Patrick G. Johnston Centre for Cancer Research, School of Medicine, Dentistry and Biomedical Science, Queen's University Belfast, Northern Ireland, UK.
Department of Cellular Pathology, Royal Victoria Hospital, Belfast Health and Social Care trust, Belfast, UK.
Mod Pathol. 2022 Apr;35(4):564-576. doi: 10.1038/s41379-021-00953-0. Epub 2021 Nov 3.
Colorectal cancer (CRC) has one of the highest cancer incidences and mortality rates. In stage III, postoperative chemotherapy benefits <20% of patients, while more than 50% will develop distant metastases. Biomarkers for identification of patients at increased risk of disease recurrence following adjuvant chemotherapy are currently lacking. In this study, we assessed immune signatures in the tumor and tumor microenvironment (TME) using an in situ multiplexed immunofluorescence imaging and single-cell analysis technology (Cell DIVE) and evaluated their correlations with patient outcomes. Tissue microarrays (TMAs) with up to three 1 mm diameter cores per patient were prepared from 117 stage III CRC patients treated with adjuvant fluoropyrimidine/oxaliplatin (FOLFOX) chemotherapy. Single sections underwent multiplexed immunofluorescence staining for immune cell markers (CD45, CD3, CD4, CD8, FOXP3, PD1) and tumor/cell segmentation markers (DAPI, pan-cytokeratin, AE1, NaKATPase, and S6). We used annotations and a probabilistic classification algorithm to build statistical models of immune cell types. Images were also qualitatively assessed independently by a Pathologist as 'high', 'moderate' or 'low', for stromal and total immune cell content. Excellent agreement was found between manual assessment and total automated scores (p < 0.0001). Moreover, compared to single markers, a multi-marker classification of regulatory T cells (Tregs: CD3+/CD4+FOXP3+/PD1-) was significantly associated with disease-free survival (DFS) and overall survival (OS) (p = 0.049 and 0.032) of FOLFOX-treated patients. Our results also showed that PD1- Tregs rather than PD1+ Tregs were associated with improved survival. These findings were supported by results from an independent FOLFOX-treated cohort of 191 stage III CRC patients, where higher PD1- Tregs were associated with an increase overall survival (p = 0.015) for CD3+/CD4+/FOXP3+/PD1-. Overall, compared to single markers, multi-marker classification provided more accurate quantitation of immune cell types with stronger correlations with outcomes.
结直肠癌（CRC）是发病率和死亡率最高的癌症之一。在 III 期，术后化疗使 <20%的患者受益，而超过 50%的患者会发展为远处转移。目前缺乏用于识别辅助化疗后疾病复发风险增加的患者的生物标志物。在这项研究中，我们使用原位多重免疫荧光成像和单细胞分析技术（Cell DIVE）评估了肿瘤和肿瘤微环境（TME）中的免疫特征，并评估了它们与患者预后的相关性。从接受辅助氟尿嘧啶/奥沙利铂（FOLFOX）化疗的 117 名 III 期 CRC 患者中制备了多达每个患者 3 个 1mm 直径核心的组织微阵列（TMA）。单个切片进行了免疫细胞标志物（CD45、CD3、CD4、CD8、FOXP3、PD1）和肿瘤/细胞分割标志物（DAPI、细胞角蛋白、AE1、NaKATPase 和 S6）的多重免疫荧光染色。我们使用注释和概率分类算法构建了免疫细胞类型的统计模型。病理学家还独立地对图像进行了定性评估，分为“高”、“中”或“低”，用于评估基质和总免疫细胞含量。手动评估与总自动评分之间存在极好的一致性（p<0.0001）。此外，与单一标志物相比，调节性 T 细胞（Tregs：CD3+/CD4+FOXP3+/PD1-）的多标志物分类与 FOLFOX 治疗患者的无病生存率（DFS）和总生存率（OS）显著相关（p=0.049 和 0.032）。我们的结果还表明，PD1-Tregs 而非 PD1+Tregs 与改善的生存相关。这些发现得到了来自 191 名接受 FOLFOX 治疗的 III 期 CRC 患者的独立队列的结果的支持，其中更高的 PD1-Tregs 与 CD3+/CD4+/FOXP3+/PD1-的总生存率增加相关（p=0.015）。总的来说，与单一标志物相比，多标志物分类提供了更准确的免疫细胞类型定量，与结果的相关性更强。
