The Significance Of Systemic Inflammatory Markers,CD15+ Neutrophil And MDA5 Expression And In Oral And Oropharygneal Cancer

Part ?,Prognostic significance of pre-operation NLR and PLR for oral and oropharyngeal carcinomaBackgroundHead and neck cancer is the sixth largest tumor worldwide.Oral,oropharyngeal and lip cancer are the most common head and neck cancer.2018 global cancer data reported 354864 new cases of oral cavity and lip cancer and 177384 deaths,oropharyngeal cancer with 92887 new cases and 51005 deaths,and the trend of rising year by year.In spite of the treatment technology of head and neck cancer is unceasing developing,especially based on local immune microenvironment and inflammatory state adjust immune treatment technology development.Improvement of 5-year survival rate of head and neck cancer is still increase slowly in the past 20 years(less then 5%).As for clinical tumor and tumor biological characteristics,accuracy cancer prognosis prediction based on the analysis of traditional TNM staging is not enough.Such as the early colorectal cancer patients with about 25%of people in the poor prognosis,and analysis of the head and neck cancer is found to have a certain proportion of patients with early prognosis.Some large data also found that many cancer patients with the same TNM staging have significant different response of radiation and chemotherapy and immunity treatment.It greatly affects the improvement of the clinical curative effect and control of side effect.Therefore,it is one of the important contents of tumor clinical research to explore and solve the incomplete information of tumor TNM stage supplementation and improve the accurate prognosis prediction of cancer patients.Studies on the relationship between inflammation and tumor progression,treatment response and prognosis prediction have found that the inflammatory status and types of many solid tumors are not only related to the progression,sensitivity of chemoradiotherapy and immunotherapy response,but also show good supplementary value for the accuracy of TNM-based prognosis prediction.A tumor-burdened host inflammatory conditions including system local inflammatory inflammatory signs and tumor microenvironment,among them,the exploration system inflammatory marker significance to the prediction of prognosis of tumor and its treatment response.It is not only one of the important content of tumor prognosis prediction research field,but also one of hot topics in the study of tumor treatment response sensitivity content.As the most abundant cell type in human peripheral blood,Polymorphonuclear(PMN)accounts for 50-70%of the circulating white blood cells and it is the first line of defense against microbial invasion.Many studies have found that systemic inflammatory markers such as PMN and corresponding ratio such as neutrophils/lymphocyte ratio(NLR),Platelet/lymphocyte ratio(PLR),etc.,not only with cardiovascular disease(CVD)events such as atherosclerotic plaque stability,the prognosis of myocardial infarction,but also related to many inflammatory disease complications occurred,such as bacterial infection,shock prediction.Other studies have found that systemic inflammatory markers are associated with immune-related diseases such as alzheimer's disease caused by multiregional neurodegeneration of the brain.Based on the relationship between inflammation and tumor progression and prognosis of,a lot of research analysis of the tumor patients with systemic inflammatory markers on its prognosis,such as NLRhi and or PLRhi and gastric cancer,colorectal cancer,liver cancer and non-small cell lung cancer and other malignant tumors of poor prognosis,with independent forecasts in meaning,and the poor prognosis of systemic inflammatory markers predict meaning can be explained by PMN promote inflammation in the tumor.Recent studies have found that the NLR level of non-small cell lung cancer is predictive of pD-1/PD-L1 blocking therapy.Studies on renal cancer have also found that NLR is not only related to prognosis,but also its dynamic changes are related to the efficacy of PD-1 inhibitors.A growing body of evidence suggests that systemic inflammatory markers are important in predicting both tumor prognosis and therapeutic response.Oral,oropharyngeal cancer and lip cancer is one of the most important head and neck cancer,but its risk factors and biological behaviour are different.Although many studies have reported NLR,PLR are closely related with the biological behavior and prognosis of head and neck squamous cell carcinoma,data about the relationship between NLR,PLR and its progress and prognosis in oral,oropharyngeal cancer and lip cancer are less and