Based on the analysis of mutation data and clinical data of the ICI-treated Miao-LUAD cohort, this study found that mutation of the BCR signaling pathway was a potential predictor of ICIs efficacy. Compared with WT group, BCR signaling MUT group had better PFS and OS. By analyzing the transcriptome data of the TCGA-LUAD cohort, we found that BCR signaling MUT group had higher immunogenicity and more CD4 + and CD8 + T cell infiltration degree, as well as higher immune-related signature scores. The results of the pathway enrichment analysis also showed that the signaling pathways of BCR signaling MUT group to promote the proliferation and metastasis of tumor cells was significantly down-regulated, and conversely the signaling pathways for inhibiting the proliferation and metastasis of tumor cells was significantly up-regulated. These conclusions support our initial hypothesis, that is, the mutation state of the BCR signaling pathway can be used as a potential biomarker to predict the efficacy of ICIs therapy for LUAD patients.
TME is a complex system mainly composed of tumor cells, surrounding immune and inflammatory cells, tumor-related fibroblasts, nearby interstitial tissues, capillaries, and various cytokines and chemokines.(Dougan and Dougan 2017) Before ICIs baseline treatment, TILs infiltrated in the TME; in particular, CD8 + T cells were reported to be positively correlated with ICIs treatment efficacy,(Jacquelot et al. 2017; Zhang et al. 2021; Huang et al. 2021) and CD8 + T cells were considered to be specific anti-tumor T cells. They can produce IFN-γ, TNF and granzyme B through the specific combination of T cell receptors with tumor cells, thus killing tumor cells.(Reiser and Banerjee 2016) The main factors that limit the function of CD8 + T cells are whether T cells can differentiate into sufficient CD8 + T cells, and whether CD8 + T cells can infiltrate into a TME smoothly.(Nolz 2015) CD4 + T cells play an important role in helping CD8 + T cells to enhance anti-tumor immunity.(Tran et al. 2014; Cui et al. 2021) Once activated by tumor-associated antigen, CD4 + T cells can promote the maturation of dentritic cells, present more antigens, and promote the differentiation of naive CD8 + cells into effector CD8 + T cells, thus increasing the number of CD8 + T cells. CD4 + T cells can also simultaneously act as cytotoxic cell by secreting IFN-γ and TNF or express receptors of TNF family factors such as FASL and TRAIL, mediating a direct cytotoxic immune response. MHC class II restricted antigen recognition of CD4 + T cells, as distinct from MHC class I restricted antigen recognition of CD8 + T cells, is an important supplementary part of antigen recognition in anti-tumor immunity. Interestingly, in the absence of CD4 + T cells, the ability of CD8 + T cells to kill tumor cells is also limited. According to a recent study, the number of memory CD4 + T cell subsets in peripheral blood before treatment is a strong predictor of the efficacy of immunotherapy for NSCLC. To sum up, the synergy between CD4 + T cells and CD8 + T cells is the key to anti-tumor immunity. In this study, we analyzed the infiltration degree of 22 kinds of immune cells in the TCGA-LUAD cohort via a CIBERSORT method. There are more CD8 + T cells and activated memory CD4 + T cells infiltrating in BCR signaling MUT group. On the contrary, the degree of Tregs, which can directly inhibit CD8 + T cells infiltration in LUAD,(Ap et al. 2013) infiltration in BCR signaling MUT group is significantly lower than that in WT group. The role of mast cells in tumor immunity has been controversial. According to epidemiological surveys, the existence of mast cells in TME is negatively correlated with the progress of lung cancer.(Sinnamon et al. 2008; Hodges et al. 2012) Mast cell-derived TNF can kill tumor cells;(Liu et al. 2011) however, the humanized-mouse melanoma model proves that tumor-infiltrated mast cells are related to the tolerance of anti-PD-1 antibody treatment, which is a negative factor for immunotherapy efficacy.(R et al. 2021) In our results, there are significant differences in the degree of mast cell infiltration between the BCR signaling MUT group and WT group, but both only for a small number. Further research is required to determine the role it plays. In general, the anti-tumor immune microenvironment clearly activated in BCR signaling MUT group can explain why this group has significantly prolonged PFS and OS.
Immunogenicity of tumors is considered to be a prerequisite for exerting anti-tumor immunity.(Wang et al. 2019) Based on the analysis of RNA expression data and mutation data, and after comparing TMB, NAL and DDR-related pathways mutations of BCR signaling MUT group and WT group, which are representative markers for evaluating immunogenicity, it was found that BCR signaling MUT group had higher immunogenicity. In addition, correspondingly, the results of immune-related characteristic scores show that the BCR signaling MUT group has a higher IFN-γ score. IFN-γ secreted by TILs activates cellular immunity and induces cell cycle arrest and death of cancer cells.(Schoenborn and Wilson 2007) Furthermore, the role of macrophages in anti-tumor immunity varies according to their phenotype, while IFN-γ can induce the differentiation of type I macrophages, and make it secrete IL-12 to induce an anti-tumor immune response.(Ostrand-Rosenberg et al. 2012) Studies have shown that IFN-γ secreted by T cells, stimulated by tumor antigens, can spread widely, and play an important role in clearing metastatic tumor cells.(Hoekstra et al. 2020) A recent study has proven that IFN-γ activates the immune response by activating the tumor intrinsic STING pathway in NSCLC, which in turn is related to the increase of DNA damage,(Della Corte et al. 2020; Xiong et al. 2022, p.) consistent with our previous results that BCR signaling MUT group had higher DDR-related pathways mutations.
