Abstract

Purpose

   The advent of immune checkpoint inhibitors (ICIs) is a revolutionary
   breakthrough. However, without the selection of a specific target
   population, the response rate of ICI therapy in lung adenocarcinoma
   (LUAD) is low, so a clinical challenge has arisen in effectively using
   biomarkers to determine which patients can benefit from ICI therapy.

Methods

   In this study, patients were divided according to whether or not
   nonsynonymous mutations were present in the BCR signaling pathway, and
   univariate and multivariate Cox regression models were established
   based on a LUAD cohort treated with ICIs (Miao-LUAD). Then the
   relationship between the mutation status of the BCR signaling pathway
   and the prognosis of immunotherapy was examined. Finally, data from The
   Cancer Genome Atlas (TCGA) LUAD cohort, the Rizvi-LUAD, the
   Samstein-LUAD, and the Zhujiang Hospital of Southern Medical University
   LUAD (Local-LUAD) cohort were combined, and the mutation panorama,
   immunogenicity, tumor microenvironment (TME) and pathway enrichment
   analysis between the BCR signaling pathway mutant group (BCR signaling
   MUT) and the BCR signaling pathway wild group (BCR signaling WT) were
   comprehensively compared.

Results

   It was found that, compared with the BCR signaling WT, the BCR
   signaling MUT had a significantly improved progression-free survival
   (PFS) rate and overall survival (OS) rate, higher immunogenicity (tumor
   mutational burden, neoantigen load, and DNA damage response signaling
   mutations), and anti-tumor immune microenvironment.

Conclusion

   These results revealed that the mutation state of the BCR signaling
   pathway has potential as a biomarker to predict the efficacy of ICIs in
   LUAD.

   Keywords: non-small cell lung cancer, B cell receptor, immune
   checkpoint inhibitor, biomarker, microenvironment

Introduction

   Among all cancer-related deaths, those caused by lung cancer rank first
   both globally and in China, posing a significant threat to human
   health.[36]^1^,[37]^2 Lung adenocarcinoma (LUAD) is the main
   histological type in NSCLC, accounting for 40% of total lung cancer
   instances. Although surgery is the first choice of treatment for
   patients with early-stage LUAD, more than 70% of patients have reached
   advanced stages by the time they see a doctor. The 5-year survival rate
   of patients with advanced LUAD under traditional chemotherapy and
   radiotherapy is less than 5%.[38]^3 The advent of immune checkpoint
   inhibition (ICI) therapy has brought about revolutionary changes in the
   treatment of solid tumors including lung cancer. The ICIs target the
   PD-1 or the PD-L1 axis, thus restoring the anti-tumor activity of T
   cells.[39]^4 In the treatment of NSCLC, the use of ICIs alone or
   together with chemotherapy has demonstrated remarkable curative
   effects, and it has already become a standard treatment program for
   patients with advanced NSCLC. According to the results of a Phase III
   clinical trial,[40]^5 the 5-year survival rate of NSCLC patients who
   have previously received platinum therapy is increased by more than 5
   times (13.4% vs 2.6%) after PD-1 inhibitor treatment, compared with the
   docetaxel treatment group. However, due to factors including the
   exhaustion of T cells, the defect of antigen presentation, the
   influence of other immune checkpoint molecules and damage to the IFN-γ
   signaling pathway, the effective rate of ICI therapy rarely exceeds
   40%.[41]^6–8 Therefore, it is important to apply clinical biomarkers to
   screen potential beneficiaries of immunotherapy.

