背景：
皮肤黑色素瘤（cutaneous melanoma,CM）是一种由黑素细胞恶变引起的恶性肿瘤，在皮肤肿瘤中恶性程度最高，2020年全球新增病例超过320 000例，死亡病例超过57 000例。尽管在皮肤肿瘤中，黑色素瘤仅占4%，但目前已经认为是世界上第19大常见肿瘤，严重威胁人类的生命和健康。在黑色素瘤治疗过程中，早期诊断和早期治疗尤为重要。但放疗、化疗和手术切除等传统的癌症治疗，在皮肤黑色素瘤患者的长期预后中差异并不显著，针对皮肤黑色素瘤的治疗仍是充满困难与挑战的。新兴的免疫治疗疗法给晚期恶性黑色素瘤患者提供了新的希望，但是免疫检查点阻断（ICB）的治疗仅对部分黑色素瘤患者有效，治疗效果的参差不齐成为了目前临床及基础研究亟待解决的问题。因此，发现和探索用于患者分层的生物标志物是必须的，同时可以据此建议最有效的治疗方案，这也是目前研究的工作重点。
目的：
1.初步筛选皮肤黑色素瘤免疫治疗潜在生物标志物。
2.初步分析HLA-DMA与皮肤黑色素瘤的免疫相关性。
3.初步验证HLA-DMA预测皮肤黑色素瘤免疫治疗反应的作用。
方法:
1.分析了基因表达综合(GEO)数据库中的两个黑色素瘤免疫治疗数据集(GSE91061和GSE100797）和肿瘤免疫功能障碍和排除（TIDE）数据库中黑色素瘤免疫治疗数据集PRJEB23709，并提取了交叉候选基因作为黑色素瘤ICB治疗的潜在生物标志物。
2.进一步对交叉候选基因进行GO富集分析，分析交叉候选基因的生物学过程（BP）。
3.使用肿瘤免疫单细胞中心（TISCH）数据库研究交叉候选基因的细胞类型图谱，通过Human Protein Atlas初步评估候选基因表达量，筛选目的基因。
4.使用癌症基因组图谱（TCGA）数据集分析目的基因与恶性黑色素瘤免疫相关性。
5.使用恶性黑色素瘤组织芯片（TMA），进行免疫组织化学染色（IHC），同时基于TCGA数据库和肿瘤免疫图谱数据库(TCIA)进一步分析，验证目的基因预测黑色素瘤免疫治疗反应的作用。
结果:
1.对三个免疫治疗数据集进行差异基因分析后，获得了34个交叉候选差异表达基因（包括CXCR6、HLA-DPA1、NLRC5、CIITA、CD80、SLFN12L、HLA-DRA、HLA-DOA、CD96、HLA-DRB1、CD83、LYZ、HLA-DMA、PSTPIP1、PTPN22、HLA-DPB1、DOCK8、HLA-DQA1、BTK、KLHDC7B、CD1D、CXorf21、IL21R、HLA-DOB、ZMYND15、PCED1B、STX11、CTSH、LGALS2、GAB3、HLA-DMB、SLC7A9、TNF、HLA-DR）。
2.交叉候选基因的生物学过程（BP）注释集中在各项免疫过程中，包括干扰素-γ的正调节、T细胞增殖的正向调控、适应性免疫反应、免疫反应、抗原的加工和呈递、T细胞活化的正向调控、通过MHC Ⅱ类的外源性肽抗原的抗原加工和呈递、通过MHC Ⅱ类的多肽或多糖抗原的抗原加工和呈递、参与了免疫球蛋白介导的免疫反应的免疫球蛋白的产生、肽抗原与MHC Ⅱ类蛋白复合物的组装。
3.在这些候选基因中，发现SKCM组织中,人类白细胞抗原-DMA(HLA-DMA)在肿瘤细胞和免疫细胞均有表达。
4.HLA-DMA与炎症肿瘤微环境(TME)相关：HLA-DMA高表达组，大量趋化因子、配对受体、MHC分子、免疫抑制剂、免疫刺激剂显著上调；肿瘤浸润性免疫细胞（TIICs）分析结果提示，HLA-DMA高表达组，许多TIICs分子显著上调；同时免疫浸润分析结果提示HLA-DMA与B细胞、CD8+Ｔ细胞、CD4+Ｔ细胞、巨噬细胞、中性粒细胞、树突状细胞、上皮样细胞、NK细胞、癌症相关成纤维细胞（CAF）呈正相关。
5.免疫组化结果提示，HLA-DMA在肿瘤组织中表达明显高于正常组织，高表达HLA-DMA的肿瘤组织中PD-L1表达升高；且HLA-DMA与PD-L1表达呈正相关，这都说明HLA-DMA可以预测黑色素瘤对免疫治疗的反应。
结论:
本研究主要鉴定了潜在生物标志物预测皮肤黑色素瘤免疫治疗反应。HLA-DMA与皮肤黑色素瘤炎症肿瘤微环境（TME）相关，这提示HLA-DMA是有前景的生物标志物，这可能有助于皮肤黑色素瘤免疫治疗反应的预测。
 

Background:
Cutaneous melanoma (CM) is a malignant tumor caused by malignant transformation of melanocytes. It is the most malignant tumor among skin tumors, with more than 320000 new cases and more than 57000 deaths in 2020 worldwide . Although melanoma accounts for only 4% of skin tumors, it is now considered to be the 19th most common tumor in the world, posing a serious threat to human life and health. Early diagnosis and treatment are especially important in the treatment of melanoma. However, traditional cancer treatments, such as radiotherapy, chemotherapy and surgical resection, do not differ significantly in the long-term prognosis of patients with CM, and the treatment of cutaneous melanoma is still difficult and challenging. Emerging immunotherapies offer new hope for patients with advanced malignant melanoma, but treatment with immune checkpoint blockade (ICB) is effective only in a subset of patients with melanoma; The uneven therapeutic effect has become an urgent problem to be solved in clinical and basic research. Therefore, the discovery and exploration of biomarkers for patient stratification is imperative, along with the ability to recommend the most effective treatment options, which is also the focus of current research.
