infection [5]. The exact molecular mechanism of gastric cancer is unknown, and for this reason, it is considered to be a complex multi-step process due to interactions between environment and genetics. Helicobacter pylori infection, Epstein-Barr virus, and dietary habits are considered to be major environmental factors [6]. Chronic inflammation is an important feature in cancerous tissue, and mast cells have an important role in this inflammation site [7]. The relationship between the immune system and the development of cancer has been studied for a long time but is still not fully understood [8]. Gastric cancerous tissue has a combination of stomach tumoral cells and a variety of immune cells, which may exist in the tumoral mass or enter from other tissues [9]. Tumor outcomes are determined not only by the intrinsic characteristics of the cancer cells as well as by the interactions among tumoral cells and their microenvironment. Tumor-infiltrating immune cells are of great interest, but mast cells are less considered. Current investigations have emphasized the effect of mast cells on cancer cell properties and invasion, but the possible impact of mast cell infiltration remains controversial [10]. Human mast cells form a highly heterogeneous cell population with different morphologies, mediators, and surface receptors [11]. Mast cells are a kind of differentiated myeloid cells and have roles in allergic reactions (especially type 1), and in native and developed immunity response by recognizing and eradicating pathogens as well as releasing active immune mediators [12]. However, the exact function of mast cells in tumor formation and growth remains controversial: mast cell-derived mediators can perform pro-tumor functions, induce tumor growth and spread, or anti-tumor functions, or limit tumor growth [13]. Previous studies have determined that mast cell infiltration levels are increased in several kinds of tumor tissues of lymphoma and breast cancer which are related to the probability of mast cell function in the growth of the tumor [14]. Mast cells constitute a small subpopulation of tumor microenvironment (TME) immune cells in tumoral tissue. Mast cells gather both in the tumoral mass and the adjacent tissues. Mast cells affect TME transformation and progression by interrelating with other components of the TME. However, it remains controversial whether mast cells relate to tumor development or activate an immune response to the tumor [15]. The act of mast cells in gastric adenocarcinoma has been a part of important attention. Mast cell density may be involved in the expansion and prognosis of gastric cancer. We wanted to evaluate the association of mast cell density with the potential for lymph node metastasis and other histopathological findings in gastric cancer to predict aggressive behavior.

In this cross-sectional study, 40 tissue specimens from patients with gastric adenocarcinoma who underwent radical gastrectomy at Sina Hospital during 2022-2023 were subjected to immunohistochemical (IHC) staining using the monoclonal antibody against mast cell tryptase after histopathological examination and determination of tumor pathological characteristics. The association between IHC expression of mast cell tryptase and demographic and histopathological factors, particularly lymph node metastasis, was statistically analyzed. Tissue sections 4 μm thick were prepared from paraffin blocks on glass slides and autoclaved at 60°C overnight to perform IHC staining. The specimens were then immersed in xylenol three times for 15 minutes each, 100 percent alcohol twice for 10 minutes each, and finally 96 percent alcohol twice for 10 minutes. The sample was then cleaned with distilled water before being sterilized for 20 minutes at 95 ℃ and 2 bar pressure with 1 point 5 ml of EDTA buffer (pH 8). The slices were once more washed twice or three times in distilled water before being left out at room temperature for 20 minutes. Next, a peroxidase solution was used to block the internal peroxidase for 10 minutes. After being in contact with the mast cell tryptase monoclonal antibody (DAKO, USA) for 10 minutes, the samples were then washed with TBS buffer and a secondary antibody was added. The samples were once more washed with TBS buffer after 30 minutes, and the DAB chromatin was then exposed to the samples for 10 minutes. After being dehydrated, the sample is prepared for mounting and microscopic examination. The samples were lastly cleaned under running water, stained with hematoxylin, and placed in bicarbonate for 30 seconds. The IHC expression of mast cell tryptase and mast cell density (average in 10 high power fields (hpf)) were associated with demographic and histopathological factors, particularly lymph node metastasis, and statistical analysis was performed after the stained slides were examined by two pathologists. In tumor tissue, mast cell tryptase staining in cytoplasmic infiltrating immune cells is considered positive (Figure 1). Also, the study has been reported in line with the STROCSS 2021 criteria [15]. This study was approved by the Research Ethics Board of IR.TUMS. SINAHOSPITAL.REC.1401.061. Statistical analysis A P value of 0.05 or less was regarded as significant for all analyses, which were conducted using the statistical package for social sciences software (SPSS version 26; IBM Company). Baseline data were checked for normality using the Kolmogorov-Smirnov test and probability plots. Continuous data were shown as mean±SD, whereas categorical variables were reported in percent (%). Levene’s test for equal variance and Fisher’s exact test were used, respectively, to compare continuous and categorical variables using an independent t-test.

