Skin cutaneous melanoma (SKCM) is one of the most aggressive malignancies, causing about 80% of deaths in skin cancer (Schadendorf et al., 2018). Nearly 50% of cutaneous melanoma harbor activating V600E mutations in BRAF, which is considered a prognostic indicator of tumor proliferation, metastasis, recurrence as well as an effective target for SKCM treatment. The major factor limiting the clinical benefit of BRAF inhibitor are short response duration, off-target effect and drug resistance (Ribas et al., 2019a). There is also evidence that oncogenic BRAF can modulate the ability of the immune system to recognize SKCM cells. Activating mutations in the BRAF gene activate the mitogen-activated protein kinase (MAPK) pathway, which contributes to immune escape by recruiting regulatory T cells, reducing antigen presentation, and inhibiting the release of IFN-γ and TNF-α (Ascierto and Dummer, 2018). A series of immunotherapy strategies such as anti-PD-1, anti-CTLA4 and MAGE-A3 have been applied in SKCM and result in improvement in patient survival (Bajor et al., 2018). In addition, the combination of BRAF inhibitors and anti-PD-1 has shown significant improvement in SKCM treatment response (Ascierto et al., 2019a). These results suggest that we may be able to improve the survival outcome of BRAF mutated patients by regulating their immune response and tumor microenvironment. The tumor microenvironment (TME) consists of a variety of immune cells and stromal cells, including fibroblasts, endothelial cells, extracellular matrix, cytokines, chemokines and receptors (Vigneron, 2015). Due to the complexity of TME and a lack of a rational mechanistic basis, it is urgent to investigate the tumor microenvironment and identify prognostic biomarkers in BRAF mutated SKCM patients (Gnanendran et al., 2020).

ESTIMATE algorithms have been developed to calculate tumor purity in various cancers based on the specific gene expression signature of immune and stromal cells (Yoshihara et al., 2013). In this current work, we applied the expression data of BRAF mutated SKCM cohorts and ESTIMATE algorithm to extract a list of tumor microenvironment associated genes. Most of the genes were found to be related to better survival outcomes in BRAF mutated SKCM patients. Importantly, we estimated the proportion of immune cells based on gene expression profiling in BRAF mutated samples. Finally, we identified 4 hub genes associated with prognosis and immune cell infiltration in BRAF patients.

Skin cutaneous melanoma (SKCM) is one of the dead cancers with high malignant metastasis and mortality rates (Siegel et al., 2020). Identification of oncogenes provides novel insights into the progression of cancer therapy. BRAF oncogene was found in more than 50% of skin cutaneous melanoma as well as other cancers such as colorectal cancer and papillary thyroid cancer (Pollock and Meltzer, 2002; Rajagopalan et al., 2002; Cabanillas et al., 2020). The majority of researches claimed that BRAF mutation was often associated with high risk of metastasis, recurrence and poor survival outcomes (Ascierto et al., 2019b). BRAF inhibitor was considered the foundation of BRAF mutated melanoma treatment, and have demonstrated success and enhanced patient survival. However, only about 33% of patients benefit from target therapy in 5-year overall survival and the major limitations include short response duration, development of drug tolerance, and off-target effects (Robert et al., 2019). Recently, a growing group of researches showed that oncogenic BRAF can decrease the ability of the immune system to recognize melanoma cells. And the inhibition of BRAF can restore tumor immune recognition (Boni et al., 2010). Previous studies based on mice demonstrated that BRAF inhibitor response durations in vivo were significantly longer when melanoma cell lines were grown in immunocompetent mice compared to immunocompromised (Smalley, 2020). Besides, BRAF inhibition was associated with increased infiltration of CD4⁺T, CD8⁺T cells and reduced levels of myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs). While the depletion of CD4⁺ and CD8⁺ T cells significantly blunted the BRAF inhibitors response (Ribas et al., 2019b). Over the past years, the combination of immune therapy (anti-PD-1, anti-CTLA-4) and target therapy (BRAF inhibitor) has achieved an impressive improvement of the patients’ survival (Erkes et al., 2020). All results above suggested that the tumor microenvironment and immune effects play a vital role in SKCM therapy. However, the exploration of BRAF mutated immune microenvironment and the identification of immune-related prognostic targets in SKCM patients are still lacking (Boussadia et al., 2018).

Tumor immune microenvironment (TME) is described as significantly affecting the cancer treatment and prognosis (Hinshaw and Shevde, 2019). The ESTIMATE has been applied in glioma, renal cell carcinoma and gastrointestinal tumors, showing the validity of this algorithm in estimating tumor purity (Alonso et al., 2017; Jia et al., 2018). Therefore, ESTIMATE algorithm was applied to identify immune-related prognostic genes that contributed to patients’ overall survival by investigating the TME. Our research showed that both of the immune and stromal scores were inversely correlated with Breslow depth and AJCC stage, which have been considered as classical prognostic factors for SKCM. As shown in the Kaplan-Meier survival curve that patients with a higher immune score had longer overall survival time than those with a lower immune score in the BRAF mutated SKCM.

Through the DEGs analysis, we found that there were 990 overlapped genes which both upregulated in the immune and stromal groups. Function annotation indicated that immune response, defense response, positive regulation of immune system process and cytokine binding were important biological processes of the 990 overlapped genes. Pathway analysis demonstrated that the majority of the overlapped genes served a role in chemokine signaling pathways, cytokine-cytokine receptor interaction and cell adhesion molecules. As expected, dysregulation of immune function had a significant impact on the microenvironment of BRAF mutant SKCM patients. Among them, chemokine signaling is mainly involved in the recruitment of various immune cells, and their dysregulation may be an important reason for reducing the level of immune infiltration and leading to poor prognosis in patients with BRAF mutation. Based on our results and previous researches, it is conceivable to hypothesize that chemokines and immune response play a vital role in the regulation of SKCM TME (Huang et al., 2020; Li et al., 2020).

