Introduction
Cutaneous melanoma (CM) is a highly malignant skin cancer that originates from melanocytes. Over the past few decades, its incidence among white people has increased dramatically, with 23,0000 new cases worldwide each year (World Health Organization) [
1]. Immunotherapy is an indispensable and important treatment for CM [
2]. In recent years, CM immunotherapy has made exciting progress and ushered in a new era for CM therapy. Compared with conventional chemotherapy, immunotherapy can cause an unprecedented, sustained response in patients with advanced cancer. However, this reaction occurs only in a relatively small number of patients, and the effect varies greatly among CM patients. These clinical challenges drive researchers to identify new tools to predict which patients are inherently resistant to targeted therapy and immunotherapy. This can better guide clinical management of patients and promote the rational use of clinical resources.
Copper is a mineral nutrient that is involved in cell proliferation and death pathways [
3]. The link between copper and cancer has been well established, and many studies have shown that tumors require higher levels of copper than healthy tissues. Cancer cells also have a higher demand for copper than non-mitotic cells. It has been reported that the concentration of copper in tumors and serum is increased in both animal models and patients with breast, lung, gastrointestinal, oral, thyroid, gallbladder, gynecologic, and prostate cancer [
4,
5]. Copper can also promote tumor angiogenesis and lead to tumorigenesis, growth, and metastasis. Cuproptosis is a mode of cell death induced by copper ions that is dependent on the accumulation of copper ions. When the cellular concentration of copper ions reaches a certain level, they regulate cell death through targeting acylated proteins in the TCA cycle [
6]. CRGs affect tumorigenesis, invasion, and metastasis in a manner similar to ferroptosis and pyroptosis genes. cuproptosis is closely related to the progression of cancer and is a potential new therapeutic target for the targeted killing of cancer cells [
4,
7].
The interaction between immune regulation and tumor cells in the tumor microenvironment (TME) can modulate the effect of immunotherapy. The TME plays an important role in these interactions via suppressing or enhancing the immune response. Emerging evidence suggests that copper overload and cuproptosis lead to immune dysfunction through the creation of reactive oxygen species (ROS). For example, ROS may contribute to the release of damage-related molecular patterns (DAMP), significantly regulating the immune response [
8,
9]. Therefore, the molecular characteristics of CRGs may provide important insight into the characteristics of the TME and the potential mechanism of CM. Delineation of cuproptosis patterns and their inherent mutation patterns, immunotherapeutic effects, TME differences, and prognostic effects in patients with CM will provide new insights into the mechanisms of occurrence, development, and potential effects of immunotherapy on CM.
In this study, we systematically studied the expression of CRGs, somatic mutations, and CNV patterns in patients with CM using CM data from the TCGA and GEO databases. Two different modes of cuproptosis were identified in patients with CM, and differences in the TME and immunotherapy between the two modes were compared. To better guide clinical management, we constructed a CRSS and applied it to eight immunotherapy cohorts containing 502 CM patients. Results showed good risk stratification and prediction of the effects of immunotherapy. Finally, we combined four clinical features (age, sex, AJCC stage, CRSS) to construct a prognostic model. Results showed that the prognostic capability and stability of the combined model was greatly improved compared to each single variable alone.
Discussion
Immunotherapy is one of the most important treatments for patients with advanced CM. Immune activity and TME status of CM patients plays a decisive role in the effect of immunotherapy. Cuproptosis is a newly identified mode of cell death. Many studies have confirmed that CRGs are closely related to tumor immunotherapy and TME status. To date, there have been no studies focused on the relationship between cuproptosis and TME in CM, or the effect on immunotherapy.
In this study, we systematically divided TCGA-SKCM patients into two groups based on the 10 most classical CRGs using unsupervised cluster analysis. The immune score, the abundance of immune cell infiltration, the expression of immune checkpoints, TMB, and other TME indices in group A were significantly higher than in group B. In addition, the effect of immunotherapy and overall prognosis in group A were significantly better than in group B. We also evaluated the sensitivity of the two groups of CM patients to common immunotherapeutic drugs.
To explore the causes of the differences in prognosis and construct a risk prediction model, we performed difference analysis using univariate and multivariate COX regression analyses. We identified 10 genes that were most likely to lead to significant differences in immunotherapy efficacy and prognosis between the two groups. The CRSS composed of 10 the CRGs accurately stratified the risk of CM patients and showed that immune cell infiltration abundance, TMB, immunotherapy sensitivity, and immunotherapeutic effect in patients with high CRRS were significantly higher than in those with low CRRS. CRSS accurately predicted immunotherapeutic effect in 11 cohorts, including eight CM immunotherapy cohorts and three other tumor immunotherapy cohorts. In addition, the prognostic nomogram model constructed using CRSS and clinicopathological features was more accurate and stable than CRSS or AJCC stage alone.
