Cellularly resolved molecular pathway assessment in biopsies via spectral imaging

描述（由申请人提供）： 这是一个快速通道应用程序，可在癌症活检中提供可靠的细胞分辨分子通路评估，以协助药物开发和提供患者特定的预后和治疗指导（“个性化医疗”）。组织的主题是，适当的分析单位应该是单个细胞，而不是平均肿瘤提取物。为此，新技术将与谨慎的方法开发相结合。光谱成像和先进的图像分析工具将允许在完整组织切片的细胞和亚细胞水平上进行多靶点免疫组织化学（IHC）和/或免疫荧光（IF）检测。CRi开发的图像处理和机器学习工具提供了自动化和复杂的定量选项。多重染色方案将产生独立的，潜在的化学计量标记与组合的IHC和IF。将仔细评估所有潜在标志物对组织处理变化的敏感性;有些可能是稳健的，适用于存档组织研究，其他则太不稳定。将测试超声辅助固定法保留此类不稳定表位用于前瞻性获得的组织的能力。在逐个细胞的基础上，将在组织切片中检测到四种或更多种途径相关蛋白，即使是共定位的并且具有光谱重叠的标记。还将跟踪途径分子的协调亚细胞位置，同时评估细胞表面受体（例如，EGFR、VEGF、Her 2-neu）、下游信号传导蛋白和磷蛋白（例如，pAKT，pERK），核蛋白（例如，ER，Ki 67），和新的球员，如蛋白质折叠介质（如BIP 1）。 该项目将结合联合收割机优化的组织方案，多重IHC/IF试剂盒和独特的机器学习图像分析，可用于自动化区域检测和标记定量。所有这些都依赖于CRi的多光谱成像方法，用于在组织切片中逐个细胞和细胞隔室的基础上评估多种分析物。我们的合作者将为早期方法开发提供小动物肿瘤模型，临床癌症活检中途径蛋白的多重免疫组化标记，从靶向途径的临床药物试验中获得存档和前瞻性获得的组织，高度信息化的存档组织微阵列，获得验证的激活特异性抗体，超快速组织固定和生物统计学支持。“可交付成果”将是一套适合临床使用的产品，可以在完整组织背景下提供急需的基于单细胞的通路状态的有效信息，以支持药物开发工作并提供分子重点的患者护理。 意义和奠定叙事：在单个临床组织制备中，在逐个细胞的基础上定量评估多个分子靶标和途径的能力在当前的个性化医疗工具箱中缺失，其缺乏将新药和候选药物与特定患者匹配的良好手段。传统的分子病理学方法通常限于对给定组织切片的单一免疫组织化学（IHC）测试或昂贵且耗时的蛋白质组学或表达阵列方法（其不能直接报告癌细胞群和亚群中的通路激活状态）。多重IHC（包括免疫荧光）结合优化的样品处理方案以保留途径蛋白和先进的图像分析将能够明确检测活性信号传导途径，从而有利于药物研究选择患者进行更好的靶向试验和监测反应，以及诊断，治疗选择和监测反应的临床实践（即，theranostics）。

DESCRIPTION (provided by applicant): This is a Fast-Track application to provide reliable, cellularly resolved molecular pathway assessment in cancer biopsies to assist pharmaceutical drug development and provision of patient-specific prognosis and therapy guidance ("personalized medicine"). The organizing theme is that the appropriate unit of analysis should be the individual cell as opposed to averaged tumor extracts. To this end, novel technologies will be coupled with careful methods-development. Spectral imaging and advanced image analysis tools will permit multi-target immunohistochemical (IHC) and/or immunofluorescence (IF) detection, at the cellular and subcellular level in intact tissue sections. CRi-developed image processing and machine-learning tools provide automation and sophisticated quantitation options. Multiplexed staining protocols will yield independent, potentially stoichiometric labeling with combined IHC and IF. The sensitivity of all potential markers to variations in tissue handling will be carefully assessed; some may be robust and suitable for archival tissue studies, others will be too labile. Ultrasound-assisted fixation will be tested for its ability to preserve such labile epitopes for use in prospectively acquired tissues. Four or more pathway-related proteins will be detected in tissue sections, on a cell-by-cell basis, even if co-localized and with spectrally overlapping labels. The coordinated subcellular location of the pathway molecules will be also tracked, with simultaneous assessment of cell-surface receptors (e.g., EGFR, VEGF, Her2-neu), downstream signaling proteins and phosphoproteins (e.g., pAKT, pERK), nuclear proteins (e.g., ER, Ki67), and novel players such as protein-folding mediators (e.g. BIP1). The project will combine optimized tissue protocols, multiplexed IHC/IF reagent kits, and unique machine- learning image analysis that can be used to automate region-detection and label-quantitation. All these depend on CRi's multispectral imaging approaches for assessing multiple analytes on a cell-by-cell and cell- compartment basis in tissue sections. Our collaborators will provide small-animal tumor models for early methods development, multiplexed immunohistochemical labeling of pathway proteins in clinical cancer biopsies, access to archived and prospectively acquired tissues from pathway-targeting clinical drug trials, highly informative archival tissue microarrays, access to validated, activation-specific antibodies, ultra-fast tissue fixation, and biostatistics support. The "deliverable" will be a suite of products suitable for clinical use that can provide much-needed valid information on single-cell-based pathway status in an intact tissue context to support pharmaceutical drug development efforts and provide molecularly focused patient care. Significance and lay narrative: The ability to quantitatively evaluate multiple molecular targets and pathways on a cell-by-cell basis, in a single preparation of clinical tissue, is missing from the current toolbox of personalized medicine, which lacks good means of matching novel drugs and drug candidates to specific patients. Conventional molecular pathology methods are typically limited to a single immunohistochemical (IHC) test on a given tissue section or to expensive and time-consuming proteomics or expression-array approaches (which cannot directly report out pathway activation status in cancer cell populations and subpopulations). Multiplexed IHC (including immuno-fluorescence) combined with optimized sample handling protocols to retain pathway proteins and advanced image analysis will enable the unambiguous detection of active signaling pathways, benefiting pharmaceutical research in the selection of patients for better targeted trials and in the monitoring of response, and clinical practice for diagnosis, therapy selection, and monitoring response (i.e., theranostics).