Study On The Detection Of Plasma Soluble Podoplanin Levels In Patients With Lung Cancer And Breast Cancer And Its Clinical Application Value

Background:Malignant tumors,second only to cardiovascular and cerebrovascular events,rank second in morbidity and mortality in Chinese residents.As a developing country,the incidence of different cancers in China is slightly different from that in developed countries,but lung cancer still occupies the most important position,and the incidence rate of breast cancer is significantly higher among women worldwide.Although,with the advancement of medical technology,we can carry out surgical treatment,interventional treatment,and chemoradiotherapy for lung cancer patients,but with little effect,the 5-year survival rate of lung cancer patients in China is only 19.7%.Many patients were already in the stage of disease progression when they were diagnosed,and they missed the best time for treatment,which increases the difficulty of treatment.Therefore,we need the lung cancer screening method which is easier to get and popularize to the basic medical units,so as to improve the diagnosis rate of early lung cancer and treat it as soon as possible.We shift our attention to the podoplanin(PDPN),which has a small amount of expression in normal body and an up-regulated content in tumor tissue.PDPN is a small molecule sialic mucin that exists in many mammals.In normal human tissues,it is mainly expressed on the lymphatic endothelial tube,and there is a small amount on bone tissue and alveolar cells It plays an important role in embryonic lung development and kidney development.Recently,many scholars have used PDPN monoclonal antibody D2-40 to stain a variety of human malignant tumor specimens and noticed that PDPN is highly expressed in tumor tissues and metastatic tissues,such as melanoma,glioma,and osteosarcoma.By acting on platelets,PDPN increases the possibility of tumor metastasis and related thrombosis.We already developed two strains of monoclonal antibodies SZ-163 and SZ-168 derived from mouse against PDPN,but the double-antibody sandwich enzyme-linked immunosorbent(ELISA)test kit has some problems,such as low production,so it is difficult to mass-produce and promote their use.However,it has been found that the plasma soluble PDPN level of cancer patients is significantly higher than that of normal people.Objective:To prepare a high-efficiency and stable monoclonal antibody,which can be established a stable ELISA Kit with SZ-168,and apply it to detacting breast cancer patients and lung cancer patients,to test whether sPDPN can play a role in the diagnosis of breast cancer patients and lung cancer patients.Methods:(1)CHO-hPDPN,PDPN polypeptide,and CHO cell-clad plate were used to screen the recovered SZ-163 primary hybridoma cells.Positive cell lines were obtained by 3 times of monoclonalization.A large amount of ascites was produced by the principle of induction in vivo.The antibody was purified by affinity chromatography.(2)Indirect ELISA and Western Blot(WB)were used to observe whether the obtained antibodies could recognize and bind to CHO-hPDPN,PDPN polypeptide,CHO cells,and melanoma cells C8161 with PDPN on the surface.(3)The obtained new antibody SZ-163-8C12-3 were respectively plated,and the plasma collected from female stem cell donors and breast cancer patients collected from the First Affiliated Hospital of Soochow University was added,recombinant human PDPN full-length polypeptide was used as a standard,Peroxidase-labeled SZ-168(SZ-168-HRP)as second antibody,and explore the best linear range,minimum detection limit,biological detection limit and sensitivity of the new kit.And the two measurements were compared using Pearson correlation analysis and Bland-Altman consistency analysis.Acceptance of P<0.05 was considered statistically significant.Collect invasive breast cancer specimens from the Department of Pathology,hematoxylin-eosin staining(HE staining),and immunohistochemical staining(IHC)with anti-PDPN monoclonal antibody D2-40,combined with other clinical parameters,observe the staining results.