Abstract
Background: Esophageal carcinoma is a malignancy with a poor prognosis and new treatment modalities must be sought. One possibility that has been tested in patients with malignant melanoma is treatment which aims towards boosting the immune system. In the present study, we investigated the role of mast cells in patients with esophageal carcinoma. The intention was to determine whether a higher number of mast cells is associated with better survival and may thus be a marker for future immunotherapeutic studies. Materials and Methods: A total of 61 archived tumor samples were retrieved of patients having received treatment due to esophageal carcinoma at the Department of Oncology, Uppsala University Hospital, Sweden. The tissue specimens were fixed in formalin, embedded in paraffin and sectioned in 3 μm-thick sections. The monoclonal antibody G3, recognizing the mast cell-specific protein tryptase was used, and the avidin—biotin—horseradish peroxidase complex (ABC/HRP) and diaminobenzidine (DAB) techniques were used to visualisze tryptase-positive cells. The positive cells were counted in 10 randomly selected high power fields. Results: When the number of mast cells was investigated in conjunction with relapse, no correlation was found (p=0.38). The number of mast cells was also not associated with survival (p=0.96). Using a pre-defined cut-off value of 31, no significant changes in survival was found (p=0.79) between patients with mast cell numbers above this value compared to those with numbers below. Conclusion: We conclude that mast cells do not seem to be related to prognosis in patients with esophageal carcinoma.
Esophageal carcinoma is the seventh most common cause of cancer-related death in the Western World and the incidence of esophageal adenocarcinoma is increasing (1).
The major challenge in the treatment of esophageal carcinoma is to reduce the risk of local recurrence. However, within the first year after surgery (or curatively intended radiation therapy), the majority of these patients relapse and the median survival is dismal (2, 3). Since currently available treatments seldom cure these patients, new treatment modalities must be sought for. One possibility that has been tested in patients with malignant melanoma is treatment which aims towards boosting the immune system (4). One of the major players in the immune system is the mast cell (5) Several studies have been published in which the role of mast cells in different malignancies was studied, as well as their role in angiogenesis. Mast cells have been shown to have antitumor functions (6, 7) and Henderson et al. demonstrated that mast cells are involved in the peroxidase system, the biological products of which are able to function as antitumor compounds (8, 9). In the present study, the role of mast cells was investigated in patients with squamous cell carcinoma as a pilot study of whether there seems to be a correlation with survival or clinical parameters.
Materials and Methods
Between 1990 and 2000, 126 patients were recorded as having received treatment due to esophageal carcinoma at the Department of Oncology, Uppsala University Hospital, Sweden. from these patients, a total of 61 tumor samples were retrieved and were investigated for the analysis of mast cells. The excluded patients did not have sufficient tumor material for the present analysis.
The following clinical parameters were recorded: age; gender; localization, grouped into an upper (15-24 cm), a middle (25-34 cm) and a lower (35-46 cm) part of the oesophagus; performance status at first admittance; smoking; histology and tumor stage at first admittance defined as localized or metastatic disease.
Immunohistochemistry. The tissue specimens were fixed in formalin, embedded in paraffin and sectioned in 3 μm-thick sections. The monoclonal antibody G3, recognizing the mast cell-specific protein tryptase was provided by Dr L. B. Schwartz, MCV, Richmond, VA, USA (10). The avidin—biotin—horseradish peroxidase complex (ABC/HRP) and diaminobenzidine (DAB) techniques were used to visualise the tryptase-positive cells. The positive cells were counted in 10 randomly selected high power fields under light microscopy by one author (Figure 2).
Clinical characteristics of the included patients as well as the median number of mast cells and univariate analysis of investigated clinical parameters.
Statistics. The survival functions were estimated with the Kaplan-Meier product limit method and the median survival time estimated with linear interpolation of the survival function. The log-rank test was used to compare survival in two groups. The backward stepwise logistic regression method was utilized for univariate statistical interpretation of the data. Cox regression was utilized for the multivariate analysis. Throughout this study, a 5% significance level was used in the statistical tests.
Results
Tumors from a total of 61 patients with squamous cell carcinoma of the esophagus were investigated for the presence of mast cells. Patient demographics and statistical analysis concerning differences between median mast cell counts and clinical variables are shown in Table I. Mast cells were detected in all patients. Table II shows the correlation analysis in which the mast cells have been investigated in conjunction with clinical parameters. When the number of mast cells were investigated in conjunction to relapse, no correlation was found (p=0.38). The number of mast cells was not associated with survival (p=0.96). Using a pre-defined cut-off value of 31 (as decided by the authors based on clinical observations from other solid tumors, data not published), no significant changes in survival was found (p=0.79) (Figure 1).