C-Type Lectin Domain Family 2 Member B (CLEC2B) encodes a member of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily. Members of this family share common protein folding and have a variety of functions such as cell adhesion, intercellular signal transduction, glycoprotein conversion, and roles in inflammation and immune responses. CLEC2B encodes a type 2 transmembrane protein that functions as a cell activation antigen [
37]. However, the research on CLEC2B and CM is currently lacking. In this study, compared with normal tissues and CM patients with low CRRS, CLEC2B was significantly downregulated in CM patients and CM patients with high CRRS. Moreover, univariate (HR = 0.690) and multivariate (HR = 0.810) COX regression analyses showed that CLEC2B was an independent protective factor for patients with CM. Therefore, CLEC2B may inhibit the occurrence and development of CM by enhancing immune surveillance.
Fc Gamma Receptor Iia (FCGR2A) encodes a member of the immunoglobulin Fc receptor gene family found on the surface of many immunoreactive cells. The protein encoded by this gene is a cell surface receptor present on phagocytes such as macrophages and neutrophils and is involved in the phagocytosis and clearance of immune complexes. Following binding to IgG, FCGR2A initiates cellular responses against pathogens and soluble antigens. FCGR2A also promotes phagocytosis of opsonized antigens [
38]. Mutations or deletions of FCGR2A can lead to significant resistance to immunotherapy in a variety of tumor types including colon cancer, breast cancer, and leukemia [
39‐
41]. The protein encoded by Alpha-2-Macroglobulin (A2M) is a protease inhibitor and cytokine transporter. A2M uses decoy and trap mechanisms to inhibit broad-spectrum proteases including trypsin, thrombin, and collagenase, and inhibits inflammatory cytokines, thereby disrupting the inflammatory cascade [
42]. Lindner et al. found that A2M inhibits the malignant properties of astrocytoma cells by impeding beta-catenin signaling [
43]. There are currently no mechanistic studies exploring the link between A2M, FCGR2A, and CM. In the present study, A2M and FCGR2A were simultaneously upregulated in CM and downregulated in CM patients with a high CRRS relative to patients with a low CRRS. Further, univariate and multivariate COX regression analyses suggested that both A2M (HR = 0.860) and FCGR2A (HR = 0.820) were independent protective factors in CM patients. Therefore, we hypothesize that FCGR2A and A2M may enhance the efficacy of immunotherapy and improve prognosis by enhancing immune activity and immune lethality in CM patients.
Brevican (BCAN) encodes a member of the chondroitin sulfate proteoglycan glycan family that is specifically expressed in the central nervous system. This protein is regulated during development and may play a role in cell adhesion. BCAN is highly expressed in glioma and may promote the growth and cell motility of brain tumor cells [
44,
45]. However, there is no reported research on BCAN and CM. In our study, the expression of BCAN in the tumor and high CRRS groups was significantly higher than in normal tissues and the low CRRS group. In addition, univariate (HR = 1.09) and multivariate (HR = 1.06) COX regression analyses showed that BCAN is an independent risk factor in patients with CM. Therefore, we speculate that BCAN may promote the development of CM by affecting cell adhesion.
This study had some limitations. First, the data were retrospective, and a prospective cohort would be needed to validate our model. Second, the expression of each gene in our model was based on skin tissues. Development of biomarkers based on urine or blood samples would be more appropriate for clinical application. Finally, future studies should include in vivo and in vitro experiments to verify the specific mechanisms of each molecule in CM.
In the future, we will first retrospectively collect biological samples and clinical data of past and prospective CM patients in our hospital, and construct an external validation cohort to validate the accuracy of our signature in predicting the effect of immunotherapy, as well as the accuracy of our model in predicting the prognosis of CM patients. Then, biological specimens and detailed clinical data of CM patients admitted to our hospital will be prospectively collected under the condition of ethical review and informed consent signed by patients. The required sample size was calculated according to the effect value, test level (α) and test efficacy (1-β) equivalence of the previous retrospective study results. The experimental group and the control group were matched by age, sex, clinical staging, and other basic characteristics. A 5-year follow-up will be conducted to record the status of CM patients after immunotherapy, as well as the time to death. We wound calculate the accuracy of our signature in predicting immunotherapy effect in CM patients and the ability of model scores to predict prognosis in CM patients at a follow-up time of 6 months, 1 year, 3 years, and 5 years.
Overall, we first attempted to developed a new cuproptosis-related scoring system that can stratify the risk of CM patients and predict their prognosis, thus potentially affecting immunotherapeutic choices. Moreover, we constructed a prognostic nomogram model to more efficiently guide clinical decision making.
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