Molecular patterns of resistance to immune checkpoint blockade in melanoma [scRNA-Seq]

Immune checkpoint blockade (ICB) has improved outcome for patients with metastatic melanoma but not all benefit from treatment. Several immune- and tumor intrinsic features are associated with clinical response at baseline. However, we need to further understand the molecular changes occurring during development of ICB resistance. Here, we collected biopsies from a cohort of 44 melanoma patients after progression to anti-CTLA4 or anti-PD1 monotherapy. Genetic alterations of antigen presentation and interferon gamma signaling pathways were observed in approximately 25% of ICB resistant cases. Anti-CTLA4 resistant lesions had a sustained immune response, including immune-regulatory features, as suggested by multiplex spatial and TCR clonality analyses. One anti-PD1 resistant lesion harbored a distinct immune cell niche, however, anti-PD1 resistant tumors were generally immune poor with non-expanded TCR clones. Such immune poor microenvironments were associated with melanoma cells having a de-differentiated phenotype lacking expression of MHC-I molecules. In addition, anti-PD1 resistant tumors had reduced fractions of PD1+ CD8+ T cells as compared to ICB naïve metastases. Collectively, these data show the complexity of ICB resistance and highlight differences between anti-CTLA4 and anti-PD1 resistance that may underlie differential clinical outcomes of therapy sequence and combination. Single cell transcriptomics from four samples that contain B cell rich regions, two of which having progressed on Immune Checkpoint Blockade, generated by single cell RNAseq. *************************************************************** Authors state that due to Swedish and Danish laws, the patient consent, and the risk that the sequencing data contains personally-identifiable information and hereditary mutations, we cannot deposit the short sequencing read data in a public access repository ***************************************************************

免疫检查点阻断（Immune checkpoint blockade, ICB）已显著改善转移性黑色素瘤患者的预后，但并非所有患者均可从治疗中获益。基线状态下，多种免疫固有及肿瘤固有特征与临床应答相关。然而，目前学界仍需进一步阐明ICB耐药发生过程中的分子变化机制。

本研究收集了由44例黑色素瘤患者组成的队列中，经抗CTLA-4（anti-CTLA4）或抗PD-1（anti-PD1）单药治疗后进展的患者的活检样本。约25%的ICB耐药病例中可检测到抗原呈递与干扰素γ信号通路的遗传改变。多重空间成像及T细胞受体（T cell receptor, TCR）克隆性分析结果显示，抗CTLA-4耐药病灶存在持续的免疫应答，包括免疫调节相关特征。1例抗PD-1耐药病灶呈现独特的免疫细胞微环境，但总体而言抗PD-1耐药肿瘤的免疫浸润较弱，且TCR克隆未发生扩增。此类免疫贫瘠的微环境与黑色素瘤细胞的去分化表型相关，此类细胞不表达主要组织相容性复合体I类（MHC-I）分子。此外，与未接受过ICB治疗的转移灶相比，抗PD-1耐药肿瘤中PD1+ CD8+ T细胞的比例显著降低。

综上，本研究数据揭示了ICB耐药的复杂性，并凸显了抗CTLA-4与抗PD-1耐药之间的核心差异，这些差异可能是治疗序贯及联合方案临床结局不同的潜在分子基础。本研究针对4个包含B细胞富集区域的样本开展了单细胞转录组测序，其中2个样本来自经免疫检查点阻断治疗后进展的患者，测序采用单细胞RNA测序（single cell RNAseq）技术。

作者声明：受瑞典、丹麦相关法律法规限制，加之患者知情同意要求，且测序数据可能包含个人可识别信息及遗传性突变，因此无法将短读长测序数据（short sequencing read data）提交至公共访问数据库（public access repository）。