多重免疫荧光成像（mIF）可为肿瘤免疫表型提供多个免疫标记物的全面定量和空间信息。然而，由于预分析异质性，对来自多个机构的临床试验样本进行大规模应用具有一定挑战性。本研究报告了迄今为止最大的临床试验样本多参数免疫表型研究的分析方法。我们分析了来自QUASAR 2和SCOT两个临床试验的240多个机构收集的3,545个结直肠癌组织微阵列（TMA）点中的12,592个点，这些点通过mIF染色进行了CD4、CD8、CD20、CD68、FoxP3、泛细胞角蛋白和DAPI的标记。 TMA切片进行了多光谱成像，并通过基于细胞和基于像素的标记物分析进行了分析。我们开发了一种自适应阈值方法来考虑TMA分析中的片间和片内强度变化。应用这种方法有效地减轻了片间和片内强度变化，改善了与使用单一全局阈值方法相比的图像分析结果。我们的mIF分析方法得出的CD8数据与后续章节中由单峰染色免疫组织化学CD8数据相关联，表明了我们方法的有效性（Spearman等级相关系数介于0.63和0.66之间，p≪0.01），与当前的黄金标准分析方法相比。细胞基础和像素基础分析结果之间的相关性评估确认了两种分析方法的等价性（ρ>0.8，p≪0.01，除了上皮区域中的CD20）。这些数据表明，我们的自适应阈值方法可以通过数字病理学对mIF染色的临床试验TMA数据集进行大规模的精密免疫表型分析。©2023年作者。《病理学杂志：临床研究》由英国和爱尔兰病理学会和John Wiley & Sons Ltd.出版。

Multiplex immunofluorescence (mIF) imaging can provide comprehensive quantitative and spatial information for multiple immune markers for tumour immunoprofiling. However, application at scale to clinical trial samples sourced from multiple institutions is challenging due to pre-analytical heterogeneity. This study reports an analytical approach to the largest multi-parameter immunoprofiling study of clinical trial samples to date. We analysed 12,592 tissue microarray (TMA) spots from 3,545 colorectal cancers sourced from more than 240 institutions in two clinical trials (QUASAR 2 and SCOT) stained for CD4, CD8, CD20, CD68, FoxP3, pan-cytokeratin, and DAPI by mIF. TMA slides were multi-spectrally imaged and analysed by cell-based and pixel-based marker analysis. We developed an adaptive thresholding method to account for inter- and intra-slide intensity variation in TMA analysis. Applying this method effectively ameliorated inter- and intra-slide intensity variation improving the image analysis results compared with methods using a single global threshold. Correlation of CD8 data derived by our mIF analysis approach with single-plex chromogenic immunohistochemistry CD8 data derived from subsequent sections indicates the validity of our method (Spearman's rank correlation coefficients ρ between 0.63 and 0.66, p ≪ 0.01) as compared with the current gold standard analysis approach. Evaluation of correlation between cell-based and pixel-based analysis results confirms equivalency (ρ > 0.8, p ≪ 0.01, except for CD20 in the epithelial region) of both analytical approaches. These data suggest that our adaptive thresholding approach can enable analysis of mIF-stained clinical trial TMA datasets by digital pathology at scale for precision immunoprofiling.© 2023 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.