Analytic and Clinical Validation of a 7-plex MIF Assay for Predictive Response of Advanced NSCLC to Anti-PD-1 based Therapy

项目总结 PD-L1膜(细胞表面)在预处理活检组织中表达最多 常用的方法与抗PD-1治疗反应的可能性相关。总的发现是 PD-L1的表达与抗PD-1治疗的肿瘤反应之间的关系已被研究 在数以万计的多种肿瘤类型的患者中得到证实，这些患者接受了抗- PD-(L)1。然而，尽管PD-L1的表达因对抗PD-(L)1的应答而丰富，但它不是 足够了。此外，虽然病理学家展示了对肿瘤细胞进行评分的良好重复性 (TC)PD-L1的显色免疫组化和光学显微镜表达，其重复性较差 用于免疫细胞(IC)PD-L1表达的评分。 其他已被证明改善PD-L1生物标志物的相关特征包括 PD-1与PD-L1的亲和力、CD8+FoxP3+细胞密度、CD68和肿瘤标志物 免疫染色，后者有助于识别PD-L1在ICs和TCS上的共表达， 分别进行了分析。一种捕捉所有这些的定量多重免疫荧光分析 已开发的功能包括PD-L1、PD-1、CD8、FoxP3、CD68，一种肿瘤标记物 (非小细胞肺癌的细胞角蛋白AE1/3)、泛膜标记物和 DAPI。通过这种分析，有可能列举关键的细胞亚群及其共表达 配置文件。还可以包括空间参数，包括PD-1和PD-1之间的距离 PD-L1，以前没有包括在预测性或预后的外科病理学中 以样本为基础的分析。该MIF检测方法具有更高的敏感性和特异性 多发性肿瘤中抗PD1治疗与单独检测PD-L1表达的比较 类型，包括黑色素瘤、非小细胞肺癌和默克尔细胞癌等。 这项建议的目的是进行MIF染色分析的站点间验证 和四个主要学术站点(约翰·霍普金斯，马里兰州)之间的关联锁定算法 安德森、耶鲁大学和普罗维登斯波特兰医疗中心)。以下是分析 将使用来自250名晚期非小细胞肺癌患者的验证、发现和验证队列 确定最终化验参数(包括阈值)，与以下临床结果相关联 抗PD-1为主的治疗。这项研究的成果是一种精炼的、多元化的生物标志物检测方法 对抗PD-1抗体的反应/抵抗力已在多个学术站点得到验证。这个 结果将是一种多重IF检测，适合于针对以下目标的高级开发 临床实施。虽然NSCLC是当前赠款提案的重点，但初步 结果表明，该检测方法在许多其他实体瘤类型中也有很大的应用价值。

PROJECT SUMMARY PD-L1 membranous (cell surface) expression in pretreatment biopsies is the most commonly used correlate of the likelihood of response to anti-PD-1 therapy. The general finding of an association of PD-L1 expression with tumor response to anti-PD-1 therapy has been substantiated across tens of thousands of patients with numerous tumor types treated with anti- PD-(L)1. However, while PD-L1 expression enriches for response to anti-PD-(L)1, it is not sufficient. Additionally, while pathologists demonstrate good reproducibility for scoring tumor cell (TC) PD-L1 expression by chromogenic IHC and light microscopy, they have poor reproducibility for scoring PD-L1 expression on immune cells (IC). Other related features which have been shown to improve on the PD-L1 biomarker include the proximity of PD-1 to PD-L1, the density of CD8+FoxP3+ cells, and CD68 and tumor marker immunostains, the latter of which help identify co-expression of PD-L1 on ICs and TCs, respectively. A quantitative multiplex immunofluorescence assay which captures all of these features has been developed and includes PD-L1, PD-1, CD8, FoxP3, CD68, a tumor marker (cytokeratin AE1/3 for non-small cell lung carcinoma, NSCLC), a pan-membrane marker and DAPI. Through this assay, it is possible to enumerate key cellular subsets and their co-expression profiles. It is also possible to include spatial parameters, including the distance between PD-1 and PD-L1, which have not previously been included in predictive or prognostic surgical pathology specimen-based assays. This mIF assay has increased sensitivity and specificity for response to anti-PD1 therapy when compared to the assessment of PD-L1 expression alone in multiple tumor types, including melanoma, NSCLC, and Merkel cell carcinoma, amongst others. The purpose of this proposal is to perform inter-site validation of the mIF staining assay and associated lock-down algorithm amongst four major academic sites (Johns Hopkins, MD Anderson, Yale University, and Providence Portland Medical Center). Following analytical validation, discovery and validation cohorts from 250 patients with advanced NSCLC will be used to establish final assay parameters (including thresholds), linked to clinical outcomes following anti-PD-1-based therapy. The deliverable of the study is a refined, multiplex biomarker assay for response/resistance to anti-PD-1 that has been validated across multiple academic sites. The result will be a multiplex IF assay that is suitably staged for advanced development aimed at clinical implementation. While NSCLC is the focus of the current grant proposal, preliminary results suggest that this assay will also have great value in numerous other solid tumor types.