The Infiltration And Significance Of Inflammatory Cells In Nasopharyngeal Carcinoma

BackgroundWith the development of molecular biology and immunology, recently, it is found that tumor microenvironment which is closely connected with the tumor, plays an important roleon tumor evolution. The tumor microenvironmentis the part between the tumor and adjacent normal tissues, including extracellular matrix, soluble molecules and tumor stroma cells such as immune inflammatory cells, fibroblasts, endothelial cells, pericytes, etc. There are complex cross talks and interactions between tumor cells and the microenvironment.Theseinteractions change or even reverse the tumor biological characteristics, have important influence in the tumor epigenetics, immune escape, metastasis potential, and so on.Since, in the 19th century, the Virchow for the first time put forward the hypothesis that there was a relationship between inflammation and cancer,anumber of epidemiological and molecular biology experiments have confirmed the hypothesis, indicated about 25%of human tumors are associated with chronic inflammation. In solid tumors, there are many inflammatory cells such as neutrophils, mast cells, natural killercells, dendritic cells, tumor associated macrophage and tumor infiltrating lymphocytes. These t inflammatory cells as an important component of the immune microenvironment, do not exist in isolation, but as a complex multicellular population, and between them orcancer cells and other cells, the interaction directly or indirectly affect the development of tumor. Cell culture and transplantation model studies have proved the paracrine interactions between stroma and tumor have promoted tumor cell proliferation, invasion, tumorigenicity and metastasis. At present, tumor-promotinginflammatory cells include macrophages, mast cells, neutrophils, T lymphocytes and B lymphocytes.These inflammatory cells and inflammatory factors they secreted compose the tumor immunosuppression microenvironment, and inhibit the NK cells and T cells activity, play an important role inthe proliferation, invasion and metastasis of tumor cells. Researchs showed that immune active cells’ species, density, functional status and distribution in the tumor microenvironment,these four aspects comprehensive immunological factors, are prognostic factorsindependent of TNM stage. Therefore,understanding the characteristics and roles of inflammatory cells in the tumor microenvironment, is crucial to tumor diagnosis, staging, targeted therapy, prevention of recurrence and metastasis and understanding of the pathogenesis of tumor.Nasopharyngeal carcinoma (NPC) is a malignancy that occurses in the epithelial lining of the nasopharynx. Histologically, NPC has a high background ofreactive lymphocytes, and used to be calledlymphoepithelioma.Tumor infiltrating inflammatory cells played an important rolein the development, invasion or metastasis of nasopharyngeal carcinoma.Earlier studies have shown that there were many inflammatory cells in NPC microenvironment, such as T lymphocytes and B lymphocytes, macrophages, dendritic cells, etc, CD3+ T lymphocytes were the main component, B lymphocyteswere relatively fewer, NK cells were the fewest, and between different individuals, the distribution, degree type, etc.ofinflammatory cells, were different. However, due to the limitation of sample size and the research methods, these researchs failed to fully confirm the relationship between TIL with the prognosis, or got the opposite results.The relationship between immune cells and nasopharyngeal carcinoma, is still controversial, and lack in-depth, systematic and comprehensive studies, especially tumor infiltrating mast cells and neutrophils, which has not been reported.The mechanism bout these tumorinfiltrating inflammatory cells in the tissue of nasopharyngeal carcinoma is still not clear.This study through immunohistochemical staining first comprehensivelyresearched the distribution and infiltration of inflammatory cells in nasopharyngeal carcinoma, combing with clinical data, discussed the relationship between inflammatory cells, pathological characteristic, clinical stage and prognosis, and evaluated the predictive value of tumor infiltrating inflammatory cells.In order to further study the role of tumor infiltrating inflammatory cells, wealso detected the microvascular density (MVD) in nasopharyngeal carcinoma tissues, found out the relationship between inflammatory cells and angiogenesis,and discussed the effect of tumor inflammatory cells in tumor metastasis.Part I Distribution and prognostic significance of tumour-infiltrating inflammatory cellsin nasopharyngeal carcinomaTo investigate the distribution and prognostic significance of tumour-infiltrating inflammatory cells in nasopharyngeal carcinoma (NPC), and evaluate the predictive value of tumour-infiltrating inflammatory cells.Materials and MethodsA total of 177 nasopharyngeal carcinoma specimens were collected fromNanfang Hospital between 2007 to 2011. Adequate clinical follow-up data were available in these patients. CD56+ NK cells, FOXP3+ regulatory T cells (Tregs), CD8+ cytotoxic T cells (CTLs), CD3+ T cells, CD20+ B lymphocytes, CDla+ immature dendritic cells, CD83+ mature dendritic