尽管免疫检查点抑制剂（ICI）疗法显着改善了转移性黑色素瘤患者的预后，但许多患者并未受益。由于不良事件可能很严重，因此耐药性的生物标志物将很有价值，特别是在辅助治疗中。我们使用 NanoString GeoMx® 对来自 ICI 治疗的转移性黑色素瘤病例的 53 个治疗前标本进行了高复数数字空间分析 (DSP)。我们同时询问了四个分子区室中的 77 个靶标，这些分子区室由肿瘤的 S100B、巨噬细胞的 CD68、白细胞的 CD45 和非免疫基质细胞定义为所有三个区室标记均为阴性但 SYTO 13 呈阳性的区域。对于 DSP 验证，我们确认了结果通过定量免疫荧光 (QIF) 获得一些免疫标记物，例如 CD8、CD4、CD20、CD68、CD45 和 PD-L1。在单变量分析中，38 个变量与结果相关，其中 14 个变量在多变量调整后仍然显着。其中，CD95 在发现免疫治疗 (ITX) 队列和具有类似特征的独立验证队列中使用多重免疫荧光进一步得到验证，显示高水平的 CD95 与较短的无进展生存期之间存在关联。我们发现基质中的 CD95 与 ICI 耐药性相关。经过进一步验证，该生物标志物对于选择不会从免疫治疗中受益的患者可能具有价值。© 2023 作者。经泰勒许可出版

Although immune checkpoint inhibitor (ICI) therapy has dramatically improved outcome for metastatic melanoma patients, many patients do not benefit. Since adverse events may be severe, biomarkers for resistance would be valuable, especially in the adjuvant setting. We performed high-plex digital spatial profiling (DSP) using the NanoString GeoMx® on 53 pre-treatment specimens from ICI-treated metastatic melanoma cases. We interrogated 77 targets simultaneously in four molecular compartments defined by S100B for tumor, CD68 for macrophages, CD45 for leukocytes, and nonimmune stromal cells defined as regions negative for all three compartment markers but positive for SYTO 13. For DSP validation, we confirmed the results obtained for some immune markers, such as CD8, CD4, CD20, CD68, CD45, and PD-L1, by quantitative immunofluorescence (QIF). In the univariable analysis, 38 variables were associated with outcome, 14 of which remained significant after multivariable adjustment. Among them, CD95 was further validated using multiplex immunofluorescence in the Discovery immunotherapy (ITX) Cohort and an independent validation cohort with similar characteristics, showing an association between high levels of CD95 and shorter progression-free survival. We found that CD95 in stroma was associated with resistance to ICI. With further validation, this biomarker could have value to select patients that will not benefit from immunotherapy.© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.