Chemokines and cytokines secreted by immune cells in TME, such as IL-12, IFN-γ, CXCL-9, and CXCL-10, are important components of LUAD anti-tumor immunity.(Lin et al. 2021) However, In our ssGSEA results, cytokines and chemokine produce pathways related to immunosuppression, such as IL-10, IL-1β, CCL2 and CXCL2, were significantly down-regulated in BCR signaling MUT group, functions of these factors which are secreted by cancer cells and cancer-related immune cells are completely opposite with those mentioned at the beginning.(Paval et al. 2022) These factors recruit tumor-associated macrophages (TAMs), Tregs and Myeloid-derived suppressor cells (MDSCs) into the TME, helping tumor cells escape detection by the immune system and promoting the development and metastasis of tumors.(Shimizu et al. 2010; Srivastava et al. 2012) There is evidence that IL-10 secreted by cancer cells activated type II macrophages, the latter promoting tumor growth by inhibiting T cells. Tumor cells can also recruit a large number of Tregs and MDSCs for themselves by secreting IL-10 and TGF-β and up-regulating the expression of CXCR2 ligand.(Shimizu et al. 2010; Zaynagetdinov et al. 2012) Both Tregs and MDSCs are notorious immunosuppressive cells, which jointly inhibit the infiltration and function of CD8 + T cells and mediate the adaptive immune tolerance of tumors. Anti-tumor immunity can be further weakened through the up-regulation of IL-10, which can promote the differentiation of native CD4 T cells into Tregs.(Zdanov et al. 2016) IL-1β is a pro-inflammatory factor, and in the NSCLC mouse model, IL-1β derived from mast cells and tumor-associated neutrophils has been proven to be beneficial to tumor growth.(McLoed et al. 2016; Lilis et al. 2019) The effects of IL-1β are significantly related to a poor prognosis for NSCLC.(Kim et al. 2013) At the same time, a drug clinical trial using IL-1β inhibitor showed that, after the secretion of IL-1β is inhibited, the mortality rate related to NSCLC decreased significantly.(Ridker et al. 2017a, b) In addition, in the study of the mouse model of pleural cancer with lung cancer metastasis, researchers found that the vicious circle of IL-1β and CCL2 working together explained the occurrence and development of malignant pleural effusion in NSCLC.(Giannou et al. 2015; Agalioti et al. 2017; Marazioti et al. 2018)
In addition, in our GSEA results, some classic signaling pathways that promote tumor occurrence and development were significantly down-regulated in BCR signaling MUT group,(Akiri et al. 2009; Ivy et al. 2009; Vanhaesebroeck et al. 2010; Xi and Chen 2014) such as signaling by WNT in cancer and constitutive signaling by aberrant PI3K in cancer. It is worth noting that the pathways related to tumor angiogenesis, which is the basis for the survival and metastasis of tumor cells, were also significantly down-regulated in BCR signaling MUT group.(Chinchilla et al. 2010; Desai and Adjei 2016; Hu et al. 2022) Tumor angiogenesis in solid tumors can also prevent tumor-specific T cells from infiltrating TME.(Bellone and Calcinotto 2013) Finally, the analysis revealed that the signal pathways related to tumor inhibition were significantly up-regulated in MUT group, such as positive regulation of pathway restricted SMAD protein phosphorylation;(Markowitz and Roberts 1996) activated SMAD protein inhibited the activation of TGF-β signal pathway, which was reported to be related to the enhancement of tumor invasiveness and metastasis in advanced NSCLC. Correspondingly, in ssGSEA results, BCR signaling MUT group had lower scores of TGF-β signaling pathway. FoxO signaling pathway not only inhibits the abnormal expression of Fox family proteins, but also, and consequently, inhibits the role of these proteins in promoting tumor growth.(Katoh et al. 2013) In addition, we also found that the MUT group had lower scores in ssGSEA, a signaling pathway related to fatty acid metabolism, which was related to immune failure and tumor metastasis.(Zhao et al. 2019) To sum up, the enrichment results show that the MUT group has a pathway expression environment that is not conducive to tumor growth and metastasis. These results may indicate the weakness in the viability of tumor cells in LUAD patients in BCR signaling MUT group, compared with WT group.
Generally speaking, our study comprehensively analyzed the relationship between the mutation state of BCR signaling pathway and the prognosis of LUAD patients treated with ICI, from the perspectives of survival analysis, gene mutation, immune infiltration and pathway enrichment, and deduced the related mechanism; however this study still has several limitations. First, because the survival data of the LUAD cohort treated with ICI is very limited, the relationship between the BCR signaling pathway and ICI treatment survival was only able to be explored in the Miao-LUAD cohort. Secondly, we did not carry out related cell experiments and animal experiments to further verify our results; this is an area of research that we hope to supplement in the future. Thirdly, because of the quantity and complexity of mutation sites in the BCR signaling pathway, we did not explore the specific molecular mechanism between mutation and ICI efficacy, but we compared the TME differences between the wild group and the mutant group, and formed a hypothesis for the mechanism, using immune infiltration analysis and pathway enrichment analysis combined with previous literature reports. Finally, we hope that our findings can provide assistance for future cancer mechanism investigation.