cells, neutrophil elastase+ neutrophilswere assessed by immunohistochemical staining. Data were obtained by calculating the mean number of positively stained cells per 400 x high-power microscopic fields in 5 separate HPFs. All data analysis was carried out with the SPSS 13.0 software.Measurement data was expressed as median and interquartile range (IQR) based on the skewed distribution.The associations between inflammatory cells and clinicopathological characteristics were analyzed by Pearson chi2 test. Spearman’s rank correlation test was applied to study the correlation between differentinflammatory cells. Survival curves were plotted according to the Kaplan-Meier method,and analyzed by the log-rank test. Univariate and multivariate analyses were performed using the Cox proportional hazard models.Two-tailed P< 0.05 was judged to be significant.Results1.Among the 177 patients, the median follow-up time was 51 (31-60 months),4 years’overall survival rate was 65.9%, progression-free survival rate was 58.7%.2. Tumor infiltrating inflammatory cells,such as neutrophil elastase+ neutrophils,CD3+ T lymphocytes, were mainly distributed in the tumor stroma, CD8+ CTLs and FOXP3+ Tregswere visible in cancer nest and stroma, CDla+immature dendritic cellswere mainly distributed in the nest.3. Tumor infiltrating inflammatory cells mainly were T lymphocytes, CD8+ CTLsaccounted for 60.11%(47.13%-71.12%) in T lymphocytes, FOXP3+ Tregsaccounted for 23.20%(16.34-32.05%), CD56+ NK cellswere the fewest.In tumor infiltrating dendritic cells, CDla+ immature dendritic cells were the main part.4. Spearman correlation analysis showed that the positive relationships between inflammatory cells were common.5. The densities of CD3+ T lymphocytes, CD20+ B lymphocytes, neutrophil elastase+ neutrophils and CD83+ mature dendritic cells infiltrating were positively associated with tumor pathological classification (P< 0.05).The densities of FOXP3+ Tregs, neutrophil elastase+neutrophils and tryptase+ mast cells infiltrating were positively correlated with clinical stage and regional lymph node metastasis (P< 0.05), and the density of CD56+ NK cellswas negatively related with partial tumor invasion classification (T stage).6. In univariate analysis, Patients with low densities of FOXP3+ Tregs, CD8+ CTLs, neutrophils and mast cellshad higher overall survival and progression-free survival, while with low densities of CD la+ immature dendritic cells and CD56+ NK cells had lower overall survival and progression-free survival (P< 0.05).7.The multivariate Cox proportional hazardsanalysis indicated thatCD8+ CTLs, FOXP3+Tregs were the independent risk factors for OS, not PFS. The density of mast cells was the independent risk factors for OS and PFS. The density of CD56+ NK cellswas an independent favorable factor for OS and PFS.ButThe density of CD1a+ immature dendritic cells and neutrophils were not the independent prognostic factors for OS and PFS.8. CD56+ NK cells combined with other inflammatory cells were independent prognostic factorsfor OS and PFS (P<0.01), and the ROC analysis had confirmed that the predictive ability was superior to a single type of inflammatory cell.ConclutionsOur findings characterise the immune response in NPC and identify inflammatory cell types thatinfluence the outcome of NPC patients. InNPC, tumor infiltrating Tregs, CTLs, mast cells, neutrophils are closely associated with poor prognosis, CDla+ immature dendritic cells and CD56+ NK cellsare associated with better prognosis. Moreover, the combination of CD56+ NK cells and other inflammatory cells mayserve as a better predictive biomarker for the prognosis of nasopharyngeal carcinoma.Part II The relationshipbetween tumour-infiltrating inflammatory cells and angiogenesis in nasopharyngeal carcinomaTo investigatethe relationshipbetween tumourinfiltrating inflammatory cells and angiogenesis in nasopharyngeal carcinoma.Materials and MethodsThe specimens were the same as the above. Immunohistochemistry for microvessel density (MVD) was performed. All data analysis was carried out with the SPSS 13.0 software.The associations between tumourinfiltrating inflammatory cells and clinicopathological factors were analyzed by Pearson chi2 test. Spearman’s rank correlation test was applied to study the correlations between different inflammatory cells and MVD. Survival curves were plotted according to the Kaplan-Meier method,and analyzed by the log-rank test. Univariate and multivariate analyses were performed using the Cox proportional hazard models.Two-tailed P< 0.05 was judged to be significant.Results1.MVD in nasopharyngeal carcinoma was positively correlated with N stage and clinical stage. Patients with higher MVD, had significant lymph node metastasis, advanced clinical stage and worse OS and PFS.2. Spearman correlation analysis showed that there were significantly positive correlations between MVD, mast cells and NK cells. The correlation between MVD, and mast cells was closer. No significant correlation was found between MVD and other inflammatory cells.ConclusionsAngiogenesis is closely associated with poor prognosis and mast cells in nasopharyngeal carcinoma. Moreover, mast cells may promote tumor metastasis, by facilitating tumor angiogenesis.