differences.This study included 421 cases of qilu hospital of shandong university as a queue,the above three kinds of head and neck cancer using multivariate analysis,including preoperative NLR and PLR inflammatory markers as a system,analyzes its relationship with survival and recurrence,and patients of NLRhi and PLRhi subgroup analysis,and further explore some patients were analyzed in radiation and chemotherapy after the intervention of the difference between survival and recurrence relations with NLRhi and PLRhi.The results of this study provide new cohort analysis results and new findings for the prognostic value and significance of systemic inflammatory markers NLR and PLR in patients with oral cancer,oropharyngeal cancer and lip cancer,and also provide new basis for the prognostic prediction research and stratified treatment management of the above three kinds of head and neck cancer.Research purposesThe relationship between systemic inflammatory markers NLR and PLR and clinicopathological indicators of oral,oropharyngeal and lip cancers was analyzed.The predictive value and independent prognostic significance of NLR and PLR in these three head and neck cancers were analyzed.The relationship between NLR and PLR and recurrence and metastasis of these three kinds of head and neck cancers was analyzed.The subgroup analysis of NLRhi and PLRhi patients was conducted to explore the difference between the predictive value of survival after further chemoradiotherapy intervention and recurrence and metastasis of these patients,so as to provide a new basis for the study of prognosis prediction and stratified treatment management of oral cancer oropharyngeal cancer and lip cancer.Research methods1.From January 2006 to December 2015,a total of 421 patients who were pathologically diagnosed as oral,oropharyngeal or lip cancer in Qilu Hospital of Shandong University(Jinan,China)were included in the study.Clinic-pathologic characteristics,including age,sex,smoking or alcohol drinking history,tumor site,pathological type and histological grading,were retrieved from the database of Qilu Hospital of Shandong University.TNM definition and clinical stage classification were defined according to American Joint Committee on Cancer Stage(Seventh Edition).2.The optimal cut-off values of NLR and PLR were determined using the ROC curve.The value on the curve conforms to the most approximate lygitindex(sensitivity/0.44+specificity/0.84-1)corresponding to the optimal critical value,and is analyzed hierarchically.3.421 patients were analyzed with NLR and PLR correlation with clinical pathology data.There were relapse rates,recurrence sites,overall survival time(OS)and relapse-free survival time(RFS)in 195 patients with a final follow-up of more than 1 year.Correlation analysis and survival analysis were performed on the above clinical pathological data.4.A subgroup analysis was conducted on NLRhi and PLRhi patients to explore the significance of survival and prognosis after further chemoradiotherapy intervention in the subgroup,and to compare the difference in recurrence and metastasis rate of patients with or without chemoradiotherapy5.All analyses were performed with SPSS version21.0(IBM,Chicago,Illinois.USA),the survival curves were produced with GraphPad Prism 7(GraphPad,Inc.,San Diego,California.USA).Chi-square test was applied to determine the differences of the unordered categorical variates.Spearman's rank correlation coefficient,rs(ranged form-1 to+1),was utilized to quantify the correlation of ordinal variates.Kaplan-Meier method was used to estimate overall and relapse-free survival and the log-rank test was used to analyze the differences between groups.Univariate and multivariate(forward stepwise regression)COX proportional-hazards models were applied to identify the factors affecting overall and relapse-free survival.All P values and confidence intervals presented in this report are two-sided.P value of<0.05 is considered as statistical significance.Results1.NLRhi value was positively correlated with larger tumor size(P<0.001)and later staging(P=0.002).There was no significant correlation between NLR elevation and age grouping,gender,smoking or drinking,histological grade(squamous cell carcinoma only),presence of cervical lymph node metastasis.2.PLRhi was significantly correlated with tumor diameter(P<0.001)and postoperative stage(P=0.008).In addition,PLRhi in the male group was higher(P=0.03),and in the smoking or drinking group(P=0.02).There was no significant correlation between PLR elevation and age,histological grade,and cervical lymph node metastasis.3.The results of univariate analysis indicated that NLRhi group was lower