   At present, many studies have revealed potential biomarkers for
   predicting the efficacy of ICI therapy such as PD-L1 expression and
   nonsynonymous tumor mutation burden (TMB).[42]^9 However, some studies
   have found that NSCLC patients can benefit from nivolumab immune
   checkpoint therapy regardless of the expression level of PD-L1.
   Furthermore, the expression of PD-L1 is a dynamic process. It can be
   induced by IFN-γ secreted by tumor infiltrating lymphocytes (TILs),
   which can be affected by the process of treatment.[43]^10 At present,
   TMB, which is documented as being positively correlated with the OS of
   ICI therapy, has uneven detection quality, and there is no unified
   cutoff standard.[44]^11 Some studies have even shown that high TMB does
   not suggest a positive ICIs curative effect.[45]^12 Neither PD-L1 nor
   TMB alone is sufficient as a predictor of the efficacy of ICIs.
   Therefore, more practical biomarkers are needed to screen the
   population of LUAD patients under ICI therapy.

   The B cell receptor (BCR) is a membrane immunoglobulin that transmits
   signals downstream by binding extracellular antigens and ligands, thus
   regulating the proliferation, activation, differentiation, cell
   selection and apoptosis of B cells.[46]^13 B cells infiltrated in a
   tumor microenvironment are thought to have a dual function in tumor
   promotion and anti-tumor immunity.[47]^14 B cells activated by the BCR
   signaling pathway can act as antigen-presenting cells, presenting
   antigens to the cell surface, and then activating tumor-specific CD8+ T
   cells, thus enhancing anti-tumor immunity.[48]^15 It is also reported
   that plasma cells differentiated from a B cell can infiltrate tumors
   and play an anti-tumor role by producing the tumor-specific antibody
   IgG1.[49]^16 However, an overactivated BCR signaling pathway is also
   considered an important contributing factor in the occurrence and
   development of B-cell-derived malignant tumors such as chronic
   lymphocytic leukemia and diffuse large B-cell lymphoma.[50]^17^,[51]^18
   Clinically, there are many inhibitors of BCR and related pathways, such
   as Bruton’s tyrosine kinas inhibition.[52]^19 However, at present, the
   relationship between the BCR signaling pathway and LUAD is not clear,
   and more research is needed on the relationship between the mutation of
   this pathway and the efficacy of ICIs. Therefore, we hope to explore
   the relationship between the mutation state of the BCR signaling
   pathway and the efficacy of ICIs through analysis of clinical, genome
   and transcriptome information.

   In this study, a TCGA-LUAD data set, Samstein-LUAD, Rizvi-LUAD and
   Miao-LUAD ICIs-treated data sets, as well as Local-LUAD data sets were
   analyzed; the prognosis of ICI treatment was compared after grouping
   according to the nonsynonymous mutation status of the BCR signaling
   pathway; the relationship between BCR signaling pathway mutation and
   ICI treatment efficacy from the gene level to the tumor
   microenvironment level was comprehensively analyzed using the
   bioinformatics method; and hypotheses were formed about the related
   mechanism.

Materials and Methods

LUAD Sample Collection

   Three data sets of LUAD patients treated with ICIs were collected. The
   Rizvi-LUAD group included 186 advanced LUAD patients treated with
   anti–PD-(L)1 monotherapy alone or in combination with anti–cytotoxic
   T-cell lymphocyte-4.[53]^20 The Miao-LUAD group included 47 advanced
   LUAD patients treated with anti-PD-1, anti-PD-L1, anti-CTLA-4, or a
   combination of these therapies.[54]^21 The Samstein-LUAD included 266
   LUAD patients who received at least one dose of immunotherapy
   (atezolizumab, avelumab, durvalumab, ipilimumab, nivolumab,
   pembrolizumab, or tremelimumab).[55]^22 Both ICIs-treated cohorts
   included mutation data from before they had received ICI treatment and
   prognosis data. In addition, the LUAD dataset was downloaded from the
   TCGA database using the TCGA biolinks R package,[56]^23 which included
   mutation data, expression data and clinical data of 511 patients.

   70 LUAD samples were collected from Zhujiang Hospital of Southern
   Medical University, and targeted sequencing was used (HapOncoTM680
   Panel) to obtain mutation data. (see [57]Supplementary Table 1 for
   Panel on targeted sequencing). The written informed consent of all
   participants was obtained, and this study was approved by the Zhujiang
   Hospital Research Ethics Committee of Southern Medical University.

