Exploration Of The Efficacy And New Treatment Strategies Of Radiotherapy Combined With Immunotherapy For Non-small Cell Lung Cance

Part 1.Efficacy and safety of radiotherapy combined with immunotherapy for metastatic non-small cell lung cancerBackgroundIn recent years,the application of immunotherapy has greatly improved the prognosis of patients with advanced non-small cell lung cancer(NSCLC).However,most patients will develop resistance to immunotherapy sooner or later after receiving immunotherapy,and the options for later-line treatment are limited and the efficacy is poor.How to discover a better treatment strategy is of great clinical significance,among which radiotherapy combined with immunotherapy shows certain application prospects,but the specific combination strategy,benefits and safety of radiotherapy combined with immunotherapy in patients with metastatic non-small cell lung cancer remain inconclusive.MethodsThis study was a retrospective clinical study,including patients with metastatic NSCLC who received immune checkpoint inhibitors(ICI)combined with or without chemotherapy in Peking Union Medical College Hospital from January 2015 to November 2022.Patients were divided into with-radiotherapy group and noradiotherapy group.The efficacy of immunotherapy,survival and incidence of immunerelated adverse events(irAEs)between two groups were analyzed.ResultsA total of 352 patients with metastatic NSCLC were included in this study,including 265 patients in the no-radiotherapy group and 87 patients in the with-radiotherapy group.There was no significant difference in baseline clinical characteristics between the two groups(p>0.05).The objective response rate(ORR)of the no-radiotherapy group and the with-radiotherapy group were 40.0%and 43.7%respectively(p=0.544).The disease control rate(DCR)was 88.3%and 87.4%respectively(p=0.849).In terms of survival analysis,the median progression-free survival(PFS)and median overall survival(OS)of patients in the with-radiotherapy group showed a trend of prolongation,but the difference was not statistically significant(median PFS 10.13 months vs 15.97 months,p=0.95;median OS 27.97 months vs 40.33 months,p=0.22).As for safety,there was no significant difference in the incidence of any-grade irAEs and grade>=3 irAEs between the two groups,but the incidence of immunotherapy-associated pneumonia was higher in the with-radiotherapy group(5.7%vs 13.8%,p=0.013).ConclusionIn patients with metastatic non-small cell lung cancer,the safety of immunotherapy combined with radiotherapy(with or without chemotherapy)is acceptable,although the incidence of ICI-associated pneumonia is higher.There was no statistically significant difference in survival between the with-radiotherapy and no-radiotherapy groups.Part 2.The expression and prognostic value of adenosine pathwayrelated biomarkers in non-small cell lung cancerBackgroundAdenosine is a metabolite produced abundantly in the tumor microenvironment,dampening immune response in inflamed tissues via adenosine A2A receptor(A2AR)which is widely expressed on immune cells,inhibiting anti-tumor immune response accordingly.Therefore,blocking adenosine signaling pathway is of potential to promote anti-tumor immunity.The production of adenosine is mainly mediated by exonucleosidase CD39 and CD73,among which CD73 is the key rate-limiting enzyme that catalyzes the production of adenosine.This study aims to evaluate the expression of adenosine pathway-related markers such as CD39,CD73,and A2AR in non-small cell lung cancer(NSCLC)and analyze the prognostic value of these markers.MethodsThe pathological tissue samples of NSCLC patients who underwent surgical treatment in Peking Union Medical College Hospital from January 2009 to December 2012 were collected.Tissue microarrays were established and the clinical and prognostic data of these patients were collected.The expression of adenosine pathway markers and related cytokines in NSCLC tissues was evaluated by immunohistochemistry and quantified by H score,and the high expression group and low expression group were divided according to the median of H score.The distribution and proportion of macrophages M1 and M2 in NSCLC tissues were detected by immunofluorescence.Gene mutations in each tissue sample were detected by high-throughput sequencing.The prognostic differences between CD73 and A2AR high expression group and low expression group were compared