详细的原发性黑素瘤空间免疫表型分析揭示了与患者预后相关的免疫细胞亚群。

Detailed spatial immunophenotyping of primary melanomas reveals immune cell subpopulations associated with patient outcome.

机构信息

Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia.

Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia.

出版信息

Front Immunol. 2022 Aug 8;13:979993. doi: 10.3389/fimmu.2022.979993. eCollection 2022.

Abstract

While the tumor immune microenvironment (TIME) of metastatic melanoma has been well characterized, the primary melanoma TIME is comparatively poorly understood. Additionally, although the association of tumor-infiltrating lymphocytes with primary melanoma patient outcome has been known for decades, it is not considered in the current AJCC melanoma staging system. Detailed immune phenotyping of advanced melanoma has revealed multiple immune biomarkers, including the presence of CD8+ T-cells, for predicting response to immunotherapies. However, in primary melanomas, immune biomarkers are lacking and CD8+ T-cells have yet to be extensively characterized. As recent studies combining immune features and clinicopathologic characteristics have created more accurate predictive models, this study sought to characterize the TIME of primary melanomas and identify predictors of patient outcome. We first phenotyped CD8+ T cells in fresh stage II primary melanomas using flow cytometry (n = 6), identifying a CD39+ tumor-resident CD8+ T-cell subset enriched for PD-1 expression. We then performed Opal multiplex immunohistochemistry and quantitative pathology-based immune profiling of CD8+ T-cell subsets, along with B cells, NK cells, Langerhans cells and Class I MHC expression in stage II primary melanoma specimens from patients with long-term follow-up (n = 66), comparing patients based on their recurrence status at 5 years after primary diagnosis. A CD39+CD103+PD-1- CD8+ T-cell population (P2) comprised a significantly higher proportion of intratumoral and stromal CD8+ T-cells in patients with recurrence-free survival (RFS) ≥5 years those with RFS <5 years (p = 0.013). Similarly, intratumoral B cells (p = 0.044) and a significantly higher B cell density at the tumor/stromal interface were associated with RFS. Both P2 and B cells localized in significantly closer proximity to melanoma cells in patients who remained recurrence-free (P2 p = 0.0139, B cell p = 0.0049). Our results highlight how characterizing the TIME in primary melanomas may provide new insights into how the complex interplay of the immune system and tumor can modify the disease outcomes. Furthermore, in the context of current clinical trials of adjuvant anti-PD-1 therapies in high-risk stage II primary melanoma, assessment of B cells and P2 could identify patients at risk of recurrence and aid in long-term treatment decisions at the point of primary melanoma diagnosis.

摘要

虽然转移性黑色素瘤的肿瘤免疫微环境(TIME)已经得到了很好的描述,但原发性黑色素瘤的 TIME 相对了解较少。此外,尽管几十年来一直知道肿瘤浸润淋巴细胞与原发性黑色素瘤患者预后相关,但目前的 AJCC 黑色素瘤分期系统并未考虑这一点。对晚期黑色素瘤的详细免疫表型分析揭示了多种免疫生物标志物,包括 CD8+T 细胞的存在,可预测免疫治疗的反应。然而,在原发性黑色素瘤中,缺乏免疫生物标志物,并且尚未广泛描述 CD8+T 细胞。由于最近的研究将免疫特征与临床病理特征相结合,创建了更准确的预测模型,因此本研究旨在描述原发性黑色素瘤的 TIME,并确定预测患者预后的因素。我们首先使用流式细胞术对新鲜 II 期原发性黑色素瘤中的 CD8+T 细胞进行表型分析(n=6),鉴定了富含 PD-1 表达的 CD39+肿瘤驻留 CD8+T 细胞亚群。然后,我们对来自具有长期随访的患者(n=66)的 II 期原发性黑色素瘤标本进行 Opal 多重免疫组化和基于定量病理学的 CD8+T 细胞亚群免疫特征分析,比较了基于 5 年原发性诊断后复发状态的患者。在无复发生存(RFS)≥5 年的患者中,CD39+CD103+PD-1-CD8+T 细胞群体(P2)在肿瘤内和基质中 CD8+T 细胞中的比例显著更高 那些 RFS<5 年的患者(p=0.013)。同样,肿瘤内 B 细胞(p=0.044)和肿瘤/基质界面上更高的 B 细胞密度与 RFS 相关。在无复发生存的患者中,P2 和 B 细胞都更接近无复发的黑素瘤细胞(P2 p=0.0139,B 细胞 p=0.0049)。我们的结果强调了描述原发性黑色素瘤中的 TIME 如何为免疫系统和肿瘤之间的复杂相互作用如何改变疾病结局提供新的见解。此外,在目前高危 II 期原发性黑色素瘤辅助抗 PD-1 治疗的临床试验背景下,评估 B 细胞和 P2 可以识别有复发风险的患者,并有助于在原发性黑色素瘤诊断时做出长期治疗决策。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0999/9393646/619c79c25066/fimmu-13-979993-g001.jpg

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