Correlation analysis investigating the role of mast cell-numbers and clinical parameters in patients with esophageal cancer.
Discussion
In the present study, we report that mast cells do not seem to be associated with prognosis in patients with esophageal carcinoma. We were unable to distinguish any clinical correlation of mast cells in the tumors, contradicting the role of modulating the immune system in patients with esophageal carcinoma. Patients with esophageal carcinoma are in general subjected to preoperative chemoradiotherapy followed by surgery, or in inoperable cases, curatively intended chemoradiotherapy. Despite these intense treatment modalities, survival rates are poor and this has increased the awareness of other available treatment modalities for this patient category. Immune-modulating treatments have much interest for patients with malignant melanoma and there are at least three approaches to boost antitumor CD8 T-cell responses in these patients (5). In patients with esophageal carcinoma, Toh et al. (11) injected autologous tumor-activated lymphocytes into patients with distant metastases from esophageal carcinoma, with clinically beneficially results. However, the numbers of studies in this field are limited for esophageal carcinoma patients. One of the major players in the immune system is the mast cell and this cell-type has been shown to regulate different functions in the inflammatory process (12) in which they seem to regulate, amongst other things, vascular functions (13). When the mast cells are stimulated, they release the contents of their granules, thereby activating leukocytes (14). Since tumor cells are surrounded by inflammatory cells, the role of the mast cells has gained an increasing interest in the field of tumor biology (15) and the mast cell has emerged as candidate for mediating tumor promotion (16). The issue of vascular invasion has been studied by several authors and it has been shown that mast cell granules produce or attract, amongst other things, histamine (17), basic fibroblast growth factor (18) and heparin (19). All these factors have been attributed to the promoting of tumor angiogenesis and, furthermore, mast cell tryptase was recently found to be a potent angiogenic factor, thus confirming these results (20). Moreover, in lymphomas, mast cells are suggested to cooperate in the induction of angiogenesis associated with disease progression (21). Antitumor effects have been reported in studies on breast cancer (22) as well as colorectal neoplasias (23). Concerning esophageal carcinoma, Tomita et al. (24) showed that in their material from 48 patients with squamous cell carcinoma of the esophagus, there was a significant correlation between mast cell count and microvessel density. These studies suggest that mast cells might release substances intervening with and possibly affecting the tumor cell, thus causing tumor death. Other studies, however, have shown that mast cells enhance tumor proliferation in vitro (25, 26). In the present work, the number of mast cells within the primary tumor was not associated with survival (p=0.96). Nor was there any correlation with relapse (p=0.36). To further diminish the role of mast cells, no clinical correlation was found (Table II). In a study by Elpek et al., 53 patients with squamous cell carcinoma of the esophagus were investigated regarding correlation of mast cells and angiogenesis. The authors concluded that a high number of mast cells in conjunction with increased angiogenesis is associated with poorer prognosis (27). The role of mast cells is not fully conclusive for patients with esophageal carcinoma. In conclusion, in the present study, to our knowledge the largest in which the role of mast cells has been investigated in patients with esophageal carcinoma, we did not find any clinical correlations or associations of mast cell numbers with relapse or survival, implicating a limited role of the immune system in this tumor type, possibly negating there being a future role of immunotherapy for these patients.
Survival analysis for all patients based on a cut-off of 31 mast cells.
Immunohistochemical staining of mast cells in a patient with esophageal carcinoma.
Acknowledgements
The Authors are in debt to the Foundations Stiftelsen vid Onkologiska klinikens forskningsfond and Stiftelsen Gävle Cancerfond for providing funds for the present study. Furthermore, the Authors would like to express their gratitude to Martin Dreilich MD, Ph.D., Micael Strandell MD, as well as Rose-Marie Amini MD, Ph.D., for assistance in the evaluation of the clinical charts, as well as in the evaluation of the mast cells. The Authors also express their gratitude to Patrik Hesselius Ph.D. for statistical guidance.
Footnotes
- Received October 30, 2009.
- Accepted December 14, 2009.
- Copyright© 2010 International Institute of Anticaner Research (Dr. John G. Delinassios), All rights reserved
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