The Chromosome 11q13.3 Amplification Induces Acquired Resistance To Anti-PD-1 Antibody In Cancer Immunotherapy

Background and ObjectionAs a comprehensive treatment method,tumor immunotherapy has been recommended by NCCN guidelines for the treatment of a variety of tumors.But overall,the objective response rate of ICIs in cancer patients is low,only about 20%[1].However,some patients with effective initial response,after a period of disease control,gradually appear tumor recurrence and metastasis,which is considered to be the acquired resistance to cancer immunotherapy[2].It limits the clinical application and efficacy of ICIs.Therefore,pre-screening the beneficiaries of immunotherapy and finding a treatment method to overcome acquired drug resistance will be of great significance for patients with advanced cancer.Our clinical and basic experimental data show that the combined amplification of the gene FGF3/FGF4/FGF19/CCND1 located at the 11q13.3 locus of chromosome can induce acquired resistance to anti-PD-1 antibodies,which is regarded as a negative predictor of the efficacy of anti-PD-1 antibody therapy in patients with advanced metastatic esophageal squamous cell carcinoma.However,the role of FGF3/FGF4/FGF19/CCND1 co-amplification in the acquired resistance of anti-PD-1 antibodies is rarely reported,and its mechanism remains to be clarified.MethodsWe analyzed the clinical cases of acquired drug resistance after receiving anti-PD-1 antibody treatment and the database information of TCGA and MSKCC to clarify the amplification status of FGF3/FGF4/FGF19/CCND1 and the changes level of immune cell infiltration and activation.We used anti-PD-1 antibody to construct the acquired drug-resistant mouse subcutaneous tumors model,and estimated the expression of FGF3/FGF4/FGF19/CCND1 by Western Blot and Q-PCR.We used flow cytometry techniques,HE staining and immunohistochemistry experiments to analyze the changes of the proportion of immune cell populations under different conditions to reflect the anti-tumor immunity.Finally,we used the esophageal cancer,lung adenocarcinoma and melanoma datasets of the TCGA database to do functional enrichment analysis of the differential genes between FGF3/FGF4/FGF19/CCND1 co-amplification and non-co-amplification to explore the potential activated pathways induced by quadruple gene amplification.And we used Western Blot and Q-PCR to verify the activation of related pathways and cytokines.ResultsClinical cases information showed that genes FGF3/FGF4/FGF19/CCND1 and FGFR1 amplification occurred after the patients receiving anti-PD-1 antibody treatment developed acquired resistance,and TCGA and MSKCC databases suggests that in most cancers,the FGF3/FGF4/FGF19/CCND1 quadruple genes are in the form of co-amplification and are associated with poor tumor prognosis.Tumor immune analysis showed that compared with tumors without quadruple genes co-amplification,quadruple genes co-amplified tumors had significantly lower anti-tumor immue activity.Western Blot and Q-PCR resaults showed that the expression of quadruple genes in drug-resistant tumors is significantly higher than that in treatment-sensitive tumors.Vivo and vitro experiments indicate that although single anti-PD-1 antibody can not suppress acquired drug-resistant tumors,it combined with FGFR inhibitor erdafitinib treatment can significantly inhibit the proliferation of drug-resistant tumors in immune-complete mice.Flow cytometry technology resault suggests that the combination therapy significantly up-regulates the infiltration and function of CD4+and CD8+T lymphocytes in immune-complete mice,and significantly down-regulates the ratio of regulatory T cells,exhaustive CD4+and CD8+ T cells.Functional enrichment analysis indicated that the quadruple genes amplification caused the activation of the WNT/β-catenin immunosuppressive signaling pathway and relative cytokines in tumors,FGFR inhibitors combined with anti-PD-1 antibodies can significantly inhibit the activation of this pathway and reverse the occurrence of drug resistance.ConclusionThe combined amplification of FGF3/FGF4/FGF19/CCND1 can induce the up-regulation of PD-1+Treg cell infiltration and reduce the infiltration of killer T lymphocytes,in the tumor microenvironment by activating the FGFR-WNT/β-catenin signaling pathway,thereby inducing resistance The acquired resistance of PD-1 antibody,and the combination of FGFR inhibitor can reverse the occurrence of resistance and enhance the therapeutic effect of anti-PD-1 antibody.