than that of the NLRlow group in OS(P=0.02)and RFS(P=0.009).There were no significant differences of OS and RFS between higher PLR group and lower PLR group.Gender,age,and histological differentiation had no significant effect on the overall survival time(OS)and relapse-free survival time(RFS)of patients with oral,oropharyngeal and lip cancer.OS(P=0.02)and RFS(P=0.02)of larger tumor diameter was significantly shorter than that of smaller tumor diameter.OS(P=0.01)and RFS(P=0.01)with shorter clinical stage were shorter than those with earlier stage.RFS with lymph node metastasis was shorter than none lymph node metastasis(P=0.03)but not in OS.Univariate survival analysis OS of larger tumor diameter was significantly shorter than that of smaller tumor diameter(P=0.02).OS(P=0.01)and RFS(P=0.005)with shorter clinical stage were shorter than those with earlier stage.DFS with lymph node metastasis was shorter than none lymph node metastasis(P=0.025)but not in OS.4.Relapse rate:The overall recurrence rate(P=0.03)and in situ relapse rate(P=0.02)were significantly higher in the NLRhi group,while there was no significant difference in the PLR groups.5.NLRhi and PLRhi subgroup analysis:35 NLRhi patients with postoperative radiotherapy or chemotherapy and no radiation and chemotherapy have no significant difference in OS and RFS.Relapse rate of patients with postoperative chemoradiotherapy is significantly lower than who did not have chemoradiotherapy(P=0.027).66 PLRhi patients with postoperative radiotherapy or chemotherapy and no radiation and chemotherapy have no significant difference in OS and RFS.Relapse rate of PLRhi patients have no significant difference between the postoperative chemoradiotherapy group and no radiation and chemotherapy group.Conclusion1.Oral,oropharyngeal and lip cancer are the most important head and neck cancer.But the risk factors and biological behavior and prognosis of these three cancers are different and not unified.This study include a large cohort study of 421 patients of oral,oropharyngeal and lip cancer.Preoperative NLR is found to be an independent risk factor by survival analysis to predict the prognosis of head and neck cancer.PLR had no significant effect on survival prognosis significance.2.Our study found that NLRhi was associated with a high risk of recurrence,especially in situ recurrence,while PLR was not significantly associated with the risk of recurrence,suggesting that NLR may predict the recurrence of head and neck cancer.It provides a predictive basis for evaluating the risk of postoperative recurrence.3.NLR and PLR are both positively correlated with tumor size and TNM stage.In the subgroup analysis,the recurrence rate of NLRhi patients after chemoradiotherapy intervention was significantly reduced,suggesting that NLR and PLR may provide valuable data basis for the stratified treatment management of head and neck cancer.Part ? Analysis of the relationship between CD15+neutrophil infiltration and NLR in oral and oropharyngeal carcinoma and the significance of MDA5 expression on its clinicopathological indicators and prognosisBackgroundFormation and regulating mechanism of inflammatory microenvironment of tumor is one of the main research contents for its prognostic and treatment significance based on targeting the immune microenvironment.In recent years,with the composition of the tumor microenvironment and its prognosis and therapeutic response prediction research in the field of development and further,a large number of studies have shown that microenvironment of many solid tumors including head and neck tumor exists a variety of immune cells and has high degree of heterogeneity.Researches of cancer immune microenvironment and their heterogeneity and the infiltration characteristics and its prognostic value is one of the important contents.In recent years,the CD15+neutrophils are Prereactive cells of natural immune response.Their infiltration characteristics,local natural immune responses and tumor progression control and its prognostic value are appreciated.Many researches have confirmed PMN involved in multiple physiological and pathological and physiological processes of inflammatory diseases such as tissue remodeling,angiogenesis,embryo and tissue inflammation.It is the key cell to the body's natural immune response.Tumor cells infiltration of PMN,also known as tumor related neutrophils(TANs)is an important component.It can infiltrate in the tumor microenvironment of numberous cancers include renal cell carcinoma,lung cancer,breast cancer,stomach cancer melanoma in colorectal cancer and head and neck cancer and many other solid tumor.were TANs infiltration had high heterogeneity,and