| Objective:Lung cancer has the highest morbidity and mortality among malignant tumors in the world,which is a serious threat to human physical and mental health.Therefore,the early diagnosis of lung cancer is of great significance for the treatment and prognosis of patients with this malignancy.At present,there have been many biomarkers available for the clinical diagnosis of lung cancer,but their sensitivity and specificity are not high enough for early diagnosis.On the basis of previous work,this study has designed several tumor-associated antigens selected based on their expression in lung cancer,and carried out detection of plasma IgG antibodies against tumor-associated antigens with ELISA developed in-house with single or combined peptide antigens in order to find biomarkers with high sensitivity and specificity for the early diagnosis of non-small cell lung cancer and provide some guidance for the treatment of this disease as well.Methods:1.Designing of peptide antigens:Full-length amino acid sequences of HER2,MUC1,VEGFR1,ABCC5,MYC,TNF-? and POU5F1 were retrieved from the NCBI database,and were used to design linear peptide antigens by a computational epitope prediction software(http://www.iedb.org);a CD25-derived peptide antigen was designed and proved to be closely related to the diagnosis and prognosis of non-small cell lung cancer in our previous study.A total of 9 distinct test kits were made in-house including 7 tests with single peptide antigens(HER2,MUC1,VEGFR1,ABCC5,MYC,TNF-? and POU5F1)and 2 tests with combinded antigens(HER2-MUC1-VEGFR1 and CD25-MUC1-VEGFR1).2.Detection of natural autoantibodies in plasma:A total of 211 patients who were newly diagnosed with non-small cell lung cancer(NSCLC)were recruited as the experimental group,and at the same time,200 cases of healthy subjects were also recruited as the control group.An enzyme-linked adsorption assay(ELISA)was developed in-house to detect plasma IgG antibodies against linear peptide antigens.The comparision of natural autoantibody levels was made between the patient group and the control group and between subgroups divided according to gender,age,tumor histological type and tumor stages.3.The screening of plasma:The plasma rich and low in natural antibodies against HER2,MUC1,and VEGFR1 peptides were selected from 150 healthy donors by the in-house ELISA.The plasma sample from the top one healthy donor with the highest IgG levels was assigned as positive plasma group,and the plasma sample from the bottom one healthy donor with the lowest IgG levels was assigned as negative plasma group.These plasma samples were used for the work on cell culture.4.The effects of natural autoantibodies on the proliferation of NSCLC-derived cells:Two NSCLC-derived cell lines,A549 and NCI-H2122,were treated with plasma rich and low in natural antibodies.To determine the effect of plasma natural antibodies against HER2,MUC1 and VEGFR1 on the proliferation of these two NSCLC cell lines,cell counting kit 8(CCK-8)was applied to test the cell viability.5.The expression of HER2,MUC1 and VEGFR1 mRNA in NSCLC-derived cells:The total RNA was extracted from A549 and NCI-H2122 cells and the expression of HER2,MUC1 and VEGFR1 mRNA was detected by real-time quantitative PCR.Results:1.Peptide antigen fragments were successfully designed and synthesized with the following sequence:HER2: H-kllallppgaastqvctgtdm klrlpasp-OHMUC1: H-crynltisdvsvsdvpfpfsaqsgah-OHVEGFR1: H-dlklsctvnkflyrdvtwillrtvnnrtmhysi-OHABCC5: H-tstsgthrdredskfrrtrplecqdal-OHMYC: H-rvkldsvrvlrqisnnrkcfellptpplsps-OHTNF-?: H-cqlqwlnrranallangvelrdnqlv-OHPOU5F1: H-keleqfakllkqkritlgytqadvgltc-OHCD25: H-iyhfvvgqmvyyqcvqgyralhrgpaesve-OH2.Natural autoantibody levels in NSCLC patients and healthy controls:The levels of natural IgG antibodies against VEGFR1,HER2-MUC1-VEGFR1 and CD25-MUC1-VEGFR1 were significantly lower in patients with NSCLC than in healthy controls(P < 0.001).The levels of natural IgG antibodies against MUC1,VEGFR1,HER2-MUC1-VEGFR1 and CD25-MUC1-VEGFR1 were significantly lower in female patients compared with female controls(P < 0.001)while the levels of plasma IgG for HER2-MUC1-VEGFR1 and CD25-MUC1-VEGFR1 were significantly lower in male patients compared with male controls(P < 0.001).When the subjects were divided into two groups by the age of 60,namely group < 60 and group ?60,both age groups had a significantly lower levels of plasma IgG for HER2-MUC1-VEGFR1 and CD25-MUC1-VEGFR1 