(4)Plasma was collected from normal medical examiners,patients with pneumonia and lung cancer patients in Fuzhou General Hospital of Nanjing Military Region,and tested with a kit made by the new antibody to compare the sPDPN levels in the three groups.Analyzing whether sPDPN can distinguish lung cancer patients from other populations?What is the diagnostic value?Is it an independent predictor of lung cancer?The level of sPDPN in patients with pneumonia?All accepted P<0.05 as statistically significant.Results:(1)Positive cell lines were successfully screened by three times of monoclonalization of primary cells and a stable positive hybridoma cell—8C12-3 was obtained.About 2ml of ascites/mouse was collected,and purified SZ-163-8C12-3 antibody of 3mg IgG/ml ascites was purified,which was 8 times the original SZ-163 yield.(2)ELISA results suggest that the titers of SZ-163-8C12-3 anti-CHO-hPDPN and PDPN polypeptides are 62.5ng/ml and 125ng/ml,and it cannot react with CHO cells.In the WB test,SZ-163-8C12-3,like SZ-168,can recognize PDPN on the surface of natural C8161 cells,while nromal mouse IgG cannot.(3)With SZ-163-8C12-3 as a plated antibody,a standard curve of R2=0.995 can be made.The best linear range of the SZ-163-8C12-3 plated kit is 0.9765625ng/ml-250ng ml,the lower limit of detection is 0.65ng/ml,the biologicallimit of detection is 0.91ng/ml,and the sensitivity is 0.65ng/ml.The sPDPN content of the normal control group and breast cancer group was(1.14±0.22)ng/ml and(22.23±2.68)ng/ml,P<0.001.The results of Pearson correlation analysis and Bland Altman consistency analysis showed that the results of SZ-163 kit were consistent with those of SZ-163-8C12-3 kit.Combined with HE staining results,IHC results suggest that PDPN is highly expressed in the tissues of some breast cancer patients.Breast cancer patients are classified according to age,clinical stage,degree of differentiation,and whether there is lymph node metastasis.Lymph node metastasis,estrogen receptor-negative,and progesterone receptor-negative patients had a higher PDPN-positive rate than patients with stage ?-?(P=0.022),no lymph node metastasis(P=0.042),and estrogen receptor-positive(P=0.010),progesterone receptor positive(P=0.037).The tissue PDPN-positive group had significantly higher sPDPN content than the negative group[(23.74±2.71)ng/ml vs(21.09±1.89)ng/ml,P=0.003],and the ki67 labeling rate was also higher than the negative group[(57.14±12.54)%vs(21.54±8.86)%,P<0.001].(4)The plasma levels of sPDPN were(5.17±2.81)ng/ml and(19.96±6.26)ng/ml in the pneumonia group and lung cancer group respectively,Compared with the normal group(1.37±0.55)ng/ml.The levels of sPDPN in the lung cancer group were higher than those in the pneumonia group(P<0.001)and the normal control group(P<0.001).The level of sPDPN in pneumonia group was higher than that in control group(P<0.001).In patients with lung cancer,the level of sPDPN in patients with late clinical stage,low differentiation of tumor cells and metastasis was significantly higher than that in patients with early stage(P=0.010),good differentiation(P=0.010)and no metastasis(P=0.015).The diagnostic efficacy of sPDPN was better than CEA and CYF211,which had the largest area under curve(AUC)of 0.986,sensitivity of 0.914 and specificity of 0.992,and was an independent predictor of lung cancer with or of 4.148.In pneumonia patients,the level of sPDPNwas correlated with the content of procalcitonin(PCT),the Pearson correlation coefficient was 0.798,P<0.05,but not with hsCRP(Pearson coefficient=0.217,P=0.122).Conclusion:(1)An anti-CHO-hPDPN cell,anti-PDPN PLAG,mouse monoclonal antibody 8C12,which does not react with CHO cells and can recognize PDPN in natural tumor cells,was successfully prepared.(2)SZ-163-8C12-3 can be used with SZ-168 to make a good soluble PDPN kit for human plasma.(3)There are high levels of sPDPN in the plasma of breast cancer patients,and high expression of PDPN in tumor tissues of some patients,which can be initially distinguished from late clinical stage,with lymph node metastasis,estrogen receptor negative,progesterone receptor negative,high levels of sPDPN and ki67 Patients with high labeling rates.(4)There is a high level of sPDPN in the plasma of lung cancer patients.Compared with CEA and CYF211,sPDPN has the largest area under the curve,the highest sensitivity and specificity for the diagnosis of lung cancer,and is an independent predictor of lung cancer.The clinical application value of sPDPN as a screening index for lung cancer is suggested,and the clinical stage,differentiation degree and metastasis can be initially evaluated.(5)The level of sPDPN in the plasma of patients with pneumonia is also also increased(less than that of patients with lung cancer),which is related to PCT and may be related to inflammatory reaction in vivo.