免疫组化技术一直被视为理解治疗相关蛋白质表达模式、识别预后和预测生物标志物的黄金标准。在肿瘤治疗的靶向治疗中，患者的选择已成功依赖于标准的显微镜检查法，例如单标记亮场色素免疫组化法。虽然这些结果很有前途，但是除了少数例外，分析单种蛋白质不再提供足够的信息来有效地得出治疗反应的概率。更丰富多样的科学问题促进了高通量和高阶技术的发展，以调查生物标志物表达模式和肿瘤微环境中细胞表型之间的空间相互作用。这种多参数数据分析历来被保留在缺乏免疫组化所提供的空间环境信息的技术中。过去十年间，在多通路荧光免疫组化技术和改进的图像数据分析平台的技术发展和发现中，强调了某些生物标志物之间的空间关系对于理解患者对免疫检查点抑制剂的响应可能性的重要性。同时，个体化医疗已推动临床试验设计和实施的变革，旨在使药物开发和癌症治疗更加高效、精准和经济。在免疫肿瘤学中的精准医学正被数据驱动的方法引导，以获得有关肿瘤及其与免疫系统动态相互作用的洞察力。这点特别必要，考虑到涉及一个以上免疫检查点药物、和/或将其与传统癌症治疗联合使用的试验数量的快速增长。随着多通路方法（例如免疫荧光）将免疫组化技术的边界推向前进，了解此技术的基础及如何将其作为受监管的测试用于识别单种和联合治疗的反应前景变得至关重要。为此，本工作将关注：1）开发临床多通路免疫荧光分析的科学、临床和经济要求；2）Akoya Phenoptics工作流程的特点，以支持预测性测试，包括设计原则、验证和验证需求；3）监管、安全和质量考虑因素；4）通过实验室自主开发测试和受监管的体外诊断设备应用多通路免疫组化。版权所有 © 2023 Locke and Hoyt.

Immunohistochemistry has long been held as the gold standard for understanding the expression patterns of therapeutically relevant proteins to identify prognostic and predictive biomarkers. Patient selection for targeted therapy in oncology has successfully relied upon standard microscopy-based methodologies, such as single-marker brightfield chromogenic immunohistochemistry. As promising as these results are, the analysis of one protein, with few exceptions, no longer provides enough information to draw effective conclusions about the probability of treatment response. More multifaceted scientific queries have driven the development of high-throughput and high-order technologies to interrogate biomarker expression patterns and spatial interactions between cell phenotypes in the tumor microenvironment. Such multi-parameter data analysis has been historically reserved for technologies that lack the spatial context that is provided by immunohistochemistry. Over the past decade, technical developments in multiplex fluorescence immunohistochemistry and discoveries made with improving image data analysis platforms have highlighted the importance of spatial relationships between certain biomarkers in understanding a patient's likelihood to respond to, typically, immune checkpoint inhibitors. At the same time, personalized medicine has instigated changes in both clinical trial design and its conduct in a push to make drug development and cancer treatment more efficient, precise, and economical. Precision medicine in immuno-oncology is being steered by data-driven approaches to gain insight into the tumor and its dynamic interaction with the immune system. This is particularly necessary given the rapid growth in the number of trials involving more than one immune checkpoint drug, and/or using those in combination with conventional cancer treatments. As multiplex methods, like immunofluorescence, push the boundaries of immunohistochemistry, it becomes critical to understand the foundation of this technology and how it can be deployed for use as a regulated test to identify the prospect of response from mono- and combination therapies. To that end, this work will focus on: 1) the scientific, clinical, and economic requirements for developing clinical multiplex immunofluorescence assays; 2) the attributes of the Akoya Phenoptics workflow to support predictive tests, including design principles, verification, and validation needs; 3) regulatory, safety and quality considerations; 4) application of multiplex immunohistochemistry through lab-developed-tests and regulated in vitro diagnostic devices.Copyright © 2023 Locke and Hoyt.