CHARACTERISTICS OF LEUKOCYTE INFILTRATION IN STOMACH TUMORS
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1
Acad. E.A. Wagner Perm State Medical University, Russia
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2
I.I. Mechnikov Research Institute for Vaccines and Sera, Russia
Stomach cancer incidence and mortality occupies one of the first places in the world. The study of etiology and pathogenesis of this disease and development of new treatments are topical problems of modern medicine. Tumors abundantly are infiltrated by leukocytes – neutrophils, macrophages and lymphocytes. To date, we obtained evidence that cancer pathogenesis is based on the local immune reactions caused by genetic and exogenous factors [1, 2]. Processes of initiation, promotion and metastasis of malignant tumors are significantly influenced by inflammatory cells located close to tumor. Immune cells affect tumor using various mechanisms, such as activation of matrix metalloproteinases, growth factors and angiogenesis. Infiltrating leukocytes produce different factors that stimulate formation of stroma (growth factors, interleukin-1, tumor necrosis factor-ά, fibronectin, various types of collagen, etc.) and different proteolytic enzymes [3]. Malignant tumors are able to induce and preserve a cancer microenvironment around itself that promotes their survival. Cancer cells of immune microenvironment include inflammation and tumor stromal tissue with blood and lymphatic vessels [4]. Malignant cells and immune cells ambiguously affect each other. White blood cells are able to inhibit both tumor progression and, on the contrary, contribute to it. In turn, tumor has a different effect on cell infiltration. The ratio of cells in inflammatory infiltrate is influenced by various factors: the stage, localization and histological type. On the early stages of malignant growth the predominant infiltrate cells are those with antitumor activity, and later – protumorigenic ones [3]. Many findings suggest the crucial role of immune cells in the tumorigenesis, but information about the mechanisms of interaction of inflammatory cells and the tumor is ambiguous and requires further detailed analysis.
Purpose - to study the cellular composition of leukocyte infiltrates of stomach wall affected by tumor process.
Materials and methods
Biopsies of 25 operated patients (11 men and 14 women) with various stages of gastric adenocarcinoma were studied. The age of patients ranged from 47 to 88 years.
All patients were divided into 4 groups depending on the degree of tumor differentiation. The first group consisted of 12 patients with a low degree of differentiation, the second group consisted of 7 patients with adenocarcinoma of moderate degree differentiation, two patients with high, and 4 patients had different degrees of differentiation.
Samples of material were embedded in paraffin. 5-6 microns sections were stained with hematoxylin and eosin. Infiltration of tumor nodules in the mucosa, submucosa and muscular layer of the gastric wall was evaluated by morphometric techniques. To assess the expression of markers of T-lymphocytes, B-lymphocytes, and macrophages immunohistochemical studies were performed using reagents for CD 3, CD 20, and CD 68 with ovidin-biotin method according to the standard protocol (DAKO, Denmark). Visualization and photography of stomach microsections were carried out using a microscope (Micros, Austria) digital camera and ScopePhoto computer program (10 visual fields in each slide). To determine the number of inflammatory cells expressing CD3, CD20 and CD68 markers, we counted the number of positively stained cells per area unit. Measurements of leukocyte concentrations length and area were performed using the BioVision program. Statistical analysis was performed using the EXEL software package.
Results
The collection of 850 digitized micrographs of inflammatory infiltrates in the stomach wall affected by tumor process was created. It was found that immune cells diffusely infiltrated tumor sites in the stomach wall and also form clusters in the lamina propria and submucosa. Many sections had lymphoid follicle-like aggregates with light centers. In four cases (16%) cell concentrations were not found. Follicle-like clusters consisted mainly of cells expressing the antigen of B-lymphocytes (CD20). The average area of focal infiltrates varried from 0,019mm2 to 0.244 mm2. Study of the expression of T-lymphocytes antigens (CD 3) revealed that the average density of cells infiltrating tumors is 21.2 in moderate differentiation degree and 25.9 cells per area unit in low degree (p <0.05).
The analysis of the expression of B-lymphocytes antigens (CD 20) showed statistically significant differences in the density of cells infiltrating the tumor with moderate and low grade (15.5 and 10.4 per area unit, respectively) (p <0.05).