According to the study, there were 40 patients with gastric cancer with an average age of 63 years (minimum age 39 years and maximum age 84 years), of which 75% were men and 25% were women. In all patients, 80% of the tumors were intestinal type and 20% were signet ring. The lymphovascular invasion occurred in 50% of tumors. Additionally, 58% of tumors showed no neural invasion and 42% showed neural invasion. Patients had an average of 5 lymph nodes that were affected (the range was 0 to 32). In addition, the tumor tissue had an average mast cell density of 8/10 hpfs (the minimum and maximum values were 2/10 hpfs and 30/10 hpfs, respectively). Tumor sizes ranged from 1 to 11 centimeters. Baseline characteristic data was summarized in Table 1. This study discovered no statistically significant relationship between mast cell density and lymphovascular invasion (p=0.299), the number of involved lymph nodes (p=0.454), or tumor stage (p=0.366). Further research revealed no connection between mast cell density and neural invasion (p=0.227), tumor size (p=0.455), or tumor type (p=0.55). Table 1: Baseline characteristics of study subjects. | Gender, % | | | Male | 30 (75.0) | | Female | 10 (25.0) | | Histologic subtype | | | intestinal | 32 (80.0) | | Signet-ring | 8 (20.0) | | Histologic grade | | | I | 12 (30.0) | | II | 11 (27.5) | | III | 17 (42.5) | | Tumor location | | | Antrum | 24 (60.0) | | Body | 3 (7.5) | | Cardia | 11 (27.5) | | Fundus | 2 (5.0) | | Neural invasion, % | 17 (42.5) | | Lymphovascular invasion, % | 20 (50.0) | | Tumor stage, % | | | I | 3 (7.5) | | II | 7 (17.5) | | III | 23 (57.5) | | IV | 7 (17.5) | | Mean size, cm | 4.62±2.78 | | Total lymph nodes dissected (mean±SD) | 17.7±9.5 | | Involved lymph nodes dissected(mean±SD) | 4.9±6.7 | | Mean mast cell tryptase infiltrating cells | | | 8/ 10hpfs | Figure 1: Histopathological examination shows gastric adenocarcinoma by H&E staining and immunohistochemical staining of mast cell tryptase in infiltrating cells in the tumor (right).

