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

项目摘要 预处理活检中PD-L1膜（细胞表面）表达最多， 抗PD-1治疗反应可能性的常用相关性。一般结论 PD-L1表达与肿瘤对抗PD-1治疗的反应之间的关系， 在数万名患有多种肿瘤类型的患者中证实， PD-（L）1.然而，虽然PD-L1表达富集了对抗PD-（L）1的应答，但它不是 足够了。此外，虽然病理学家证明了肿瘤细胞评分的良好再现性， (TC)显色IHC和光学显微镜检测PD-L1表达，重复性差 用于对免疫细胞（IC）上的PD-L1表达进行评分。 已显示可改善PD-L1生物标志物的其他相关特征包括 PD-1与PD-L1的接近度、CD 8 + FoxP 3+细胞的密度以及CD 68和肿瘤标志物 免疫染色，后者有助于识别IC和TC上PD-L1的共表达， 分别一种定量的多重免疫荧光检测， 已经开发了包括PD-L1、PD-1、CD 8、FoxP 3、CD 68、肿瘤标志物 （细胞角蛋白AE 1/3用于非小细胞肺癌，NSCLC），一种泛膜标记物， DAPI。通过该测定，有可能列举关键细胞亚群及其共表达。 数据区.还可以包括空间参数，包括PD-1和PD-2之间的距离。 PD-L1，以前未被纳入预测或预后外科病理学 基于生物素的测定。该mIF测定具有增加的灵敏度和特异性，以响应 与在多发性肿瘤中单独评估PD-L1表达相比，抗PD 1治疗 类型，包括黑色素瘤、NSCLC和默克尔细胞癌等。 本提案旨在对mIF染色试验进行研究中心间验证 以及在四个主要学术站点（Johns霍普金斯，MD）之间的相关锁定算法 安德森，耶鲁大学，和普罗维登斯波特兰医疗中心）。以下分析 将使用来自250例晚期NSCLC患者的验证、发现和验证队列 建立最终检测参数（包括阈值），与以下临床结局相关 抗PD-1治疗该研究的交付成果是一个完善的，多重生物标志物测定， 对抗PD-1的应答/耐药性已在多个学术研究中心得到验证。的 结果将是一种多重IF检测，适合于高级开发，旨在 临床实施。虽然非小细胞肺癌是目前赠款提案的重点， 结果表明，该测定法在许多其它实体瘤类型中也具有很大的价值。

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.