| Background:Esophageal cancer is among the most common cancer type in our country and represents a major health problem. Squamous cell carcinoma accounts for more than 90% of esophageal cancer cases in Chinese patients. Surgery, radiotherapy and chemotherapy are the main treatment modalities. Esophageal squamous cell carcinoma (ESCC) still shows a dismal prognosis with a 5-year survival rate less than 15%. The current prognostic model is mainly based on pathological parameters such as histological subtype, tumor size and pathological tumor-node-metastasis (pTNM) classification, which are urgently needed to be improved by the integration of new prognostic biomarkers. In addition, traditional treatment modalities such as surgery or chemoradiation can not control the disease effectively. It is urgent to identify new therapeutic targets.Immune reaction was proposed as the seventh hallmark of cancer. The importance of interaction between neoplastic cells and inflammatory cells is crucial for tumor initiation and progression. Leukocyte infiltration is one main characteristic of almost all malignant tumors and the major constituents of these infiltrates include tumor-associated macrophages, neutrophils, mast cells, NK cells, lymphocytes and so on. The infiltration of immune cells was on one hand able to eliminate cancer cells through cytoxic reaction, but on the other hand some immune cells lost their antitumor ability or even promote cancer invasion and metastasis by secretion of protease and inflammatory factors.Neutrophils, which represent the 50%-70% fraction of total circulating leukocytes, make up a significant portion of the leukocyte infiltration in a wide variety of human cancers. Neutrophils are rich in lysosomes which contain phosphatase and acid hydrolase, and could release several inflammatory factors. Although commonly encountered within tumor microenvironment, the role of neutrophils in cancer invasion and metastasis remain unclear. The lifespan of neutrophil is short, which stay in blood for a few hours, then move out of circulation and commit apoptosis 24-48 hours later. In the past, neutrophil infiltration in tumor was thought to be caused by infarction and infection of tissue and have minor effects on tumor progression. Recent studies suggested that TAN paly an important role in cancer progression. Tumor microenviroment could secret chemokines to recruit neutrophils and cytokines to prolong the lifespan of neutrophils. And the particles in neutrophils contain a great deal of proteases including serine proteases (NE, CG, PR3) and metalloproteases (MMP8, MMP9), which was closely associated with tumor invasion. In addition, TAN could activate angiogenesis in the early stage of carcinogenesis. On the contrary, neutrophils were reported to kill tumor cells through secreting chemokines and interlukines. The evidence concerning the clinical significance of TAN is limited. Jensen et al. analyzed the infiltration of CD66+neutrophils in renal cell carcinoma and indentified TAN as an independent prognostic factor. Rao et al. found that neutrophil infiltration was significantly associated with malignant phynotype and poor prognosis of colorectal cancer. Esophageal cancer develops from the lumen of esophagus and nearly all esophageal cancer accompany with inflammation, ulcer and infarction, which is associated with neutrophil infiltration. The clinical significance of TAN in esophageal cancer has yet been reported.Inflammation was normally considered as the reaction to damage stimuli by nature immune system of the host, including gathering of immune cells and relasing of inflammatory factors. As an existing damage stimulus, tumor definitely induces systemic and local immune reaction, and actively participates in cancer initiation, proliferation, metastasis and angiogenesis. An elevation in blood neutrophil-to-lymphocyte ratio was considered as an important marker of systemic inflammation, which shows prognostic effect in many kinds of tumors. However, the interaction between immune system and tumor cells mostly take place around the tumor tissue. The neutrophil-to-lymphocyte ratio in tumor tissue may reflect the local inflammatory reaction, which may also serve as an immune marker of prognostic significance. To date, the prognostic effect of tumor-infiltrating NLR has not been investigated in ESCC. Objectives:The aims of the study were to investigate the prognostic value of tumor-infiltrating (intratumoral and peritumoral) neutrophils and NLR, and to analyze the distribution of tumor-infiltrating neutrophils and CD8+lymphocytes in ESCC treated by curative resection. Materials and methods:1. Between January 1st and December 31st 2007,90 patients, who underwent potential radical surgery for ESCC in Thoracic Department, were included in the study. All the patient demographic and clinical data including age, gender, histological grade, lymph node status, pTNM stage, and adjuvant treatment were abstracted from the clinical records.2. Formalin-fixed paraffin-embedded surgical specimens were used for the double immunohistochemical analysis for CD66b and CD8.3. The analysis was performed with an Olympus IX71S1F-3 Inverted Microscope. The leukocyte number was determined as the mean value of numbers by the observers. Intratumoral leukocytes were determined as neutrophils or CD8+lymphocytes that infiltrated into cancer nests or stroma, whereas peritumoral leukocytes were the cells that distributed along the tumor-myometrial or tumor-connective tissue junction. The tumor sections were screened at low power magnification (x100), and 6 high power fields (x400) with highest number of