Expression Analysis and Significance of PD-1, LAG-3, and TIM-3 in Human Non-Small Cell Lung Cancer Using Spatially Resolved and Multiparametric Single-Cell Analysis
Expression Analysis and Significance of PD-1, LAG-3, and TIM-3 in Human Non-Small Cell Lung Cancer Using Spatially Resolved and Multiparametric Single-Cell Analysis
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DOI：
10.1158/1078-0432.ccr-18-4142
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发表时间：
2019-08-01
影响因子：
11.5
通讯作者：
Schalper, Kurt A.
中科院分区：
文献类型：
--
作者：
Datar, Ila;Sanmamed, Miguel F.;Schalper, Kurt A.
用途：确定PD-1、LAG 3和TIM-3蛋白表达在人非小细胞肺癌（NSCLC）中的肿瘤组织/细胞分布、功能关联和临床意义。使用多重定量免疫荧光，我们进行了CD 3，PD-1，LAG-3，和TIM-3蛋白在来自组织微阵列中表示的三个独立组群的> 800个临床注释的NSCLC中的表达。在The Cancer Genome Atlas NSCLC数据集中研究了标记物表达与主要基因组改变之间的关联。使用从20个切除的NSCLC收集的白细胞的质谱细胞术（CyTOF）分析，我们确定了表达PD-1、LAG-3和TIM-3的免疫细胞的水平、共表达和功能谱。结果：PD-1、LAG-3和TIM-3在肿瘤浸润淋巴细胞（tumorinfiltrating lymphocytes，TIL）中的阳性率分别为55%、41.5%和25.3%。这些标记物显示出彼此之间的显著相关性，与未接受免疫治疗患者的主要临床病理变量和生存率的相关性有限。EGFR突变腺癌中标记物的表达较低，与肿瘤突变负荷的相关性有限。在单细胞CyTOF分析中，PD-1和LAG-3主要位于T细胞亚群/NKT细胞上，而TIM-3在NK细胞和巨噬细胞中表达更高。PD-1、LAG-3和TIM-3的共表达与显著的T细胞活化（CD 69/CD 137）、效应子功能（颗粒酶-B）和增殖（Ki-67）相关，但也与促凋亡标志物（FAS/BIM）水平升高相关。LAG-3和TIM-3存在于缺乏PD-1表达的TIL亚群中，并显示出不同的功能特征。在90例PD-1轴阻滞剂治疗的晚期NSCLC患者的基线样本中，LAG-3升高与较短的无进展生存期显著相关。结论：PD-1，LAG-3和TIM-3在人类NSCLC中具有不同的组织/细胞分布，功能意义和基因组相关性。这些免疫抑制受体在TIL中的表达与显著的活化相关，但也与促凋亡T细胞表型相关。升高的LAG-3表达与对PD-1轴阻断的不敏感性相关，表明这些免疫逃避途径的独立性。
Purpose: To determine the tumor tissue/cell distribution, functional associations, and clinical significance of PD-1, LAG3, and TIM-3 protein expression in human non-small cell lung cancer (NSCLC).Experimental Design: Using multiplexed quantitative immunofluorescence, we performed localized measurements of CD3, PD-1, LAG-3, and TIM-3 protein in > 800 clinically annotated NSCLCs from three independent cohorts represented in tissue microarrays. Associations between the marker's expression and major genomic alterations were studied in The Cancer Genome Atlas NSCLC dataset. Using mass cytometry (CyTOF) analysis of leukocytes collected from 20 resected NSCLCs, we determined the levels, coexpression, and functional profile of PD-1, LAG-3, and TIM-3 expressing immune cells. Finally, we measured the markers in baseline samples from 90 patients with advanced NSCLC treated with PD-1 axis blockers and known response to treatment.Results: PD-1, LAG-3, and TIM-3 were detected in tumorinfiltrating lymphocytes (TIL) from 55%, 41.5%, and 25.3% of NSCLC cases, respectively. These markers showed a prominent association with each other and limited association with major clinicopathologic variables and survival in patients not receiv-ing immunotherapy. Expression of the markers was lower in EGFR-mutated adenocarcinomas and displayed limited association with tumor mutational burden. In single-cell CyTOF analysis, PD-1 and LAG-3 were predominantly localized on T-cell subsets/NKT cells, whereas TIM-3 expression was higher in NK cells and macrophages. Coexpression of PD-1, LAG-3, and TIM-3 was associated with prominent T-cell activation (CD69/CD137), effector function (Granzyme-B), and proliferation (Ki-67), but also with elevated levels of proapoptotic markers (FAS/BIM). LAG-3 and TIM-3 were present in TIL subsets lacking PD-1 expression and showed a distinct functional profile. In baseline samples from 90 patients with advanced NSCLC treated with PD-1 axis blockers, elevated LAG-3 was significantly associated with shorter progressionfree survival.Conclusions: PD-1, LAG-3, and TIM-3 have distinct tissue/cell distribution, functional implications, and genomic correlates in human NSCLC. Expression of these immune inhibitory receptors in TILs is associated with prominent activation, but also with a proapoptotic T-cell phenotype. Elevated LAG-3 expression is associated with insensitivity to PD-1 axis blockade, suggesting independence of these immune evasion pathways.