and further verified by The Cancer Genome Atlas(TCGA)database.For the groups with different prognosis,the difference of the mutated genes was analyzed and gene function enrichment analysis of these genes was carried out.ResultsA total of 158 NSCLC patients were included in this study,including 44 cases of stage Ⅰlung squamous cell carcinoma,46 cases of stage Ⅲa lung squamous cell carcinoma,32 cases of stage Ⅰ lung adenocarcinoma,and 36 cases of stage Ⅲa lung adenocarcinoma.The study found that the expression level of CD73 on tumor cells was higher in patients with adenocarcinoma,while the expression level of CD73 on tumor-infiltrating immune cells was higher in patients with squamous cell carcinoma.The expression level of A2AR was higher in males,patients with smoking history and lung squamous cell carcinoma.The expression level of CD73 on NSCLC tumor cells was positively correlated with CD47(p<0.001),and negatively correlated with the expression level of interleukin 2(IL-2)(p<0.05).The expression level of A2AR on NSCLC tumor cells was positively correlated with cytotoxic T lymphocyte-associated protein 4(CTLA-4)on tumor cells,A2AR and IL-2 on immune cells.No correlation was found between CD73 or A2AR expression levels and macrophage infiltration or M2/M1 ratio.In the prognostic analysis,high expression of CD73 in tumor cells was an independent risk factor(hazard ratio=2.12,95%confidence interval 1.16-3.89,p=0.01).The mutation frequency of KRAS and EGFR was higher in the CD73 high expression group,while the mutation frequency of NFE2L2 and EP300 was lower.Gene function analysis found that these genes were significantly correlated with the forkhead box O(FoxO)signaling pathway.ConclusionThere is a certain correlation between the adenosine pathway-related markers CD73/A2AR and the expression levels of other immune checkpoints such as CD47 and CTLA-4 in the NSCLC tumor microenvironment.The high expression of CD73 on tumor cells is an independent risk factor for the prognosis of patients.The gene mutation frequencies of KRAS,EGFR,NFE2L2 and EP300 were significantly different between the CD73 high expression group and the low expression group,and there was a significant correlation between these genes and the FoxO signaling pathway.Part 3.The anti-tumor efficacy and cellular mechanism exploration of radiotherapy combined with adenosine pathway blockade in NSCLCBackgroundAfter radiotherapy,the level of extracellular adenosine in the tumor microenvironment is significantly increased.Adenosine/adenosine A2A receptor(adenosine A2A receptor,A2AR)pathway blockade has become an important research direction of immune regulation after radiotherapy.This study aims to explore the anti-tumor efficacy of radiotherapy combined with A2AR blockade in non-small cell lung cancer(NSCLC)and to explore the possible cellular mechanisms involved.Methods1.The CRISPR-Cas9 system was used to construct A2AR gene knockout(A2AR-KO)mice,establish a mouse lung cancer subcutaneous tumor model,and compare the difference in tumor growth between A2AR-KO mice and wild-type mice after radiotherapy.2.The difference in the proportion of M2 in macrophages in tumor tissue was detected by flow cytometry between A2AR-KO and wild-type mice after radiotherapy.3.In the in vitro experiment,the macrophage M0 was induced and differentiated by the THP-1 cell line,and the lung cancer H1299 cell and macrophage M0 co-culture system was constructed.Differences in antitumor efficacy was detected by the the CCK-8 experiment between the radiotherapy group and the radiotherapy-A2AR inhibitor combination group.Results1.Compared with the control group,the tumor growth of mice in A2 AR-KO group was significantly inhibited after radiotherapy.2.The proportion of M2 macrophages in tumor tissues of mice in A2AR-KO group significantly decreased.3.In the in vitro cell co-culture system,compared with the radiotherapy group,there was no significant difference in the inhibitory effect of the A2AR inhibitor group after radiotherapy on tumor cells.4.The combined A2AR inhibitor group after radiotherapy had no significant effect on the migration ability of macrophages.Conclusion Radiotherapy combined with A2AR pathway blockade can further improve the antitumor efficacy in NSCLC.Macrophages do not play a leading role in enhancing the anti-tumor efficacy after A2AR pathway blockade,and may depend on the participation of other immune cells.