it has similarities and differences of the prognostic value.The latest study found that the infiltration of TANs effect the curative of immune inhibitors of checkpoint PD-1.Therefore revealing the characteristics and significance of tumor local PMN infiltration cells has become a new hotspot in the research of the inflammatory tumor microenvironment.In tumor innate immunity,the early infiltration of PMN is accompanied by the initiation of key Pattern recognition receptors(PRRs)of innate immunity.Its role and clinical significance in local inflammatory response are of great concern to Melanoma differentiation associated protein 5(MDA5)which is one of the most important pattern recognition receptors.It is not only a PRRs molecule that recognizes danger signals(pathogens,etc.)in the microenvironment and rapidly activates the inflammatory response,but also plays an important role in many inflammatory diseases.Studies have found that MDA5 expression mediated inflammatory cells infiltration regulated the development of pulmonary inflammatory diseases.Such as in the mouse model of chronic pneumonia study found that the inflammation in the progress,the lack of MDA5 can lead to a reduce in neutrophils and chemokines.Then it adjusted neutrophils and mediated the early inflammatory response can regulate the development of chronic lung inflammation.Recent studies have found that MDA5 was related to the progress of the malignant tumor inhibition and has prognostic value in pancreatic cancer and melanoma and neuroblastoma.But its expression and significance of PMN in the malignant tumor invasion are rarely reported.There are no reports about MDA5 in oral cancer.It is not yet clear whether MDA5 expression has relationship with oral local PMN infiltration and whether have prognostic value.To analyze the early response cell PMN and early response molecule MDA5 in the inflammatory environment of oral cancer and their clinical significance,and to provide new data and prevention strategies for tumor treatment and prognosis prediction based on the inflammatory microenvironment of tumor.Based on the above analysis,this study used TCGA database(https://portal.gdc.cancer.gov/)to download the expression and clinical pathological data of 288 cases of oral cancer,and the expression of CD 15 and MDA5 and its correlation with clinical significance was analyzed.Use immunohistochemical staining technique analyse 158 cases of qilu hospital which has more than one year survival data of the oral and oropharyngeal cancer patients tissue section.We analyzed the CD15 and MDA5 expression and its clinical significance,and further analyzed the tumor local CD15+neutrophils infiltrating corresponding preoperative NLR relationship and clinical significance.In order to analyse the early reaction cells and molecules based on natural response neutrophils and MDA5 in oral and oropharyngeal cancer accurately predict to provide the new data.Research purposeThe expressions of CD15 and MDA5 and clinicopathological data of 288 cases of oral cancer were downloaded from the TCGA database,and the expressions of CD15 and MDA5 and their correlation and clinical significance were analyzed.It is revealed to provide new clues and basis for improving the prognostic value of natural immune markers and the selection of treatment strategies.Based on 158 cases of oral and oropharyngeal cancer patients cohort study,using immunohistochemical method to detect CD15+neutrophils infiltrating and MDA5 expression.Analyze their expression characteristics and the relationship of clinical pathological features and prognosis significance.Further analyze the local joint NLR and CD15+neutrophil infiltration of prognostic significance,in order to early reaction cells and molecules based on natural response neutrophils and MDA5 oral and oropharyngeal cancer accurately predict provides new data,and based on neutrophils MDA5 immunotherapy strategy development provides new data to support.Research methods1.Download the clinical pathology of 288 patients with oral cancer data and CD15 and MDA5 expression data from The Cancer Genome Atlas(TCGA)database(https://portal.gdc.cancer.gov/).TCGA database of oral cancer patients CD15 and MDA5 stratified analysis according to the median mRNA expression,CD15 MDA5 relevance to clinical indicators using chi-square,survival curve and forest graph using GraphPad Prism 7.2.Collect pathological samples of 158 oral and oropharygneal cancer patients admitted and undergoing surgery for the first time from the Department of Oral Medicine of Qilu Hospital,Shandong University,January 1st 2006 to December 31th 2013,and establish a database of clinical pathology data for