Status Analysis of BCR Receptor Signaling Pathway

   The B cell receptor signaling pathway gene set
   (KEGG_B_CELL_RECEPTOR_SIGNALING_PATHWAY) comes from the MsigDB database
   ([58]Supplementary Table 2).[59]^24 The somatic mutation data of
   patients was processed (deleting synonymous mutation data and retaining
   nonsynonymous mutation data, and the nonsynonymous mutation type
   include “Frame_Shift_Del”, “Frame_Shift_Ins”, “Splice_Site”,
   “Translation_Start_Site”,“Nonsense_Mutation”, “Nonstop_Mutation”,
   “In_Frame_Del”,“In_Frame_Ins”, “Missense_Mutation”).[60]^25 Then, the
   number of gene mutations in the B cell receptor signaling pathway was
   counted in every LUAD patient. If the number of gene mutations in the B
   cell receptor signaling pathway in LUAD patients is zero, then the
   patient is a B cell receptor signaling pathway wild-type (WT).
   Otherwise, the patient is a B cell receptor signaling pathway
   mutant-type (MUT). The baseline clinical information on Miao-LUAD,
   TCGA-LUAD is shown in the [61]Supplementary Tables 3 and [62]4.

Immunogenicity, Immune Infiltration and Pathway Enrichment Analysis

   Drawing on previously published research results, Immunogenicity data
   on tumor TMB and neoantigen loads (NAL) was included, NAL of the
   TCGA-LUAD cohort was directly obtained from a published
   literature.[63]^26 TMB score of Samstein-LUAD cohort and Rizvi-LUAD
   cohort was directly obtained from the public datesets, and TMB score of
   Local-LUAD cohort and Miao-LUAD cohort was quantified by dividing the
   number of somatic mutations by 38 Mb. In addition, we collected the
   gene sets of DNA damage repair-related pathways from MSigDB,[64]^27 and
   counted the number of nonsynonymous mutations in each patient’s DNA
   damage repair (DDR)-related pathways. The immune cell analysis and the
   immune related pathway score analysis were the main types of immune
   infiltration analyses used. In the analysis of immune cells, we applied
   a CIBERSORT algorithm[65]^28 to estimate the relative abundance of 22
   types of immune cells in each patient’s TME. Immune-related scores come
   from a previously published study.[66]^26 The pathway enrichment
   analysis included a gene set enrichment analysis (GSEA), and a single
   sample gene set enrichment analysis (ssGSEA).[67]^29^,[68]^30

Statistical Test

   The Mann–Whitney U-test and Fisher’s exact test were used to evaluate
   the statistical differences between the two groups of categorical
   variables. The univariate and multivariate Cox risk proportional
   regression models and the Kaplan-Meier analysis were used to explore
   the prognosis of the B cell receptor signaling pathway for patients
   with LUAD who received ICI treatment. All data analysis and data
   visualization in this study are based on R software (Version 3.6). The
   p value is bilateral and p<0.05 has statistical difference.

Results

LUAD Patients with BCR Signaling Pathway Mutation Have Better PFS and OS
After ICI Treatment