Object:
1. Preliminary screening of potential biomarkers for immunotherapy of cutaneous melanoma. 
2. To analyze the relationship between HLA-DMA and the immunity of cutaneous melanoma. 
3. To verify the role of HLA-DMA in predicting the response to immunotherapy of cutaneous melanoma.
Method:
1.Two immunotherapy datasets (GSE91061 and GSE100797) in the Gene Expression Omnibus (Geo) database and databset PRJEB23709 in the Tumor Immune Dysfunction and Exclusion(TIDE) database were analyzed, and cross-candidate genes were extracted as potential biomarkers for ICB therapy in melanoma. 
2.Furthermore, GO enrichment analysis was performed to analyze the biological process of the cross-candidate genes. 
3. Using Tumor Immune Single Cell Hub（TISCH） database to analyze the cell type map of cross-candidate genes and screen the target genes.The expression of candidate genes was evaluated by Human Protein Atlas and the target genes were screened. 
4.The Cancer Genome Atlas (TCGA) database was used to analyze the relationship between target gene and the immunity of malignant melanoma. 
5.Using malignant melanoma TMA, immunohistochemical staining (IHC) was performed, while further analysis based on the TCGA database and the The Cancer Immunome Atlas(TCIA) validated the role of target gene in predicting melanoma immunotherapy responses.
Results:
1.After differential gene analysis of three immunotherapy datasets, 34 cross-candidate differentially expressed genes (DEGs) were obtained,includingCXCR6、HLA-DPA1、NLRC5、CIITA、CD80、SLFN12L、HLA-DRA、HLA-DOA、CD96、HLA-DRB1、CD83、LYZ、HLA-DMA、PSTPIP1、PTPN22、HLA-DPB1、DOCK8、HLA-DQA1、BTK、KLHDC7B、CD1D、CXorf21、IL21R、HLA-DOB、ZMYND15、PCED1B、STX11、CTSH、LGALS2、GAB3、HLA-DMB、SLC7A9、TNF、HLA-DR. 
2.Annotation of the biological processes of the cross-candidate genes focuses on the various immune processes, including positive regulation of interferon-gamma production, positive regulation of T cell proliferation, adaptive immune response, immune response, antigen processing and presentation, positive regulation of T cell activation, antigen processing and presentation of exogenous peptide antigen via MHC class II, antigen processing and presentation of peptide or polysaccharide antigen via MHC class II, immunoglobulin production involved in immunoglobulin mediated immune response, peptide antigen assembly with MHC class II protein complex. 
3. Among these candidate genes, Human leukocyte antigen DMA (HLA-DMA) was found to be expressed by both tumor cells and immune cells in SKCM tissues. 
4. HLA-DMA is related to the inflammatory tumor microenvironment (TME) . In the high expression group, a large number of chemokines, receptors, MHC, immunoinhibitors, and immunostimulators were significantly up-regulated. The results of tumor-infiltrating immune cells (TIICs) analysis suggested that many TIICs molecules were significantly up-regulated in HLA-DMA high expression group. HLA-DMA was positively correlated with B cells, CD8+ T cells, CD4+T cells, macrophages, neutrophil, dendritic cells, epithelioid cells, NK cells and cancer-associated fibroblasts (CAFs) . 
5. The results of immunohistochemistry showed that the expression of HLA-DMA in the tumor tissues was significantly higher than that in the normal tissues, and the expression of PD-L1 was increased in the tumor tissues with high expression of HLA-DMA. There was a positive correlation between HLA-DMA and PD-L1 expression.These results suggest that HLA-DMA can predict the response of melanoma to immunotherapy.
Conclusion:
This study primarily identified potential biomarkers to predict the response to immunotherapy for cutaneous melanoma. HLA-DMA is associated with the inflammatory tumor microenvironment (TME) in cutaneous melanoma, suggesting that HLA-DMA is a promising biomarker that may contribute to the prediction of therapeutic response in cutaneous melanoma.