cell infiltration in either intratumoral or peritumoral areas for neutrophils or CD8+ lymphocytes were selected.4. Chi-square test, Student t test, the Kaplan-Meier survival analyses were employed to analyzed the data.Results:1. Patient characteristicsMedian age was 60.5 years (range,42 to 78 years), and 80% of patients were male. Tumor locations were upper thoracic in 10 patients, middle thoracic in 48 patients, and lower thoracic in 32 patients. The median length of the tumor was 4 cm (range, 0.5-8.5). The histopathological differentiations were poor in 26 cases, moderate in 39 cases, and well in 25 cases.59 patients (65.6%) had T3/T4 tumors.33 patients (36.7%) have positive lymph nodes. The pathological stages were stage I in 18 patients, stage Ⅱ in 41 patients and stage III in 31 patients.55 patients (61.1%) received surgery alone,10 (11.1%) received postoperative chemotherapy,16 (17.8%) received postoperative radiotherapy and 9 (10%) received postoperative chemoradiation.63 patients (70%) had recurrence and 57 patients (63.3%) died during the follow-up. The estimated 1-,3-,5-year DFS and OS rates were 76%,48%,36% and 89%,56%,41%, respectively. Median DFS was 31.7 months (range,1.5 to 71.5 months). Median OS was 45.5 months (range,2.6 to 71.5 months).2. Analysis of immunohistochemical parametersThe median numbers of intratumoral and peritumoral neutrophils was 18.5 cells/HPF (range,0-387 cells/HPF) and 19 cells/HPF (range,0-247 cells/HPF), respectively. The median number of intratumoral and peritumoral CD8+ lymphocytes were 19 cells/HPF (range,0-122 cells/HPF) and 32 cells/HPF (range,0-200 cells/HPF), respectively. Infiltration of neutrophils or CD8+ lymphocyte was identified in both stroma and islet of the tumor. The density of intratumoral neutrophils was not significantly different from that of peritumoral areas (p=0.348). The density of intratumoral CD8+ lymphocytes was significantly lower than that of peritumoral area (p<0.001). The distribution of neutrophils was not correlated to that of CD8+ lymphocytes. The number of intratumoral neutrophils were also not significantly associated with that of intratumoral CD8+ lymphocytes (r= 0.005, p= 0.966), and the number of neutrophils in peritumoral areas were not significantly associated with that of lymphocytes in peritumor (r=-0.127, p= 0.232), either.3.Correlation of immunohistochemical parameters with clinicopathological parametersIncreased intratumoral neutrophils was significantly associated with lymph node metastasis (P=0.016), advanced pTNM stages (P=0.013) and recurrence (p=0.001). High intratumoral CD8+ lymphocytes were more frequently observed in elder patients (p=0.02). Intratumoral NLR was not associated with any of the clinicopathological parameters. Peritumoral neutrophil density was not associated with the studied clinicopathological parameters. Decreased peritumoral CD8+ lymphocyte density was more frequently observed in patients with single positive lymph node (p= 0.045). Increased peritumoral NLR was associated with male gender (p= 0.009), advanced T stages (p< 0.001), positive lymph node metastasis (P= 0.041) and a trend towards advanced pathological stage (p= 0.053).4. Survival analysisTo identify variables of potential prognostic significance in the patients with ESCC, univariate and multivariate analyses were further employed to investigate the impact of tumor-infiltrating neutrophils, CD 8+lymphocytes, NLR and other clinicopathological parameters on the prognosis of the 90 ESCC patients. In univariate analysis, increased intratumoral neutrophils were significantly associated with poor DFS (p< 0.001) and OS (p< 0.001). No association with DFS or OS was observed for intratumoral and peritumoral CD 8+lymphocytes, intratumoral and peritumoral NLR and peritumoral neutrophils. The 5-year DFS and OS rates for patients with increased intratumoral CD66+neutrophils were 20% and 26.7%, compared with 51.1% and 55.5% for patients with decreased intratumoral CD66+ neutrophils, respectively. Clinicopathological factors, which significantly associated with short DFS, were moderate differentiation, pT4 stage, lymph node metastasis and advanced pTNM stages. Factors, which statistically significantly associated with poor OS, were advanced T stages, lymph node metastasis, advanced pTNM stages and adjuvant radiation. A multivariate analysis revealed that upper tumor location, advanced pTNM stages and increased intratumoral neutrophils were associated with decreased DFS and advanced pTNM stages, postoperative radiation, and increased intratumoral neutrophils were independent predictors for poor OS.Conclusion:1. Infilation of CD66+ neutrophils and CD8+ lymphocytes was observed in most esophageal squamous cell carcinoma.2. Increased intratumoral neutrophils were significantly correlated with lymph node metastasis and advanced pTNM stages, and were an prognostic factor for poor DFS and OS, independent of certain well-established clinical features, including invasion depth, lymph node metastasis, and pTNM stages.3. Increased peritumoral NLR was significantly associated with depth of invasion, lymph node metastasis and a trend towards advanced pathological stage.4. Clarifying the role of neutrophil in cancer progression might aid the clinician to select more suitable therapy for the individual patients, e.g. favoring a more aggressive regimen in tumors with an increased intratumoral neutrophils or misbalance of peritumoral neutrophil and CD8+ lymphocytes. Moreover, Immunotherapy targeted to the above predictors may also help improve the prognosis of esophageal cancer. |
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