   [69]Figure 1 is a brief flowchart of this study. The Miao-LUAD cohort
   was used as our discovery cohort, and the Samstein-LUAD and Riziv-LUAD,
   TCGA-LUAD cohort and Local-LUAD cohort were used as verification
   cohorts. The detailed analysis process is showed in the flow chart.
   According to whether or not the BCR signaling pathway gene showed
   nonsynonymous mutation, the 47 patients in the Miao-LUAD cohort were
   divided into a BCR signaling MUT group (31) and a BCR signaling WT
   group (16). The results of the univariate Cox regression model showed
   that the BCR signaling pathway was a protective factor in the mutant
   group, which could predict better PFS and OS. After adjusting for
   related clinical confounding factors such as gender, pack years, TMB,
   and smoking, the results of the multivariate Cox regression model
   further confirmed that the mutation of the BCR signaling pathway can be
   used as an independent protective factor for LUAD patients receiving
   ICI treatment ([70]Figure 2A). Next, a Kaplan-Meier survival analysis
   of the Miao-LUAD cohort between BCR signaling MUT and WT groups was
   carried out to establish whether the mutant state of this pathway can
   effectively predict the prognosis of ICI treatment. The results showed
   that the BCR signaling MUT group had significantly longer PFS (Logrank
   test, HR = 0.25, 95% CI [0.03–2.07], p = 0.018) ([71]Figure 2B) and
   longer OS (Logrank test, HR = 0.48, 95% CI [0.20–1.14], p = 0.032)
   ([72]Figure 2C).[73]^21

Figure 1.

   [74]Figure 1
   [75]Open in a new tab

   Work flowchart of clinical cohort establishment and subsequent analysis
   in this study.

   Abbreviations: ICIs, immune checkpoint inhibitors; BCR signaling MUT, b
   cell receptor signaling pathway mutation group; BCR signaling WT, b
   cell receptor signaling pathway wild group.

Figure 2.

   [76]Figure 2
   [77]Open in a new tab

   The predictive value of clinical characteristics and the mutation
   status of the BCR signaling pathway for ICI efficacy. (A) Forest plot
   of the results of the univariate and multivariate Cox regression
   analyses in the Miao-LUAD cohort (ICI-treated cohort). The main portion
   of the forest plot presents the hazard ratios (HR) and 95% confidence
   intervals (95% CI). The p value represents the statistical significance
   of the variable. The HR indicates whether the factors are predictors of
   favorable (HR < 1) or poor (HR > 1) outcomes. KM survival curves for
   (B) PFS and (C) OS in 47 LUAD patients from the Miao-LUAD cohort.

   Abbreviations: BCR, b cell receptor; ICI, immune checkpoint inhibitor;
   LUAD, lung adenocarcinoma; PFS, progression-free survival; OS, overall
   survival; MUT, mutation group, WT, wild group.

Overview of Gene Mutation Between BCR Signaling MUT and WT Groups

   In the Miao-LUAD cohort, the somatic mutant genes were compared with
   the top 20 mutation frequencies across BCR signaling MUT patients and
   BCR signaling WT patients ([78]Figure 3A). It was found that in the BCR
   signaling MUT group, KRAS (52% vs 0%; p<0.05), CSMD3 (42% vs 0.06%;
   p<0.05), and COL11A1 (39% vs 0; p<0.05), the mutation frequency of the
   three genes, of which only the KRAS gene is tumorigenic, increased
   significantly. It is also worth noting that there was no statistically
   significant difference in gender or in the proportion of smokers and
   non- smokers between the MUT and WT groups, suggesting that these two
   clinical factors may not be related to the mutation of the BCR
   signaling pathway. Subsequently, mutually exclusive co-occurrence
   analyses were conducted on these 20 genes ([79]Figure 3B). We then used
   the lollipop diagram to show the mutation sites of the KRAS gene and
   COL11A1 gene which mutated only in the BCR signaling MUT group
   ([80]Supplementary Figure 1).

Figure 3.

   [81]Figure 3
   [82]Open in a new tab

   Genomic profiles of 47 LUAD patients in the Miao-LUAD cohort. (A) The
   20 genes with the highest mutation frequencies and corresponding
   clinical information. (B) Mutual exclusion co-occurrence analysis of
   the top 20 mutated genes. BCR signaling MUT, b cell receptor signaling
   mutation group, BCR signaling WT, b cell receptor signaling wild group
   (*p<0.05; **p<0.01; and ***p<0.001).

