DataSheet1_Validation of an Accurate Automated Multiplex Immunofluorescence Method for Immuno-Profiling Melanoma.PDF

Multiplex immunofluorescence staining enables the simultaneous detection of multiple immune markers in a single tissue section, and is a useful tool for the identification of specific cell populations within the tumour microenvironment. However, this technology has rarely been validated against standard clinical immunohistology, which is a barrier for its integration into clinical practice. This study sought to validate and investigate the accuracy, precision and reproducibility of a multiplex immunofluorescence compared with immunohistochemistry (IHC), including tissue staining, imaging and analysis, in characterising the expression of immune and melanoma markers in both the tumour and its microenvironment. Traditional chromogenic IHC, single-plex immunofluorescence and multiplex immunofluorescence were each performed on serial tissue sections of a formalin-fixed paraffin-embedded (FFPE) tissue microarray containing metastatic melanoma specimens from 67 patients. The panel included the immune cell markers CD8, CD68, CD16, the immune checkpoint PD-L1, and melanoma tumour marker SOX10. Slides were stained with the Opal™ 7 colour Kit (Akoya Biosciences) on the intelliPATH autostainer (Biocare Medical) and imaged using the Vectra 3.0.5 microscope. Marker expression was quantified using Halo v.3.2.181 (Indica Labs). Comparison of the IHC and single-plex immunofluorescence revealed highly significant positive correlations between the cell densities of CD8, CD68, CD16, PD-L1 and SOX10 marker positive cells (Spearman’s rho = 0.927 to 0.750, p < 0.0001). Highly significant correlations were also observed for all markers between single-plex immunofluorescence and multiplex immunofluorescence staining (Spearman’s rho >0.9, p < 0.0001). Finally, correlation analysis of the three multiplex replicates revealed a high degree of reproducibility between slides (Spearman’s rho >0.940, p < 0.0001). Together, these data highlight the reliability and validity of multiplex immunofluorescence in accurately profiling the tumour and its associated microenvironment using FFPE metastatic melanoma specimens. This validated multiplex panel can be utilised for research evaluating melanoma and its microenvironment, such as studies performed to predict patient response or resistance to immunotherapies.

多重免疫荧光染色（multiplex immunofluorescence staining）可在单张组织切片上同时检测多种免疫标志物，是鉴定肿瘤微环境（tumour microenvironment）内特定细胞群的有效工具。然而，该技术极少与标准临床免疫组织化学（immunohistochemistry, IHC）进行验证对照，这成为其向临床实践转化的一大阻碍。本研究旨在针对免疫组织化学（IHC）验证并评估多重免疫荧光染色的准确性、精密度与重现性，涵盖组织染色、成像及分析环节，以表征肿瘤及其微环境中免疫标志物与黑色素瘤标志物的表达情况。本研究对67例转移性黑色素瘤患者的福尔马林固定石蜡包埋（FFPE）组织芯片的连续组织切片，分别开展传统显色免疫组化、单重免疫荧光染色及多重免疫荧光染色。本次检测的标志物组合涵盖免疫细胞标志物CD8、CD68、CD16、免疫检查点（immune checkpoint）PD-L1，以及黑色素瘤标志物SOX10。使用Akoya Biosciences公司的Opal™ 7色试剂盒，在Biocare Medical公司的intelliPATH全自动染色机上完成玻片染色，并通过Vectra 3.0.5显微镜进行成像。采用Indica Labs公司的Halo v.3.2.181软件对标志物表达量进行定量分析。对免疫组化与单重免疫荧光染色的结果进行比较后发现，CD8、CD68、CD16、PD-L1及SOX10阳性细胞的细胞密度之间均存在极强的正相关，斯皮尔曼相关系数（Spearman’s rho）为0.750~0.927（p<0.0001）。单重免疫荧光与多重免疫荧光染色的所有标志物结果之间也均存在极强的相关性，斯皮尔曼相关系数（Spearman’s rho）>0.9（p<0.0001）。最后，对3次多重免疫荧光重复实验的结果进行相关性分析后发现，不同玻片之间具有极高的重现性，斯皮尔曼相关系数（Spearman’s rho）>0.940（p<0.0001）。综上，本研究结果证实，采用福尔马林固定石蜡包埋的转移性黑色素瘤样本，通过多重免疫荧光染色可精准表征肿瘤及其微环境的特征，证明了该技术的可靠性与有效性。本研究验证的多重标志物检测组合可用于黑色素瘤及其微环境相关研究，例如用于预测患者对免疫治疗的应答或耐药性的相关研究。