than the control group(P < 0.001)while patients of group<60 showed that plasma anti-VEGFR1 IgG levels remained significantly lower than healthy controls(P < 0.001).When patients with NSCLC were divided into two subgroups based on histological types,adenocarcinoma(n = 124)and squamous cell carcinoma(n = 87),the levels of natural IgG antibodies against HER2,MUC1,VEGFR1,HER2-MUC1-VEGFR1 and CD25-MUC1-VEGFR1 were significantly decreased in adenocarcinoma patients(P < 0.001 or P < 0.05),while the levels of natural IgG antibodies against VEGFR1,HER2-MUC1-VEGFR1 and CD25-MUC1-VEGFR1 were significantly decreased in patients with squamous cell carcinoma(P < 0.001 or P < 0.05),as compared with healthy controls.Patients with NSCLC were divided into three subgroups according to the stages of lung cancer,group A for stages I and IIA,group B for stage IIB and group C for stages III and IV.While the levels of plasma IgG for HER2-MUC1-VEGFR1 and CD25-MUC1-VEGFR1 were significantly decreased in the three groups(P < 0.001),Plasma anti-VEGFR1 IgG levels were significantly decreased in both group A(P < 0.001)and group C(P < 0.05),compared with the healthy control group.Plasma anti-MUC1 IgG levels were significantly decreased only in early stage NSCLC(group A)compared with healthy controls(P < 0.05).3.ROC curve analysis showed that the ELISA sensitivity was 19.0% for the VEGFR1 IgG assay,19.0% for the HER2-MUC1-VEGFR1 IgG assay and 42.2% for the CD25-MUC1-VEGFR1 IgG,respectively,when the specificity was set at 95.0%,in which the sensitivity of the CD25-MUC1-VEGFR1 IgG assay was as high as 49.6% in early stage of NSCLC(group A).4.Kaplan-meier survival analysis showed that plasma natural autoantibody levels were not associated with the overall survival of NSCLC patients(P >0.05).5.The cell viability was decreased in NCI-H2122 cells treated with anti-HER2 IgG positive plasma as compared with those treated with negative plasma(P < 0.05),while the cell viability was increased in A549 cells treated with any IgG positive plasma compared with IgG negative plasma(P < 0.05).The cell viability was decreased in NCI-H2122 cells treated with anti-MUC1 or anti-VEGFR1 IgG positive plasma as compared with those treated with negative plasma(P < 0.001),while the cell viability was not significantly changed in A549 cells treated with any IgG positive plasma compared with IgG negative plasma(P > 0.05).6.The mRNA expression analysis showed that NCI-H2122 cells expressed significantly higher levels of HER2,MUC1 and VEGFR1 than and A549 cells(P<0.001).Conclusions:1.The levels of plasma IgG antibodies against VEGFR1,HER2-MUC1-VEGFR1 and CD25-MUC1-VEGFR1 were significantly decreased in NSCLC patients with a diagnostic value.2.The CD25-MUC1-VEGFR1 IgG assay was more sensitive than other antibody assays with the sensitivity of 49.6% in the early stage of NSCLC patients,suggesting that the CD25-MUC1-VEGFR1 combination can be used to detect IgG antibodies for early diagnosis of NSCLC.3.The plasma anti-MUC1 IgG levels remained significantly lower in the early stage of NSCLC patients,suggesting that the MUC1 can be used to detect IgG antibodies for early diagnosis of NSCLC.The levels of plasma IgG against MUC1 were significantly decreased in female NSCLC patients,suggesting the existence of a gender difference in NSCLC immunity.4.The levels of plasma IgG against HER2 and MUC1 were significantly decreased in patients with adenocarcinoma type,which suggestied that HER2 and MUC1 IgG can be used as biomarkers for diagnosis of NSCLC of adenocarcinoma.5.The levels of plasma IgG against HER2,MUC1,VEGFR1,ABCC5,MYC,TNF-?,POU5F1,HER2-MUC1-VEGFR1 and CD25-MUC1-VEGFR1 were not associated with the overall survival of NSCLC patients,suggesting no guiding significance for judging the prognosis of patients with NSCLC.6.Plasma with high anti-HER2,anti-MUC1 and anti-VEGFR1 IgG levels was more significantly inhibited the proliferation of NCI-H2122 than plasma with low antiHER2,anti-MUC1 and anti-VEGFR1 IgG levels,which suggested that plasma natural antibody-abundant plasma has the potential for therapeutic effects on some type of NSCLC.7.The mRNA expression of HER2,MUC1 and VEGFR1 was significantly higher in NCI-H2122 cells than that in A549 cells.This may be the major reason why plasma rich in natural antibodies against HER2,MUC1,and VEGFR1 more likely to inhibit the proliferation of NCI-H2122 cells than A549 cells. |
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