The BCR Signaling MUT Group Indicates Higher Immunogenicity Compared with the
BCR Signaling WT Group

   Sufficient immunogenicity is the basis of the immune response. In order
   to explore the relationship between the mutation status of the BCR
   signaling pathway and immunogenicity, the TMB, NAL and mutation numbers
   of DNA damage repair (DDR) related pathways were compared. The results
   of TMB analysis showed that among the Local-LUAD, Rizvi-LUAD,
   Samstein-LUAD and TCGA-LUAD, the TMB of the BCR signaling MUT group was
   significantly higher than that of the BCR signaling WT group
   ([83]Figure 4A–D; all p<0.05). Furthermore, in the TCGA-LUAD, the
   analysis results on the number of DDR related pathways mutations also
   support the hypothesis that the BCR signaling MUT group had higher
   immunogenicity; that is, the BCR signaling MUT group had higher numbers
   of BER, DDR, DSB, FA, HR, SSB, NHEJ, NER and MMR pathway gene mutations
   than the BCR signaling WT group ([84]Figure 4E; all p<0.05). In
   addition, the TCGA-LUAD cohort BCR signaling MUT group had higher NAL
   than the BCR signaling WT group ([85]Figure 4F; p<0.05).

Figure 4.

   [86]Figure 4
   [87]Open in a new tab

   Comparison of TMB between the MUT and WT groups in the (A) Local-LUAD
   cohort, (B) Rizvi-LUAD cohort, (C) Samstein-LUAD cohort, and (D)
   TCGA-LUAD cohort. (E) Comparison of DDR related signaling pathways
   alterations between the MUT and WT groups in the TCGA-LUAD cohort. (F)
   Comparison of NAL between the MUT and WT groups in the TCGA-LUAD
   cohort. MUT, B cell receptor signaling pathway mutant type; WT, B cell
   receptor signaling pathway wild type; (*p<0.05; **p<0.01; ***p<0.001;
   and ****p<0.0001).

   Abbreviations: TMB, tumor mutation burden; DDR, DNA damage repair; NAL,
   neoantigen load.

Differences in the Tumor Microenvironments Between BCR Signaling MUT and BCR
Signaling WT

   Based on the mutation data and transcriptome data of the TCGA-LUAD
   cohort, a comparative evaluation was conducted to determine the
   difference between the mutation status of the BCR signaling pathway and
   the 22 kinds of immune cells infiltrated in tumors, using a CIBERSORT
   algorithm ([88]Figure 5A). The results showed that the numbers of CD8+
   T cells, activated CD4 memory T cells and activated mast cells in the
   BCR signaling MUT group were higher (all p<0.05) than in the BCR
   signaling WT group. However, the numbers of regulatory T cells (Tregs),
   monocytes, resting CD4 T memory cells, resting mast cells, and resting
   dendritic cells increased significantly in BCR signaling WT group (all
   p<0.05). Then the immune-related scores (TH2 score, number of segment
   score and Homologous recombination score) were calculated in the
   TCGA-LUAD cohort ([89]Figure 5B), and all of them were significantly
   higher in the BCR signaling MUT group (all p<0.05).

Figure 5.

   [90]Figure 5
   [91]Open in a new tab

   (A) Comparison of the proportions of immune cells estimated by the
   CIBERSORT method between MUT and WT groups in the TCGA-LUAD cohort. (B)
   Comparison of immune related scores between MUT and WT groups in the
   TCGA-LUAD cohort. The immune related scores are Th2 Cell, number of
   segment score and Homologous recombination score. (C) Results of ssGSEA
   analysis between MUT and WT groups in the TCGA-LUAD cohort (*p<0.05;
   **p<0.01; ***p<0.001; and ****p<0.0001; Wilcoxon rank-sum test).

