Clinical And Basic Studies For Immunotherapeutic Targets Of Gestational Trophoblastic Neoplasia

Backgrounds and objectivesGestational trophoblastic neoplasia(GTN)is a group disease which arises from the abnormal proliferation of placental trophoblastic cells.GTN is sensitive to chemotherapy.Low-risk patients usually receive single-agent chemotherapy with a survival rate close to 100%,while high-risk and ultra-high risk patients need multi-agent chemotherapy.Although the cure rate of high-risk GTN patients can reach 90%,the prognosis of ultra-high risk GTN patients is still poor,about 40%of the patients die from disease progression eventually.Immunotherapy has joined the ranks of surgery,radiotherapy,chemotherapy,and targeted therapy as an important pillar of cancer treatment.Previous studies have proved that PD-L1 is widely expressed in GTN tumor cells,and a few cases have reported that PD-1 inhibitors show a high response rate in treating GTN patients,but chemo-resist still occurred in some patients.Therefore,it is imperative to understand the tumor immunity pathway and seek new immune targets for GTN.V-type immunoglobulin domain-containing suppressor of T cell activation(VISTA)is the homologue of B7 family ligands,PD-L1 and PD-L2.VISTA mainly expresses in myeloid cells,monocytes,macrophages and dendritic cells.In addition to expressing in the tumor cells,VISTA is also expressed in immune cells.VISTA functions as both a ligand and a receptor separately,but these roles are not mutually exclusive.VISTA induces FOXP3+T cells,inhibits the activity of T cells,and modulates the function of antigen presenting cells.Previous studies have shown that VISTA is highly expressed in GTN tumor cells,and its expression is not related to disease stage and prognostic outcome of patients,suggesting that VISTA may be a potential target for GTN immunotherapy.The study of immunotherapy in GTN is still in preliminary stage.To analyze the therapeutic effect and further explore new targets of GTN immunotherapy,this study included following three parts:(1)efficacy and safety analysis of PD-1 inhibitor combined with apatinib in the treatment of high risk chemo-refractory/relapsed GTN;(2)analysis of the expression of immune checkpoints in the tumor infiltrating immunocytes(TIIs)of GTN;(3)the anti-tumor effects of PD-1 and VISTA blockade on choriocarcinoma in vitro and in vivo.Materials and methods1.A total of 20 patients with chemo-refractory/relapsed GTN who had failed at least two or more multi-agent chemotherapies,and had a FIGO score?7,and with an abnormal serum ?-HCG level were enrolled.Patients received camrelizumab 200 mg intravenously every 2 weeks and apatinib 250 mg orally once per day.Patients who achieved a complete response(CR)were continued with the regimen for 6 months(12 administrations)for consolidation treatment.The primary endpoint of the study was objective response rate.The secondary endpoints included safety,duration of response,and progression free survival.2.A total of 108 pathological tissue specimens were collected from patients who histopathologically diagnosed with choriocarcinoma,PSTT,and ETT,and had enough tumor samples for immunohistochemistry at Peking Union Medical College Hospital from January 2008 to December 2017.The expression levels of immune checkpoints VISTA,PD-1,LAG-3,TIM-3 and GAL-9 in TIIs were analyzed,then the correlations between the expression difference and prognosis of GTN patients were analyzed.3.The expression of VISTA and PD-L1 in choriocarcinoma cell line JEG-3 was detected by flow cytometry and cellular immunofluorescence.Effect of VISTA blockade alone or combined with PD-1 in JEG-3 cells and co-culture with T cells on the proliferation and cytotoxic ability in vitro.To explore the antitumor effect of combined blocking of PD-1 and VISTA on choriocarcinoma in humanized immune system mice.Results1.Ten patients normalized ?-HCG during treatment with camrelizumab and apatinib.In these patients,the median number of treatment cycles with ?-HCG normalization was 3(range,1-6.5).The median duration of remission was 15 months.The objective response rate was 55%(95%confidence interval[CI],31.1%-78.9%),and the CR rate was 50%(95%CI,26%-74%).Eighteen patients in the study experienced at least one treatment-related adverse effect(AE).Treatment-related grade 3 AEs occurred in 12 patients(60%),no grade 4 AE occurred.The most common grade 3 AE was hypertension(25%).One patient discontinued treatment and hospitalized because of grade 3 aspartate aminotransferase concentration increase.No treatment-related death occurred during treatment in this study.Four patients delayed treatment due to rash and fever,all with a delay<14 days.One patient permanently discontinued apatinib after developing uncontrollable hypertension.2.VISTA and GAL-9 expressed in TIIs of all GTN specimens,with moderate to strong expression in 99.1%and 94.4%patients.TIM-3 and PD-1 were expressed in 92.6%and 90.1%of TIIs,with moderate to strong expression in 45 and 56 patients,respectively.LAG-3 was expressed in 77.8%of TIIs,and moderate and strong expression were only detected in 5 patients.The expression of TIM-3 and GAL-9 in choriocarcinoma was higher than that in PSTT,but there was no significant difference between choriocarcinoma and ETT,or between PSTT and ETT.The expression of LAG-3 in ETT is lower than that of choriocarcinoma and PSTT,and there is no significant difference in the expression intensity between choriocarcinoma and PSTT.Patients with positive LAG-3 expression in TIIs have significant shorter progression-free survival.3.VISTA protein was highly expressed in the cell membrane and cytoplasm of JEG-3 choriocarcinoma cell line.The combination of blocking VISTA and PD-1 in JEG-3 cell and co-culture with T cells significantly restore the proliferation and cytotoxic ability of T cells,and reduce the proportion of FOXP+T cells.In humanized immune system mouse models,comparing with blockade of VISTA or PD-1 alone,combined blocking of VISTA and PD-1 significantly inhibited tumor growth,increased CD4/CD8 ratio and proportion of CD45RO+T cells in peripheral blood,and significantly decreased the proportion of PD-1+CD8+T cells,VISTA+CD8+T cells and FOXP3+T cells.Conclusions1.The PD-1 inhibitor camrelizumab combined with apatinib demonstrated encouraging antitumor efficacy and tolerable toxicity in patients with high-risk chemo-refractory/relapsed GTN.2.VISTA,GAL-9,PD-1,and TIM-3 were widely expressed in TIIs of GTN and had no association with the clinical outcome of patients.The positive expression of LAG-3 related to the poor prognosis,and these immune checkpoints may be potential immunotherapeutic targets for GTN.3.VISTA and PD-L1 protein was highly expressed in JEG-3 cell line.The combination of blocking VISTA and PD-1 in JEG-3 cell and co-culture with T cells significantly restore the proliferation and cytotoxic ability of T cells.In humanized immune system mouse models,comparing with blockade of VISTA or PD-1 alone,combined blocking of VISTA and PD-1 significantly inhibited choriocarcinoma tumor growth.