patients,including gender,age,history of smoking or drinking,tumor sites,The degree of separation(in which only 129 patients with squamous cell carcinoma was analyzed and described).According to the pathology and imaging basis,clinical pathology phased according to the oral and oral cancer TNM staging(AJCC 7th edition)standard,and all patients were followed by telephone.3.Immunohistochemistry:Detect the immersion of CD15+neutrophils and the expression characteristics of MDA5 in 158 specimens by using of immunohistochemistry method.Under the microscope,CD15 positive expression sits brownish-yellow particles,which are largely located in the cell's paste.CD15+neutrophils counts were performed in the high-specular field of view(HPF)hot area,and the number of positive cells in three regions was averaged as the number of CD15+neutrophils immersed in each specimen.The number of CD15+neutrophils in the three regions of the tumor,junction and matrix of all specimens was observed,and the median number of positive cells in each region was calculated as its threshold(cut out point),which was represented as CD15+hi and below the threshold represented CD15+low,and it is used for layered analysis.MDA5 is mainly coloring in tumor cells and fibroblasts,and lymphatic cells,endothelial,etc.are not colored.The expression of MDA5 in two areas of the tumor and surrounding matrix was observed,and its stainstrength was not colored,0 points;The number of stained cells(positive rate)accounted for percentage of total tumor cells:0-5%,0 points;6-25%,1 point;26-50%,2 points;51-75%,3 points;75%or more,4 points.Because there is no uniform standard for MDA5 expression intensity,the intensity and positive rate of this study were stratified:staining strength was 2-3 points,positive rate was 3-4 with high expression,dyeing strength 0-1 points,positive rate 0-2 was divided into low expression.4.According to Receiver Operating characteristic(ROC),the optimal threshold value of NLR of the 158 cases of oral and oropharyngeal cancer in this cohort was determined.The corresponding value on the curve that conforms to the most approximate dang index(sensitivity/0.44+specificity/0.84-1)was the optimal threshold value,and stratified analysis was conducted.5.Statistical methods:Using SPSS 21.0 statistical analysis,survival curve and forest graph using GraphPad Prism 7.using t-test to analyze the difference of infiltration of CD15+neutrophils in three regions,using chi-squared test to analysis expression differences of MDA5 in tumor and matrix regions.Using the optimal critical value for CD15+neutrophil leachate layering.Spearman correlation coefficients analyzed the relationship between CD15 and MDA5 and clinical pathology indicators(rs range 1 to-1).Using Kaplan-Meier survival analysis(log-rank test)for single-factor survival analysis,COX risk model for multi-factor analysis.Results1.The TCGA database analyze:Download 288 oral cancer patients from the TCGA database data to establish a cohort study,the results showed that there was no significant correlation with MDA5 and CD15 expression(P=0.729,the rs=0.020).CD15 expression and the primary tumor site(CD15hi has the highest percentage of tongue,P=0.036),histological grade(CD15hi histologic classification of patients with higher histological grade,P=0.023)and whether had radiation and chemotherapy(CD15hi more chemoradiotherapy in patients,P=0.033)significantly correlated.There was no correlation between CD15 and age,sex,smoking,drinking,TNM stage,or lymph node dissection.The proportion of female patients with high MDA5 expression was higher than that of male patients.MDA5 expression was not associated with other clinical indicators.The univariate survival analysis showed that MDA5 expression was not associated with other clinical indicators.It is showed that higher 3-year survival rate in early stage patients(P=0.024).The 3-year survival rate in CD15hi patients was higher than that in CD15low patients(P=0.012).Multivariate analysis suggested that age(P=0.025),TNM staging(P<0.001),histological grade(P=0.004)and the presence or absence of cervical lymph node dissection(P<0.001)were all associated with 3-year OS.There was no significant difference between expression of CD15,MDA5 and 3-year OS2.Based on a cohort of 158 patients with oral and oropharyngeal cancer:The infiltration CD15+neutrophils are highly heterogeneous in different areas of tumors,junctions and matrix.CD15+neutrophils in tumor matrix are tiptop(74/HPF,18-152/HPF),and lower in tumors(37/HPF,7-84/HPF)and junctions(41/HPF,11-104/HPF)are relatively low(P<0.001).3.The relationship between CD15+hi and clinical pathology indicators.CD15+hi in tumors was associated