Pathway Enrichment Analysis Between BCR Signaling MUT and BCR Signaling WT

   Based on transcriptome data of the TCGA-LUAD cohort, ssGSEA and GSEA
   were used to enrich and analyze the functional gene sets. The results
   of ssGSEA ([92]Figure 5C) showed that the ssGSEA scores of several
   cytokines and chemokines pathways related to IL-1β, IL-10, CXCL2, CXC2
   and TGF-β, were significantly lower in the BCR signaling MUT group (all
   p<0.05). The ssGSEA score of arachidonic acid and fatty acid metabolism
   pathways in the BCR signaling MUT group was also significantly lower
   than that in the WT group (all p<0.05). In addition, GSEA results
   showed that the SMAD signaling pathway and the FoxO signaling pathway
   were significantly activated in the BCR signaling MUT group (p<0.05,
   ES>0), whereas pathways of the signaling by WNT in cancer and
   constitutive signaling by aberrant PI3K in cancer were down-regulated
   in the BCR signaling MUT group (p<0.05, ES<0) ([93]Figure 6A).
   Similarly, within the BCR signaling MUT group the signal pathways
   relating to angiogenesis such as vascular endothelial cell
   proliferation, vascular endothelial growth factor signaling pathway,
   Hedgehog signaling pathway and fibroblast growth factor receptor
   signaling pathway were significantly down-regulated (p<0.05, ES<0
   ([94]Figure 6B). Finally, based on the results of the analysis, we
   hypothesized the possible underlying mechanism for the better prognosis
   in the BCR signaling MUT group ([95]Figure 6C).

Figure 6.

   [96]Figure 6
   [97]Open in a new tab

   (A and B) Comparison of GSEA analysis between MUT and WT groups in the
   TCGA-LUAD cohort. (C) Potential mechanism underlying the prognostic
   value of the BCR signaling pathway mutation.

Discussion

   In this study, it was found that nonsynonymous BCR signaling pathway
   mutation status before treatment could predict better PFS and OS after
   ICI treatment in the Miao-LUAD cohort. This was then combined with
   other published data sets and the Local-LUAD group from our own
   hospital to calculate the degree of immune cell infiltration, the
   number of TMB, NAL and DDR related pathway mutations from the
   perspective of immunogenicity and immune microenvironment, and were
   then further combined with pathway enrichment analysis. With these
   results the ability of nonsynonymous mutation status in the BCR
   signaling pathway as a predictive marker of the efficacy of ICIs was
   further validated.

   Immunogenicity of tumors is considered to be a prerequisite for
   exerting anti-tumor immunity,[98]^31 and it can be assessed via NAL,
   TMB, or DDR pathway mutation.[99]^26 The number of NAL measures the
   quantity of neoantigen in tumor cells, so as to evaluate the ability of
   the tumor to induce a tumor-specific immune response. TMB is now a
   clinically used ICI treatment predictor in LUAD, and it is documented
   that a damaged DNA repair mechanism can result in enhanced
   immunogenicity and a high TMB status.[100]^32^,[101]^33 Wang et al
   showed that DDR pathway mutations can act as potential biomarkers of
   ICI therapy in multiple tumor types.[102]^32 Furthermore, Teo et al.
   Showed that DDR pathway mutations are related to a better clinical
   response and prolonged PFS and OS in urothelial carcinoma patients who
   had received ICI therapy.[103]^33 In our study, we found that the BCR
   signaling MUT group in TCGA cohort had higher TMB, NAL and DDR-related
   pathways mutations, and the TMB was higher in all five cohorts in the
   BCR signaling MUT group.

   In addition, 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.[104]^34 Before ICI baseline treatment, TILs infiltrated in
   the TME were reported to be positively correlated with ICI treatment
   efficacy.[105]^35–37 Therefore, we analyzed the infiltration degree of
   22 types of immune cells in the TCGA-LUAD cohort via a CIBERSORT
   method. There were shown to be more CD8+ T cells and activated memory
   CD4+ T cells infiltrating in BCR signaling MUT group. CD8+ T cells were
   shown to be specific anti-tumor T cells. They can kill tumor cells by
   producing IFN-γ, TNF and granzyme B.[106]^38 CD4+ T cells play an
   important role in helping CD8+ T cells to enhance anti-tumor
   immunity.[107]^39^,[108]^40 Once activated by tumor-associated
   antigens, CD4+ T cells can promote the maturation of dendritic 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 cells 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 opposed to
   MHC class I restricted antigen recognition of CD8+ T cells, are an
   important supplementary part of antigen recognition in anti-tumor
   immunity.

