A Tumor Marker Panel Scoring System To Improve Clinical Stage In Gastric Cancer And The Exploration Of Novel Biomarkers For Gastric Cancer Based On Macrophage

Gastric cancer is one of the most common malignancies and a major health problem worldwide.As the tumor heterogeneityin clinical practice,patients with histologically similar tumors often have different clinical courses and outcomes,due to the molecular differences.Based on the severity of illnes,customizing a personalized treatment regimen for gastric cancer patients,will improve the outcome and help patients avoid unnecessary costs and side effects.In general,tumor biomarkers are mainly divided into two classes:serum tumor markers and cellular tumor markers,reflecting the occurrence and development of tumors.However,for gastric cancer,despite many prognostic,predictive,and therapeutic biomarkers are investigated to date,there is no specific serum tumor markers,and the most cellular tumor markers are still in the research stage as the existing uncertainties.So screening effective biomarkers and exploring its changes during gastric cancer progression is clinically significant.This study was divided into two parts as below:Part I:The screening of serum tumor markers and a tumor marker panel scoring system to improve clinical stage in gastric cancerObjective To investigate the correlation among tumor markers test/reference cutoff ratio score(TRRS),disease progression and clinical stages in gastric cancer.Methods 708 patients hospitalized because of gastric cancer for the first time with preoperative tumor makers[carcinoembryonic antigen(CEA),carbohydrate antigen(CA)19-9,and CA 125]measured between January 2005 and December 2014 at Zhongnan Hospital of Wuhan University were enrolled.We took the normal reference of tumor marker as a cut-off value.According to the reference cutoff value(5 ng/mL for CEA,37 U/mL for CA 19-9,and 35 U/mL for CA 125),the TRR was defined as the test value divided by the reference cut off value of each tumor marker(CEA TRR = test value/5,CA19-9 TRR = test value/37,CA 125 TRR = test value/35).Based on the related references,TRR was divided into four categories:TRR<1.00,1.00<TRR? 3.00,3.00<TRR ? 6.00 and TRR>6.00.Each category got a score which was classified as TRR score 0(TRRS0),TRR score 1(TRRS1),TRR score 2(TRRS2)and TRR score 3(TRRS3),respectively.The TRR scores of three tumor markers were analyzed and special attention was focused on the relationship between each tumor marker TRRS and clinical outcomes of gastric cancer,in order to investigate the exact quantitative relationship between tumor marker elevation times and the accuracy of the TNM stage.Results CEA,CA19-9 and CA125 test/reference cutoff ratio(TRR)levels were increased significantly according to the clinical stages,TRRS were significantly correlated with the depth of tumor invasion,lymph node metastasis,distant metastasis and clinical stage(All P<0.001).A tumor marker panel scoring system(TMSS)was devised as we defined Negative TMSS(TMSS = 0),Low TMSS(1 ? TMSS ? 3),Middle TMSS(4 ? TMSS ? 6),High TMSS(7 ? TMSS ? 9)based on the calculated TRR score.TMSS was significantly correlated with clinical stage.The later the TNM stage was,the higher the percentage of Middle and High TMSS will became.The distribution of TMSS in gastric cancer patients with High TMSS was found in advanced stages(stage ? and ?).Conclusions CEA,CA19-9 and CA125 TRRS are useful indicators for disease progression in gastric cancer patients hospitalized for the first time,the TMSS could be a useful tool for the prediction of clinical stage,especially for the advanced stage.Part ?:The exploration of novel biomarkers for gastric cancer based on macrophageObjective To explore the degree of macrophage infiltration in gastric cancer and its clinical significance,explore the dynamic changes of macrophages in gastric cancer progression,provide new biomarkers and practical guidance on clinical evaluation of the severity of gastric cancer.Methods Through searching the the Oncomine Gene Profiling Database to determine the expression difference of macrophage marker(CD68)between normal tissue and gastric cancer.Immunohistochemistry was used to detect the expression of CD68 in gastric cancer and paracancerous tissues.Kaplan Meier Plotter database was used to evaluate the prognostic significance of macrophage infiltration density in gastric cancer patients with different stages.THP-1 cells differentiated into macrophages after treatment with phorbol-12-myristate-13-acetate(PMA),then co-cultured with different proportions of gastric cancer cells,the mRNA and protein expression of macrophage associated molecular markers were detected by RT-PCR and Western blot,respectively.The cell apoptosis was detected by flow cytometry.Further,different sizes of subcutaneous xenograft nude models were established by using the gastric cancer cell line MGC-803.Next,obtained peritoneal macrophages,identified and detected mRNA expression of macrophage associated genes by RT-PCR and analysed the correlation between the mRNA expression and tumor size.Results Through online database anlysis and immunohistochemistry,we found that macrophage infiltration increased in gastric cancer tissues compared to paracancerous tissues,but there was no significant difference in the number of macrophages between the early cancer group(stage ? + ?)and the advanced cancer group(stage ? + ?).The higher macrophage infiltration was positively correlated with the prognosis of early gastric cancer patients,but negatively correlated with the overall survival of advanced gastric cancer patients,macrophage infiltration density played a different role in the predicting prognosis of gastric cancer patients with various clinical stages.After co-culture with gastric cancer cells at different time-points(12,24,48,72 and 96 h),THP-1-derived macrophages showed the similar morphology,molecular markers and cytokine expression profiles,there was no obvious time-dependent changes.With the increasing of gastric cancer cell numbers in co-culture group,the expression of CD80 and TNF decreased gradually,while M2-related moleculars(CD206,IL10,Clec7a,CD163,TGFP)almost increased gradually,macrophage gradually shifted from pro-inflammatory and killing phonptype(M1)to a M2 phenotype,which is anti-inflammatory and pro-tumor,the overall showed number-dependent changes.The correlation analysis between the expression of macrophage-associated genes and the volume of subcutaneous tumor in nude mice showed that most of the M1-related genes were negatively correlated with tumor volume,while most of the M2-related geneswere positively correlated with tumor volume.Conclusions Higher macrophage infiltration was found in gastric cancer tissues,but there was no significant difference in the number of macrophages between early and advanced gastric cancer,the infiltration density had different prognostic significance for gastric cancer patients with different stage.With the increasing of the tumor size,macrophages gradually switched from the anti-tumor M1 to the pro-tumor M2 phenotype,most of the M1-related genes were negatively correlated with tumor size,while most of the M2-related genes were positively correlated with tumor size.Monitoring the dynamic changes of macrophage phenotype may suggest different processes of gastric cancer.