The Predictive Value Of FXR For Chemo-immunotherapy In PD-L1^low/negative Non-small Cell Lung Cancer Patients

Background:Lung cancer is one of the most frequently diagnosed cancers and the leading cause of cancer-related deaths worldwide.Non-small cell lung cancer(NSCLC)accounts for～85%of the diagnosed lung cancers.Despite recent advances in surgery,radiotherapy,chemotherapy,targeted therapy,etc,the prognosis of NSCLC remains dismal and the 5-year survival rate is lower than 20%.Over the past decade,lung cancer immunotherapy has achieved significant breakthroughs.Immune checkpoint blockade(ICB)therapy,represented by monoclonal antibodies targeting the programmed cell death protein 1(PD-1)and its ligand PD-L1,have produced remarkable clinical responses and revolutionized the landscape of lung cancer treatment.Nivolumab and pembrolizumab have been approved by FDA as first-or second-line treatments for advanced NSCLC.However,due to the primary or acquired resistance and adverse effects,only a small fraction of NSCLC patients can benefit from immune-related therapies.Tumor PD-L1 expression detected by immunohistochemistry(IHC)is the first approved biomarker for predicting response to anti-PD-1/PD-Ll immunotherapy.Several clinical trials have demonstrated superior overall survival for PD-1/PD-L1 blockade in NSCLC patients with high PD-L1 expression,compared to those with low PD-L1 expression.Alternative predictive biomarkers,such as tumor mutational burden and tumor microenvironment(TME),have also been intensively investigated;however,conclusive evidence is lacking.It is noteworthy that the predictive value of PD-L1 expression was affected by multiple variables,including the different testing platforms and cut-off criteria for positivity,intra-tumoral and inter-tumoral heterogeneity,and the dynamic change of PD-L1 expression.In fact,clinical efficacy was also seen in cancer patients among the PD-L1low/negative group,suggesting that tumor PD-L1 expression alone is insufficient to recognize patients sensitive to PD-1/PD-L1 blockade.Future studies may help develop new predictors,especially in identifying potential responding candidates to anti-PD-1/PD-L1 among the PD-L1low/negative patients.Farnesoid X receptor(FXR)is a member of the nuclear receptor superfamily that is predominantly expressed in the liver and gastrointestinal tract.As a bile acid(BA)-activated transcription factor,FXR regulates expression of target genes involved in BA homeostasis,lipid and glucose metabolism.Recent studies have depicted the important role of FXR,either as an oncogene or as a tumor-suppressive gene,in the tumorigenesis of liver,colorectal,esophageal and breast cancer,etc.It was previously reported that FXR is upregulated in NSCLC,compared with pericarcinous lung tissues,and that FXR contributes to NSCLC cell proliferation via transactivating CCND1.Another study discovered an inhibitory role of FXR in PD-L1 expression in NSCLC.Critically,FXRhighPD-L1low mouse Lewis lung carcinoma(LLC)tumors were more vulnerable to anti-PD-1 therapy than mock LLC tumors.However,whether or not FXRhighPD-Lllow phenotype predicts clinical response to immune-related therapies in clinical NSCLC patients has not been investigated yet.Objectives:This study aimed to determine the predictive value of FXR for anti-PD-1-based chemo-immunotherapy in the setting of clinical NSCLC,mainly focused on the PD-L1low/negative group.In addition,the potential correlation between FXR expression and tumor-infiltrating CD8+T cells was also disclosed.Materials and Methods:Clinical data collection A total of 149 patients treated with anti-PD-1-based chemo-immunotherapy at Shandong Provincial Hospital were eventually enrolled in this study.Clinical and pathological information,including age,gender,smoking history,histologic type,TNM stage,ECOG performance status(PS),therapy lines,etc,were retrospectively obtained from the medical records.Tumor assessment was performed at baseline and every 2 cycles according to the Response Evaluation Criteria in Solid Tumors version 1.1(RECIST 1.1).On the basis of the best overall response,patients with complete or partial response were considered responders,while others with stable or progressive disease were considered non-responders.The progression-free survival(PFS)and overall survival(OS)were obtained through medical records or telephone follow-up.IHC staining and assessment All of the cases