Significant differences were also found in the density of macrophages diffusely infiltrating tumor in the lamina propria and submucosa with different degrees of adenocarcinoma differentiation. In a low degree of differentiation the number of macrophages on the average was 12.7 cells per area unit, whereas in a moderate one – 15.7 (p <0.05).The examined slides of patients with gastric cancer had a higher average density of T-lymphocytes in tumor sections than in the leukocyte clusters of lamina propria and submucosa. The level of B-lymphocytes, in contrast, was higher in clusters than in stroma of tumor.
The analysis of obtained data showed that the number of infiltrating lymphocytes and macrophages depended on the presence of focal leukocyte clusters in the stomach wall. The density of diffusely infiltrating T- and B-lymphocytes in patients with adenocarcinoma in the absence of focal cell clusters was respectively 24.9 and 16.7 per unit of area. In the presence of clusters – 15.1 T-lymphocytes and 11.2 B cells per area unit. The average number of macrophages in the absence of leukocyte aggregations was 9.4, and in the presence of them – 14.5. The dependence of the cellular composition of diffuse infiltrates on the size of focal accumulations was found. Sections where infiltrates area not exceeding 0.09 mm2 had average number of T-lymphocytes of 24.4 cells and macrophage 13.6 per area unit. Whereas in area infiltrates exceeding 0.1mm2, the average number of T-lymphocytes was 15.7 and macrophages – 16.7 to area unit.
Conclusion
Leukocyte accumulations in the lamina propria and submucosa in tumors consisted mainly of B-lymphocytes. Some of them looked like lymphoid follicles with light centers. Cells diffusely infiltrating tumor were essentially T lymphocytes.
Cellular structure, namely the number of macrophages and lymphocytes in clusters depended on the degree of tumor differentiation.
The ratio of T and B cells in different degree of differentiation also varied: in low degree the density of T cells was higher, whereas in moderate degree – the number of B cells. Macrophages vastly penetrate tumors in moderate degree of cancer differentiation than in low one. The presented data suggested that cells more extensively infiltrated the tumor in the absence of focal leukocyte concentrations than in their presence. Macrophages in contrast, were found in large quantities in the presence of focal infiltrates. Aggregation dimensions also were effect on infiltration: high size of focal infiltrates was associated with the reduced number of diffusely tumor infiltrating T cells and the increased number of macrophages.
References
1. Elinav E1, Nowarski R, Thaiss CA, Hu B, Jin C, Flavell RA. Inflammation-induced cancer: crosstalk between tumours, immune cells and microorganisms // Nat Rev Cancer. – 2013. – V. 13(11). – P. 759-71.
2. Fernandes JV, Cobucci RN, Jatobá CA, de Medeiros Fernandes TA, de Azevedo JW, de Araújo JM. The Role of the Mediators of Inflammation in Cancer Development // Pathology & Oncology Research. Springer. – 2015.
3. Sergei I. Grivennikov, Florian R. Greten, and Michael Karin Immunity, Inflammation, and Cancer // Cell. – 2010. – V. 140(6). – P. 883–899.
4. de Visser K. Coussens L. The Inflammatory Tumor Microenvironment and Its Impact on Cancer Development // Contrib Microbiol. Basel, Karger. – 2006. – V. 13. – P. 118-137.
Keywords:
tumor,
Stomach,
leukocyte infiltration,
Immunohistochemical studies,
T-Lymphocytes,
B-Lymphocytes,
Macrophages
Conference:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.
Presentation Type:
Poster Presentation
Topic:
Tumor immunology
Citation:
Kabanovskaya
IN,
Lebedinskaya
OV,
Kosareva
PV and
Akhmatova
NK
(2015). CHARACTERISTICS OF LEUKOCYTE INFILTRATION IN STOMACH TUMORS.
Front. Immunol.
Conference Abstract:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología.
doi: 10.3389/conf.fimmu.2015.05.00071
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Received:
06 May 2015;
Published Online:
14 Sep 2015.
*
Correspondence:
Ms. Olga V Lebedinskaya, Acad. E.A. Wagner Perm State Medical University, Perm, Russia, lebedinska@mail.ru