Dynamic Patterns Of CtDNA And Characteristics Of Tumor Microenvironment Of Non-small Cell Lung Cancer Under Selective RET Inhibition

ObjectiveRearranged during transfection(RET)has been proven to be an important targetable genetic driver in non-small cell lung cancers(NSCLCs).Selective RET inhibitors such as pralsetinib and selpercatinib are now standard therapies in RET fusion-positive non-small cell lung cancer(NSCLC).However,molecular features and their impact on prognosis of this subset of lung cancer were not well illustrated,and the activity of mainstay therapeutics has not currently been well compared.Here,we present treatment outcomes and a comprehensive investigation of safety of RET inhibitors in advanced RET-driven NSCLC patients.MethodsIn the first part of this study,patients diagnosed with NSCLCs with RET rearrangements were retrospectively analyzed for concomitant mutations,tumor mutation burden(TMB),PD-L1 expression,T-cell receptor repertoire and clinical outcomes with chemotherapy,immune checkpoint inhibitors and selective inhibitors.In the second part of this study,we analyzed a prospective cohort of RET fusionpositive,pralsetinib-treated advanced NSCLC patients.We examined the association between tumor response/progression and genomic aberration,baseline circulating tumor DNA(ctDNA)level,ctDNA clearance as defined by multiple metrics,and ctDNA dynamic patterns.In the third part of this study,paired samples before initiation and after progression were analyzed by targeted DNA sequencing,bulk/single-cell RNA sequencing to identify potential resistance mechanisms as well as characteristics of tumor microenvironment(TME),including cell components and their functions.ResultsAmong 129 patients with RET-rearranged NSCLC who were analyzed,41.1%had co-occurring genetic alterations by next generation sequencing,and concomitant TP53 mutation appeared most frequently.Patients with concurrent TP53 mutation had shorter overall survival than those without(P<0.05).Patients with lower peripheral blood TCR diversity had superior overall survival compared with those with higher diversity(P=0.035).An association with overall survival was not observed for PD-L1 expression nor for tumor mutation burden level.Median progression-free survival(PFS)was not significantly different across chemotherapy,ICIs and multi-kinase inhibitors.Overall survival was prolonged in patients treated with selective RET inhibitors when compared with untreated(HR:0.32,95%confidence interval[CI]0.16-0.66,p=0.001),but with no difference in those treated with immunotherapy(p=0.571)or multikinase RET TKIs(MKIs).A total of 21 patients were enrolled for ctDNA analysis.Concomitant PIK3CA activating mutations at baseline were correlated with less favorable PFS(P=0.016).Superior PFS was associated with lower baseline levels of multiple ctDNA metrics,including allele frequency-based(HR=0.24;95%CI 0.07-0.80,P=0.012),cfDNA quantity-normalized(HR=0.20;95%CI 0.05-0.71,P=0.006)and methylation-based(HR=0.09;95%CI 0.01-0.85,P=0.01)ones.ctDNA clearance at the first radiological examination was similarly correlated with favorable PFS and disease control.Additionally,three ctDNA kinetic profiles(rebound after clearance,rebound after reduction,sustained clearance)preceded by distinct progression patterns.Molecular progression was observed before radiological manifestation by a mean interval of 2.2 months.Putative genetic mechanism of resistance included RET G810C and KRAS variants.Differences in transcriptional regulation were observed in tumor cells without possible genetic mechanism of resistance,including significant activation of multiple signaling pathways related to tumor survival and up regulation of genes related to proliferation.The interaction between tumor cells and various immune cells in TME was observed in the samples before imitation and after resistance of selective RET inhibitors,and the interaction with macrophages was the strongest.After drug resistance,the infiltration and activation of antitumor macrophages and T cell subsets were negatively regulated.ConclusionsRET-rearranged lung cancers can be heterogeneous in terms of concomitant genetic alterations.Patients with concurrent TP53 mutation or high peripheral blood TCR repertoire diversity have relatively inferior overall survival in this series.Outcomes with traditional systemic therapies in general are suboptimal,while selective RET inhibitors were associated with improved overall survival.ctDNAbased surveillance enables early forecasting and monitoring of tumor response/progression for RET fusion-positive NSCLC patients receiving pralsetinib.Novel ctDNA metrics based on cfDNA quantity-normalization and DNA methylation are consistently associated with clinical outcomes and therefore hold promising as a generalizable biomarker in advanced driver-positive NSCLC.It is necessary to pay attention to the role of genomic alteration,transcriptional regulation and tumor microenvironment in drug resistance.