   On the contrary, the degree of Tregs, can directly inhibit CD8+ T cells
   infiltration in LUAD.[109]^41 Infiltration in the BCR signaling MUT
   group was significantly lower than that in the WT group. In addition,
   our results showed that there were significant differences in the
   degree of mast cell infiltration between the BCR signaling MUT group
   and the 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,[110]^42^,[111]^43 and a 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 in
   immunotherapy efficacy.[112]^44 However, mast cell-derived TNF can kill
   tumor cells.[113]^45 Further research is required to determine the role
   that it plays. Furthermore, the anti-tumor immune microenvironment
   clearly activated in BCR signaling MUT group could be a reason why this
   group has significantly better ICIs treatment efficacy.

   Chemokines and cytokines secreted by immune cells in TME are important
   components of LUAD anti-tumor immunity.[114]^46 The ssGSEA results
   showed that cytokines and chemokine produced pathways related to
   immunosuppression, such as IL-10, IL-1β, CCL2 and CXCL2, were
   significantly down-regulated in the BCR signaling MUT group.[115]^47
   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.[116]^48^,[117]^49 Evidence has
   been shown 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.[118]^49^,[119]^50 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.[120]^51 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.[121]^52^,[122]^53 The effects of IL-1β are
   significantly related to a poor prognosis for NSCLC.[123]^54 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.[124]^55^,[125]^56 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.[126]^57–59

   At last, in our GSEA results, some classic signaling pathways that
   promote tumor occurrence and development were significantly
   down-regulated in the BCR signaling MUT group,[127]^60–63 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 are basic to the survival and metastasis of tumor
   cells, were also significantly down-regulated in the BCR signaling MUT
   group.[128]^64–66 Tumor angiogenesis in solid tumors can also prevent
   tumor-specific T cells from infiltrating TME.[129]^67 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;[130]^68
   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 the
   ssGSEA results, the 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, consequently,
   inhibits the role of these proteins in promoting tumor growth.[131]^69
   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.[132]^70

   This study explored the impact of BCR signaling pathway nonsynonymous
   mutation status on the progress of LUAD patients treated with ICIs from
   the perspective of immune microenvironment (ie immune cells,
   immunogenicity and cytokines). However, this study still had several
   limitations. First, because the survival data of the LUAD cohort
   treated with ICIs is very limited, it was only possible to sufficiently
   explore the relationship between the BCR signaling pathway and the ICI
   treatment survival rate in the Miao-LUAD cohort. Secondly, due to the
   lack of information on TNM classification, Gene-altering events and the
   ICI treatment program, including factors such as whether ICI therapy
   was conducted alone or in combination with chemotherapy/radiotherapy,
   we cannot exclude the influence of these clinical factors on the
   relationship between BCR pathway mutations and ICIs prognosis. In
   future studies, we will try to collect more clinical data to explore
   the potential bias of these clinical factors. 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 recommend that
   readers interpret our findings with caution, since we did not carry out
   related cell and animal experiments to further verify our results, and
   we hope to supplement with further studies in the future.

Conclusion

   In this study, we found that there is a significant relationship
   between the mutation state of BCR signaling pathway and the therapeutic
   effect of ICIs. As a result, the BCR signaling MUT group had better PFS
   and OS. With further analysis of RNA-seq data, mutation data and
   clinical data in multiple cohorts, it was found that the MUT group had
   higher immunogenicity and a more anti-tumor activated microenvironment,
   and these results indicate that the BCR signaling MUT group has longer
   PFS and OS. To sum up, these results support the mutation status of the
   BCR signaling pathway as a potential biomarker to predict the efficacy
   of ICI therapy in LUAD patients.

Acknowledgments