Subsequently, 755 genes (76%) were found correlated with longer overall survival time. This further demonstrated the clinical value of these immune microenvironment-related genes in patients with BRAF mutations. Subsequently, 107 prognostic genes of BRAF patients were verified in GEO data set. PPI network and model analysis had identified 4 hub genes (TYROBP, CD86, CSF1R and ITGB2) in our study. Moreover, our hub genes occupied a central position in both the PPI network and Model 1, proving their core position and clinical value. They were mainly associated with oncogenic transformation, immune response and regulating of immune cells (D'Angelo et al., 2019; Casey et al., 2016). For example, related studies have shown that the activation of T cells requires costimulatory signals produced by the interaction of CD28 and CD86, which could increase the infiltration of T cells in tumor tissue and prolong the survival time of mice (Jia et al., 2022). The combination of anti-PD1 and anti-CSF1 receptor (CSF1R) antibodies induced the regression of melanoma in-driven transplanted mice (Neubert et al., 2018). ITGB2 is associated with immune infiltration of multiple immune cell subsets, such as CD45, CD8, CD4T cells, CD20B cells and so on (Kwak et al., 2021). Although there was no direct evidence for the association between TYROBP and melanoma, given the important association between these hub genes and immune infiltration, we regard them as potential therapeutic targets for patients with BRAF mutations.

Previous studied demonstrated that the imbalance of immune cell components was closely related to progressive disease and poor prognosis (Qiao et al., 2019). Therefore, we conducted a further immune infiltration analysis. As expected, CD4⁺T, CD8⁺T, CD4 naïve, Tr1, Th2 and many T cell subsets were significantly increased in immune-high group. While several cells (Monocyte, Macrophage and Gamma delta cells) have been found to be significantly decreased in immune-high BRAF mutated SKCM group. There were significant differences in immune-infiltration between the two groups, which may help to identify groups that are more responsive to BRAF inhibitors. Monocyte-lymphocyte ratio (MLR) is considered to be an important indicator of tumor prognosis (Garcia et al., 2022). It has been reported that cancer-associated Macrophage play a key role in tumor progression, angiogenesis, invasion and recruitment of immunosuppressive cells (Samain and Sanz-Moreno, 2020). Persistent immune-related gene expression and T-cell penetration were associated with clinical benefit in SKCM patients (Shoushtari et al., 2022). The infiltrating levels of various effector T cells, such as CD4⁺ and CD8⁺ T, were significantly higher in the immune-high group than in the control group. After binding to MHC class I antigens on tumor cells via T cell receptors, CD8⁺ T cells can produce granzymes and perforin to destroy cancer cells (Tsukumo and Yasutomo, 2018). It is well known that CD8⁺ T cells have an antitumor effect, and the increase of CD8⁺ T cells can significantly improve the prognosis of SKCM patients (Chen et al., 2018). It is important to note the emerging role of CD4⁺ T cells in antitumor immunity, and in particular, their functional versatility in the context of the tumor immune microenvironment. In actual tumor therapy, the single immune function of CD8⁺ cells is not enough to destroy tumor cells, as the immune checkpoint inhibitors (anti-PD-1, anti-CTLA-4) are only 30% effective (Gellrich et al., 2020). Recent studies have found that the best initiation and maturation of MHC-I-restricted CD8⁺T cells is CTL (cytotoxic T lymphocytes), which requires the response of CD4⁺T cells (Alspach et al., 2019). By secreting interferon and promoting the proliferation and lethality of CD8⁺T cells in TME, CD4⁺T cells play a vital role (Zhu et al., 2015; Borst et al., 2018). In preclinical studies, it was found that BRAF inhibition led to increased CD40L expression and IFN-γ release from CD4⁺T cells, and decreased levels of multiple cytokines including IL1, IL6, and IL10 (Ott et al., 2013). Therefore, we speculate that increasing the proportion of CD4⁺T cells to enhance the lethality of CD8⁺T cells in TME may be a potential strategy to improve the prognosis of BRAF mutated SKCM patients.

Moreover, the expression of these hub genes was related to the imbalance of multiple immune cells. For example, 4 hub genes (TYROBP, CD86, CSF1R and ITGB2) were mainly positively related to CD4⁺T, CD8⁺T, CD4 naïve, Tr1, iTreg, Tfh and many T cell in SKCM. While these hub genes could be found to be significantly negatively related to Macrophage and Gamma delta cells. These results were consistent with their previous association with longer overall survival. There are few study on the relationship between the hub genes and BRAF mutated SKCM treatment. Therefore, we had identified several immune-related prognostic biomarkers for BRAF mutated patients. Finally, we preliminarily validated the expression of hub genes in another dataset and evaluated their diagnostic value. Our results showed that all of the hub genes significantly up-regulated in immune-high group. These data provide reference for further development of treatment for patients with BRAF mutations.

We must acknowledge the limitations in this study. First, more patients should be collected in the future to expand the sample size, which is conducive to a deeper understanding of the mechanisms of BRAF mutated SKCM and immune dysfunction. Second, we have limited experimental data and further function validation is required to investigate the interaction between the prognostic genes and immune cells.

For the first time in this study, we try to explore the TME to better understand the potential prognostic immune‐related targets and mechanisms in BRAF mutated SKCM patients. This study revealed that the dysregulation of immune function and immune cells may contribute to the poor outcomes of BRAF mutated patients. It is of great significance to our further understanding of the TME and immune dysfunction in BRAF mutated SKCM.