As a solid tumor, gastric cancer includes immune cells as well as stromal, epithelial, and endothelial cells in addition to tumor cells. Infiltrating immune cells and stromal cells are two key non-tumor cells in the TME of gastric cancer. Numerous studies have demonstrated their importance for understanding the onset and progression of gastric cancer as well as its prognosis and drug resistance [16]. Mast cells are prominently found in solid tumors and show unique phenotypes depending on the tumor microenvironment. Their exact mode of communication in gastric cancer is still largely unknown. In the majority of tumors, such as thyroid [17], gastric [18, 19], pancreas [20-22], bladder [23], and colorectal [24, 25] cancers, hepatocellular carcinoma [26, 27], Merkel cell carcinoma [28], Hodgkin’s [29] and non-Hodgkin’s lymphoma [30] and plasmacytoma [31], mast cells conferred poor prognosis. Mast cells might act as an antitumorigenic factor in breast cancer [32]. These results suggest that mast cells’ involvement in cancer depends on the tumor. Mast cells in gastric cancer have only been the subject of a small number of studies. We still don’t know how mast cells affect the onset and progression of gastric cancer or how to control inflammation. LV Y, et al. discovered that patients with gastric cancer had tumors with noticeably more mast cells. Mast cell levels increased as the tumor developed and independently predicted a lower overall survival rate [33]. A recent study by Sammarco G, et al. showed gastric cancer patients have higher mast cell densities, which are connected with angiogenesis, the number of lymph nodes that have spread the disease, and the survival of these patients. By releasing angiogenic and lymphangiogenic factors, mast cells play a protumorigenic role in gastric cancer [34]. In another study by Ribatti D et al., commercially available samples from 30 patients undergoing curative gastrectomy were examined immunohistochemically using an anti-CD31 antibody to stain endothelial cells and an anti-tryptase and anti-chymase antibody to stain the mast cells. The results showed that stage IV gastric cancer has a higher degree of vascularity than other stages and tryptase and chymase-positive mast cells increase concomitantly with the degree of malignancy, even though the density of chymase-positive mast cells was significantly lower than those of tryptase-mast cell positive and is highly correlated with the degree of angiogenesis. This study demonstrated that angiogenesis and progression of gastric cancer patients are related to mast cell density. Understanding the mechanisms of gastric cancer angiogenesis provides a basis for a biological approach to the development of anti-angiogenic therapy in patients with this malignancy [35]. Zhong B et al. study showed there were more mast cells in the gastric cancer tissue than in the surrounding cancer tissue. Mast cell enrichment in gastric cancer tissue was found by flow cytometry and IHC staining, which may have a role in the growth of the disease [36]. The significant anti-tumor activity of mast cells revealed an as-yet unreported effect on upregulating tumor cell suppressor genes and downregulating pro-tumor genes [37]. Liu YL et al. study showed mast cell density was inversely related to the number of lymph node metastases and the depth of tumor invasion in patients with stomach cancer [38]. To better understand the functions of mast cells in tumoral tissue, preclinical evaluations were used in animal models with dysfunctional c-KIT. These studies also suggest that mast cells may facilitate vascularization, metastasis, and release of growth factors and enzymes from the tumor. These results thus show the possibility of new therapeutic approaches via the targeting of mast cells for precise chemotherapy or combined therapies. Animal models will make it easier to determine whether mast cells are harmful or helpful in the progression of cancer, though mouse data will always need to be properly validated in clinical patient settings [39]. In various pre-clinical models of solid tumors, mast cell-stabilizing agents like cromolyn sodium have been applied. Treatment with cromolyn slowed tumor growth in a mouse xenograft model of thyroid cancer [40]. In the peritoneal dissemination of gastric cancer, Interleukin-17 (IL-17) produced by mast cells aids in tumor fibrosis. A potential therapeutic approach to manage organ fibrosis involves preventing mast cell degranulation [41]. It appears that mast cell density is diverse in tumors that vary from all societies. In our study, the activity of these immune cells in predicting tumor histopathological and behavioral outcomes and consequent adverse cancer outcomes was completely questioned. In general, it can be assumed that in our culture the value of mast cell density in the assessment and estimation of aggressive behavior in gastric cancer is low, but it should be noted that the small sample size of the study could potentially limit the reliability of the results. Thus, further evaluation with a larger sample is strongly recommended. 5. Conclusion We need to be more accurate than tumor characteristics in predicting the prognosis of gastric cancer patients. Mast cells are frequently found in a tumor’s microenvironment, which suggests that they play a role in the development of cancer from chronic inflammation. Mast cell involvement in the solid malignancy microenvironment is still debatable. We also look forward to mast cells’ potential role in cancer immunotherapy, which could build on the success of current cancer therapies. Our data need confirmation in a larger patient cohort. Declaration of competing interest: We declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. also, we declare that we are preparing articles in our “personal capacity,” and we don’t use official representatives or articles on behalf of the government.

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Authors Metrics & Citations Metrics Article Usage 257views 135downloads Citations Download citation Elham Nazar, Elahe Farmani, Aysan Nozheh. Mast cell density in gastric cancer and its relation to aggressive behavior. Authorea. 06 March 2025. DOI: https://doi.org/10.22541/au.174126901.10018858/v1 DOI: https://doi.org/10.22541/au.174126901.10018858/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.