Effect Of PD-1 Immunotherapy On Serum CA19-9 Levels In Patients With Esophageal Cancer And Its Molecular Mechanisms

Background and ObjectivesEsophageal cancer(esophageal carcinoma)is a common digestive tract tumor in human beings,with a high malignancy and poor prognosis.Its incidence and mortality rate vary greatly among countries,while the annual new cases of esophageal cancer in China account for more than half of the world.Early esophageal cancer symptoms are not obvious,most patients are diagnosed in the late,esophageal cancer traditional treatment such as surgery,radiotherapy and chemotherapy and targeted drug therapy for early patients cure rate is very high,but for late patients,5 years low survival rate,the side effects in the treatment process,bring patients mental and physical damage,so the limitations of traditional treatment cannot be ignored,we must explore new treatment.Immunotherapy,which shows better antitumor effects in diseases such as non-small cell lung cancer,bladder cancer,melanoma,and renal cell cancer,is considered a promising strategy to eliminate malignancy,providing new research directions for cancer treatment and new implications for the treatment of esophageal cancer.Cancer immunotherapy is mainly about inducing a lasting human immune response to destroy tumor cells.Immunotherapy mainly includes Cancer vaccines,Adoptive cell transfer,Tumor infiltrating lymphocytes,and Checkpoint inhibitors.The efficacy and safety of immune checkpoint inhibitors in immunotherapy in tumor therapy have been recognized by scholars at home and abroad,among which programmed death receptor 1(PD-1)/programmed cell death ligand 1(PD-L1)inhibitors have been widely studied.With the deepening of immune-related research,PD-1/PD-L1 inhibitors have been widely used abroad in the treatment of more than a dozen tumors,such as lung cancer,intestinal cancer,liver cancer,urinary cancer,head and neck squamous cancer,melanoma and lymphoma.The role of PD-1 inhibitor immunotherapy plays an increasingly stable role in the process of tumor treatment.There are currently six PD-1 inhibitors in China,namely Nivolumab,Pembrolizumab,Toripalimab,Sintilimab,Camrelizumab,Tislelizumab,and two PD-L1 inhibitors,respectively Durvalumab and Atezolizumab.According to public data,as of 2021,there were 271 new clinical trials of new PD-1/PD-L1 inhibitors in China.According to the results of clinical trials,pembroliuzumab has been approved by the US Food and Drug Administration(FDA)for patients with advanced Esophageal Squamous Cell Carcinoma(ESCC),and navuzumab has been approved by the FDA as a monotherapy for esophageal squamous cell cancer patients.CA19-9 is a commonly used clinical tumor marker of glycoproteins,and it has been shown that it is abnormally expressed in malignant tumors such as lung cancer,ovarian cancer and breast cancer,and can be used for the adjuvant diagnosis of a variety of malignancies.As a serological marker for the clinical diagnosis of esophageal cancer,CA19-9 can accelerate the disease progression of esophageal cancer by glycosylation of protein,binding with E-selectin,enhanced angiogenesis and mediated immune response.The sensitivity and specificity of CA19-9 to diagnose esophageal cancer are 32.50%and 83 of 25%,respectively.However,the molecular mechanism studies are not clear,and this paper aims to analyze the effect of PD-1 immunotherapy on CA19-9 levels in oesophageal cancer patients,which will be analyzed from the molecular level.It has been reported that tarnosyltransferase 3(FUT3)and 1,3-galactosyltransferase peptides(B3GALT5)are important components of glycoprotein production and can promote the synthesis of CA19-9,whose genetic allele variation affects serum CA19-9 levels,and FUT3 and B3GALT5 expression can cause elevated CA19-9 expression,similarly observed in human cancer cell lines;B3GALT5 can enhance cell migration,invasion,mammary formation and epithelial to mesenchymal transition and contribute to breast cancer progression.We will validated at the gene level by detecting changes in the expression levels of two genes following immunotherapy.At the same time,we constructed cell models that overexpress the FUT3 and B3GALT5 genes on esophageal cancer cell lines to observe the apoptosis and proliferation of esophageal cancer cell lines,analyze their correlation with esophageal cancer cell progression,and in order for further validation,we did some simple mechanistic studies.PD-1 therapy plays a more and more important role in tumor treatment,but the mechanism by which PD-1 inhibits esophageal cancer is still unclear.This study aims to reveal the mechanism of PD-1 inhibiting tumor,and explore the clinical manifestations and CA199 related proteins.Method1.Collect peripheral blood(20 healthy people,18 patients with esophageal cancer treated with PD-15 34 patients with esophageal cancer)and tumor tissue specimens(10 surgical specimens of esophageal cancer treated with PD-1 and 15 surgical specimens of esophageal cancer not treated with PD-1);2.CA19-9 related genes and protein expression levels in tumor tissues of PD-1 inhibitor-treated and untreated esophageal cancer patients by real-time PCR and immunoblotting(Western blot);3.Apoptosis and proliferation analysis of FUT3 overexpressing cells,B3GALT5 overexpressing cells and untreated KYSE-30 cell lines using flow cytometry and thiblue-specific method(MTT);4.Gene expression levels and protein expression levels of B3GALT5,UGCG and ABO in KYSE-30 esophageal U T 3 treated with knockdown and overexpression using real-time PCR and Western blot,respectively;5.The expression levels of UGCG and ABO downstream of FUT3 in tumor tissues of esophageal cancer patients by real-time PCR and Western blot.Results1.Serum CA19-9 levels decline in patients with oesophageal cancer after being treated with PD-1 inhibitors,which may be an index to evaluate the effect of immunotherapy.2.After immunotherapy in esophageal cancer patients,FUT3 and B3GALT5 and downstream genes were downregulated by detecting relevant gene and protein expression in tumor tissues,suggesting that this may be responsible for the decline in serum CA19-9.3.The overexpression of FUT3 and B3GALT5 could alleviate apoptosis and promote proliferation of esophageal cancer cells,overexpression of KYSE-30 cell line showed FUT3,B3GALT5,UGCG and ABO,and KYSE-30 showed decreased levels of B3GALT5,UGCG and ABO,so it was speculated that FUT3 and B3GALT5 genes could affect CA19-9 expression and tumor progression.ConclusionCA19-9 serum level is higher in esophageal cancer patients than in healthy controls,PD-1 immunotherapy can reduce patient serum CA19-9 expression levels,CA19-9 can be used as a potential monitoring indicator for PD-1 immunotherapy;esophageal cancer patients cause tumor cell FUT3 and B3GALT5 gene downregulation and downstream genes,which may be one of the mechanisms that affect CA19-9 expression and tumor progression.