had available formalin-fixed paraffin-embedded(FFPE)specimens of primary tumors.Four-μm thick sections from each FFPE block were used for FXR,PD-L1 and CD8 IHC staining.For FXR and PD-L1,the IHC score was generated by multiplying the staining intensity and percentage,and the results were defined as low or high.For CD8,4-6 independent high-power fields(HPFs,200×)which represented the densest lymphocytic infiltrates were selected to reflect the extent of CD8+T cell infiltration.The average CD8+T cell density(cells/HPF)was calculated as the mean value of the 4-6 areas and defined low or high groups according to the median value.Statistical analysis Shapiro-Wilk method was used to test the normality of quantitative data.Comparisons between skewed distribution data were performed using Mann-Whitney U test.Categorical variables were compared using chi-square tests or Fisher’s exact test.Survival curves were estimated by Kaplan-Meier analysis,and the log-rank test was utilized to examine the differences between groups.Cox regression model was used to investigate the effects of FXR and PD-L1 expression and clinicopathological factors on survival of NSCLC patients.Statistical analyses were performed using the statistical software IBM SPSS Statistics 26.0 and GraphPad Prism 8.0.P<0.05 was considered statistically significant.Results:Baseline characteristics of patients The cohort included 1 19 men and 30 women with a higher proportion of the elderly,and most of them were smokers.Seventy-eight patients(52.3%)and 54 patients(36.2%)were classified as high FXR and PD-L1 expression,respectively.The majority of these NSCLC cases were non-squamous cell carcinoma(93/149,62.4%),of which 90 were adenocarcinoma,2 were sarcomatoid carcinoma and 1 was large cell neuroendocrine carcinoma.More than half of the patients(103/149,69.1%)were in stage IV at the beginning of anti-PD-1-based chemo-immunotherapy.In our cohort,all the patients received PD-1 inhibitors combined with chemotherapy as the first-line or higher lines with refractory progression after chemotherapy,radiation therapy or targeted therapy.Among them,the most widely used PD-1 inhibitor was Camrelizumab.Efficacy and survival analysis High FXR and PD-L1 expression levels correlated with higher objective response rates(ORR)in all patients(P=0.036 and 0.002,respectively).PD-L]high-expression patients were found to have a significant longer PFS(P=0.031).Interestingly,we found an inverse correlation between FXR and PD-L1 expression in NSCLC specimens.Subgroup analysis showed that high FXR expression was associated with a higher ORR(P=0.009),as well as longer PFS(P=0.013)and overall survival(OS)(P=0.03)in PD-L1low patients.Cox multivariate analysis revealed that high FXR expression was an independent predictor for PFS(P=0.038;HR,0.552;95%CI,0.315-0.967)and OS(P=0.029;HR,0.377;95%CI,0.157-0.905)in PD-L1low patients.Tumor microenvironment evaluation We sought to explain the predictive value of FXR on anti-PD-1-based chemo-immunotherapy in the perspective of TME.Because CD8+T cells represent the most crucial tumoricidal effector cells and the main target of PD-1/PD-L1 checkpoint pathway in TME,we examined the infiltration of CD8+T cells in NSCLC specimens.IHC evaluation revealed a statistically significant decrease of infiltrating CD8+T cells in FXRhigh NSCLC specimens.Conclusion:High FXR and PD-L1 expression levels correlated with higher ORR in all the patients treated with anti-PD-1-based chemo-immunotherapy.In addition,PD-L1 high-expression patients were found to have a significant longer PFS.There was an inverse correlation between FXR and PD-L1 expression in NSCLC specimens.High FXR expression was associated with a higher ORR,as well as longer PFS and OS in PD-L1low patients.Cox multivariate analysis revealed that high FXR expression was an independent predictor for PFS and OS in PD-L1low patients.There was a significantly inverse correlation between FXR and CD8 expression in NSCLC specimens,suggesting that the restrained tumor-infiltrating CD8+T cells,rather than the fully activated ones,are more readily to be rescued by anti-PD-1 in FXR high-expression tumors.The present study recommended the FXRhighPD-L1low signature as a promising predictor of response to anti-PD-1-based chemo-immunotherapy in PD-L1low/negative NSCLC,providing clinical evidence for the development of complementary biomarkers for immune-related therapies.