Clinical Analysis And Novel Immunotherapy Target Of Relapsed Gestational Trophoblastic Neoplasia

Backgrounds and objectivesGestational trophoblastic neoplasia(GTN)encompasses a group of gynecologic tumors arising from abnormal proliferation of placenta.Currently,limited information is available about the management,prognosis and risk factors of relapsed GTN.Furthermore,patients with relapsed GTN are more likely to develop multidrug resistance,therefore become refractory and have poor prognoses.Novel treatments are needed for these refractory patients to improve their prognoses.A preliminary study from The Charing Cross Trophoblastic Disease Center in UK reported the success of immune checkpoint inhibitor PD-1 antibody in patients with resistant/relapsed GTN.This suggested that immune checkpoint inhibitor might be effective for patients with refractory GTN,providing a new way for treatment of GTN.However,the regulatory network in tumor immune microenvironment is complex and various immune checkpoint molecules may play different roles in the network.The combined application of different immune checkpoint inhibitors may block multiple pathways in the network simultaneously,which can inhibit tumor immune escape more efficiently.Therefore,it’s necessary to explore the roles of other immune checkpoint molecules in antitumor immunity.V-domain Ig suppressor of T cell activation(VISTA)is a new member of B7 family,expressed on both tumor cells and immune cells.Previous studies have shown the expression of VISTA in melanoma,prostate cancer,ovarian cancer and other malignant tumors.These studies focused on the role of VISTA in immune response in several tumors.However,to the best of our knowledge,it’s still unclear about the expression of VISTA in GTN and the effects of VISTA on tumor biological behaviors of choriocarcinoma.The objectives of this study were as follows:1)to analyze management and prognoses of patients with relapsed GTN,to explore risk factors of GTN relapse;2)to examine the expression of VISTA in GTN and analyze the correlation between VISTA expression and clinicopathological parameters in GTN;3)to explore the effects of VISTA on tumor biological behaviors of choriocarcinoma and clarify the mechanism.Methods1)To review Peking Union Medical College Hospital GTN database from January 2004 to December 2017 and identify patients with GTN who relapsed after completion of treatment;to review the medical records of these patients to extract the clinical parameters,treatments,and outcomes at both initial presentation and relapse;to explore the risk factors of relapse and poor prognosis with statistical analysis.2)To analyze the expression of VISTA mRNA in normal tissues using HPA,FANTOM5,and GTEx database;to analyze the expression of VISTA mRNA in 32 tumors from TCGA database using cBiorporal and CCLE website;to examine the expression of VISTA protein in GTN with immunohistochemical staining and ELISA;to analyze the correlation between expression of VISTA and clinicopathological parameters in GTN with statistical analysis.3)To establish VISTA-knockdown JEG-3 cell lines by lentiviral transfection of short hairpin RNA and small interfering RNA,respectively;to examine proliferation and clone formation ability of cells with CCK-8 assay and plate clone,respectively;to examine migration and invasion of cells with Transwell models;to examine apoptosis of cells with flow cytometry;to examine expression of VISTA and Epithelial-mesenchymal transition(EMT)-related proteins with Western Blot.Results1)A total of 1827 patients with GTN achieved complete remission between January 2004 and December 2017.Among these patients,118(6.5%)patients relapsed after completion of treatment.The relapse rate for patients initially treated at our center and those referred to us was 2.7%and 14.6%,respectively.Patients who underwent salvage surgeries at relapse achieved a significantly higher complete remission rate than those who did not.The 5-year survival rate for patients with GTN relapse was 80.4%.An interval between antecedent pregnancy and chemotherapy>12 months and an interval from first chemotherapy to achieving β-hCG normalization>14 weeks were predictors of GTN relapse.Stage IV at relapse and multiple relapses were risk factors of poor prognosis for patients with relapsed GTN.2)VISTA mRNA was expressed in various normal tissues and 32 tumors.VISTA protein and PD-L1 protein were both highly expressed in the tumor tissues of GTN,including tumor cells and immune cells.VISTA protein was also highly expressed in the serum of patients with GTN.Furthermore,choriocarcinomas and placental site trophoblastic tumors showed higher expression of VISTA than epithelioid trophoblastic tumors.3)Knockdown of VISTA inhibited proliferation,cloning,invasion and migration of JEG-3 in vitro,while knockdown of VISTA didn’t have any effects on apoptosis or MTX-induced apoptosis of JEG-3.VISTA might promote invasion and migration of choriocarcinoma through EMT.Conclusions1)The relapse rate in patients initially treated at our center was extremely low;salvage surgery plays a beneficial role in the management of patients with relapsed GTN;most of patients with relapsed GTN could achieve a favorable outcome after salvage treatments.2)VISTA was highly expressed on both tumor cells and immune cells of GTN;the expression of VISTA on tumor cells was correlated with pathological type of GTN;VISTA might be a novel immunotherapy target for GTN.3)VISTA had influences on the tumor biological behaviors of choriocarcinoma,which might promote invasion and migration of choriocarcinoma through EMT.