with higher TNM staging(P=0.025),and the proportion of CD15+hi in the junction area was higher in oral cancer(P=0.023).CD15hi in the three regions was not significantly correlated with age,sex,smoking or drinking,histological grade,tumor size,or cervical lymph node metastasis.4.MDA5 expression has heterogeneity in different parts of the tumor.MDA5 is highly expressed mainly in tumor tissue,and the expression intensity and positive rate of the matrix are low.The strength of MDA5 in the tumor is higher than that of the tumor matrix(2-3 expression rate,58.2%vs19.6%,P<0.001),and the MDA5 positive rate in the tumor is higher than that of the tumor matrix(3-4,60.8%vs 14.6%,P=0.005).5.The relationship of MDA5 expression and clinical pathology indicators.Intra-tumor MDA5 high expression is related to neck lymph node metastasis positive(P=0.037),late TNM(P=0.036)and higher differentiation(P=0.017).There were no significant differences in age,sex,smoking or alcohol consumption,tumor site,histological grade and tumor size.The expression intensity and positive rate of MDA5 in tumor matrix were not significantly different from the clinical pathology index.6.Survival analysis:Single-factor survival analysis suggests the matrix CD15hi OS(median survival/month CD15+hi vs CD15+hi 31.7 vs.46.3,P<0.001)and RFS(median survival/month CD15+hi vs CD15+low 31.0 vs 43.4,P=0.001)are poor.RFS of later TNM stage is worse(median survival/month ?-? vs ?-? period 32.6 vs 41.1 P=0.039).OS and RFS in the tumor CD15+hi and the junction CD15+hi have no significant effect.Other clinical pathology indicators and MDA5 had no significant effect on OS and RFS.Multi-factor analysis suggested that CD15+hi in the matrix was an independent prognostication factor for OS(HR=1.853,P<0.001)and RFS(HR=1.664,P=0.003).6.NLRhi Combined Matrix CD15+hi Prognosis:Based on 158 patients with oral and oropharyngeal cancer,there was no significant correlation between NLRhi and intratumoral(P=0.081,Rs=0.139),interface(P=0.135,Rs=0.119)and matrix(P=0.520,Rs=0.052)CD15+neutrophil infiltration.OS(median survival time/month,33.1 vs 43.0,P=0.019)and RFS(median survival time/month,31.3 vs 41.2,P=0.019)of NLRhi are significantly lower than those of NLRlow.Combined with NLR and matrix CD15+neutrophil leachate for survival analysis suggests that OS(median survival time/month,33.3 vs.51.7,P<0.001)and RFS(median survival time/month,32.0 vs 48.8,P=0.001)are both poor.ln addition,P value was lower and the difference was more significant in the combined survival analysis.7.There was no significant correlation between CD15+hi and MDA5 expression.There was no significant difference between tumor CA15+hi and MDA5 intensity(low intensity vs.high intensity 50.0%vs.51.1%,rs=0.011 P=0.894)or positive rate(high positive rate vs.low positive rate 53.2%vs.49.0%,rs=-0.042 P=0.603).There was no significant difference between matrix CD15+hi and MDA5 intensity(low intensity vs.high intensity 49.6%vs.51.6%,rs=0.016 P=0.842)or positive rate(low intensity vs.high intensity 48.1%vs.60.9%,rs=0.090 P=0.262).Conclusion1.The infiltration characteristics of CD15+neutrophils at different sites in oral and oropharyngeal cancer were studied for the first time.It was found that CD15+neutrophil infiltration was highly heterogeneous,with the highest infiltration in the stroma.CD15+hi in the tumor was positively correlated with TNM stage,and CD15+hi in the stroma was an independent prognostic factor for OS and RFS.Suggesting that CD15+neutrophil infiltration in the stroma has an important prognostic significance in these two kinds of head and neck cancers.2.The first observational study of MDA5 expression in these two head and neck cancers revealed that MDA5 was mainly expressed in the tumor tissues.The expression of MDA5 in tumors is related to the TNM stage of histological differentiation and cervical lymph node metastasis,suggesting that MDA5 may play an important role in the process of tumor progression.3.The OS and RFS of patients with circulating marker NLRhi and/or local substrate CD15+hi were poor,and the survival difference was more significant than that of patients with single NLRhi.It suggests that combined detection of local neutrophil infiltration and circulatory inflammatory markers may be a better prognostic indicator,providing a basis for accurate prognostic prediction.4.The correlation between expression of MDA5 and infiltration of CD15+neutrophils in the two head and neck cancers was first studied.It was found that there was no significant correlation between the two marks,which presented more results in further in vitro and animal model experiments and gradually translated into clinical application.