Studies Of PD-1 Inhibitors And PD-1 Inhibitors Combined With Chemotherapy In Gestational Trophoblastic Neoplasia

Background and objectivesGestational trophoblastic neoplasia(GTN)is a group of gynecological malignancies originating from placental trophoblastic cells.Chemotherapy plays a crucial role in the treatment of GTN.The regimens of chemotherapy depend on the risk level determined by the FIGO score.Low-risk and high-risk GTN is treated with singleagent or multiagent chemotherapy respectively,with a cure rate of over 90%.However,a percentage of patients develop resistance to chemotherapy and refractory diseases.For patients with chemorefractory or relapsed diseases,salvage chemotherapy-based comprehensive treatment is usually adopted.The histological origin endows GTN with strong immunogenicity and PD-L1 is widely expressed in GTN tumors.Therefore,PD-1 inhibitors can serve as a new treatment option for chemorefractory or relapsed GTN patients.Among different subtypes of GTN,PD-1 inhibitors have exhibited a clinical response rate of 50%,ranking among the top in solid tumors.However,some refractory GTN patients struggle to respond effectively to PD1 inhibitor monotherapy and ultimately die from disease progression.Preclinical researches have shown that conventional chemotherapy could enhance endogenous immune responses through diverse mechanisms.On the one hand,chemotherapy can induce adaptive immunity by increasing tumor cell immunogenicity and enhancing T cell activation.On the other hand,chemotherapy may help restore antitumor function by destroying immunosuppressive components in the tumor microenvironment.Numerous clinical studies have confirmed the immunomodulatory effect of chemotherapy.The combination of chemotherapy and PD-1 inhibitors exhibits good anti-tumor activity and tolerable toxicity in various malignant tumors.However,there is limited data on the combination of PD-1 inhibitors and chemotherapy in highrisk chemotherapy chemorefractory/relapsed GTN patients.Therefore,exploring the efficacy and safety of PD-1 inhibitor combined with chemotherapy in GTN patients with chemorefractory or relapsed diseases,understanding the specific mechanisms of chemotherapy regulating anti-tumor immunity,and screening biomarkers for predicting the efficacy of PD-1 inhibitors in order to identify patients who will benefit most from anti-PD-1 therapy have become urgent issues that need to be addressed.This study includes the following three parts:1 Anti-PD-1 therapy plus chemotherapy versus antiPD-1 therapy alone in patients with high-risk chemorefractory or relapsed gestational trophoblastic neoplasia;2.The mechanism of MTX regulating the anti-tumor function of CD8+T cells through arachidonic acid;3.Research on heterogeneity of GTN using spatial transcriptomics technology.Materials and methods1.This multicenter,retrospective cohort study was conducted at three hospitals in China,including Peking Union Medical College Hospital(PUMCH),China Medical University Affliated to Shengjing Hospital,and Obstetrics and Gynecology Hospital of Fudan University.We included patients with high risk chemorefractory or relapsed GTN diseases(FIGO score≥7)who then received anti-PD-1 therapy combined with chemotherapy or anti-PD-1 monotherapy.Serum hCG content was determined to evaluate disease remission at intervals of 2 cycles.Study endpoints included objective response rate(ORR),duration of treatment(DOT),overall survival(OS),progressionfree survival(PFS)and safety of anti-PD-1 monotherapy compared with chemotherapy plus anti-PD-1.2.The differential metabolic products of MTX-treated JEG-3 cells were screened by untargeted metabolomics.Tumor cell-T cell co-culture system was applied to study the effect of MTX and its metabolite arachidonic acid on CD8+T cells.To confirm the phenotype of ferroptosis induced by arachidonic acid combined with IFN-γ.3.Tumor tissues were collected from 4 GTN patients and divided into complete response(CR)group and progressive disease(PD)group based on treatment responses to PD-1 inhibitors.The spatial transcriptomics analysis was performed to analyze the heterogeneity of GTN tumor,evaluate the dominant expression genes and functional characteristics of different clusters,and compare the differences in gene expression,immune cell infiltration and signal pathways among patients responding differently to PD-1 inhibitors.Results1.This work enrolled 66 cases.35 and 31 patients received anti-PD-1 therapy alone and combined with chemotherapy,respectively.The combined treatment dramatically increased the objective response rate from 62.9%(22/35)to 96.8%(30/31)(p<0.001).The median durations until complete response were 2.2(IQR,1.4-4.2)and 2.8(IQR,1.8-2.8)months in the anti-PD-1 monotherapy and combined treatment cohorts,respectively(P=0.299).The complete response rate for anti-PD-1-refractory patients to salvage chemotherapy was 84.6%(11/13),including 7(63.6%)with complete response to previously failed chemotherapy regimens.There was no significant difference in OS between the two groups of patients(HR 0.50,p=0.499),while the combination therapy group significantly improved PFS(HR 0.06,p<0.001).Grade 3-4 treatment-related adverse events with highest occurrence frequency included leukopenia(25.8%,combined treatment;11.4%,anti-PD-1 monotherapy)and increased alanine aminotransferase level(9.7%,combined treatment;5.7%,anti-PD-1 monotherapy)in all cohorts.Drug-related death was not reported in any groups.2.In vitro cell experiments have shown that low concentrations of MTX caused significant apoptosis and cytotoxic ability of CD8+T cells.Untargeted metabolomics showed that the content of arachidonic acid(AA)in the supernatant of choriocarcinoma cell JEG-3 increased significantly after MTX treatment.In the tumor cell-T cell coculture system,AA increased the expression of FABP5,fatty acid uptake,and cytotoxic ability of CD8+T cells.FABP5 inhibitor successfully reversed the enhanced cytotoxic ability of CD8+T cells induced by AA.Combined treatment of AA and PD-1 monoclonal antibody increased the cytotoxic ability of CD8+T cells and promoted IL2,IFN-y,and TNF-α secretion.AA combined with IFN-γ could induce a significant decrease in the expression of GPX4 and elevated level of the hallmarks of ferroptosis,MDA and lipid ROS in JEG-3 cells.3.All tumor regions were classified into 6 subsets.Cluster5 was rich in iCAF and apCAF,and myC AF was the most abundant component of cluster 1.Tumor clusters 2,3,4,and 6 exhibited strong spatial heterogeneity.The CR group showed higher score of CD8+Tem cells and macrophages,while the PD group owned higher degree of Treg infiltration.Compared with patients in the CR group,patients in the PD group showed significantly higher expression of HLA-G in tumor cells and downregulation in p53 and ferroptosis pathways.Conclusions1.Anti-PD-1 therapy combined with chemotherapy exhibits sustainably improved antitumor effect and tolerable toxic effects among high-risk chemorefractory or relapsed GTN cases.Patients not responding to PD-1 inhibitors can be effectively rescued with salvage chemotherapy.2.MTX can enhance the fatty acid metabolism of CD8+T cells through FABP5 pathway by promoting the secretion of arachidonic acid,and improve the killing function of CD8+T cells.Arachidonic acid combined with blocking PD-1 can significantly increase the killing effect of CD8+T cells.Arachidonic acid can co-operate with IFN-γ to induce ferroptosis in JEG-3 cells.3.Cancer-associated fibroblasts(CAF)were the most abundant immune cells in the tumor environment of GTN.Tumor cells of GTN owned with heterogeneity within the tumor.The degree of immune cell infiltration,HLA-G expression,p53 and ferroptosis pathway may help identify individuals responding to PD-1 inhibitors.