Abstract：
OBJECTIVE: Pathologists often use immunohistochemical staining of the proliferation marker Ki67 in their diagnostic assessment of melanocytic lesions. However, the interpretation of Ki67 can be challenging. We propose a new workflow to improve the diagnostic utility of the Ki67-index. In this workflow, Ki67 is combined with the melanocytic tumour-cell marker SOX10 in a Ki67/SOX10 double nuclear stain. The Ki67-index is then quantified automatically using digital image analysis (DIA). The aim of this study was to optimise and test three different multiplexing methods for Ki67/SOX10 double nuclear staining.
METHODS: Multiplex immunofluorescence (mIF), multiplex immunohistochemistry (mIHC), and multiplexed immunohistochemical consecutive staining on single slide (MICSSS) were optimised for Ki67/SOX10 double nuclear staining. DIA applications were designed for automated quantification of the Ki67-index. The methods were tested on a pilot case-control cohort of benign and malignant melanocytic lesions (n = 23).
RESULTS: Using the Ki67/SOX10 double nuclear stain, malignant melanocytic lesions could be completely distinguished from benign lesions by the Ki67-index. The Ki67-index cut-offs were 1.8% (mIF) and 1.5% (mIHC and MICSSS). The AUC of the automatically quantified Ki67-index based on double nuclear staining was 1.0 (95% CI: 1.0;1.0), whereas the AUC of conventional Ki67 single-stains was 0.87 (95% CI: 0.71;1.00).
CONCLUSIONS: The novel Ki67/SOX10 double nuclear stain highly improved the diagnostic precision of Ki67 interpretation. Both mIHC and mIF were useful methods for Ki67/SOX10 double nuclear staining, whereas the MICSSS method had challenges in the current setting. The Ki67/SOX10 double nuclear stain shows potential as a valuable diagnostic aid for melanocytic lesions.
METHODS: Multiplex immunofluorescence (mIF), multiplex immunohistochemistry (mIHC), and multiplexed immunohistochemical consecutive staining on single slide (MICSSS) were optimised for Ki67/SOX10 double nuclear staining. DIA applications were designed for automated quantification of the Ki67-index. The methods were tested on a pilot case-control cohort of benign and malignant melanocytic lesions (n = 23).
RESULTS: Using the Ki67/SOX10 double nuclear stain, malignant melanocytic lesions could be completely distinguished from benign lesions by the Ki67-index. The Ki67-index cut-offs were 1.8% (mIF) and 1.5% (mIHC and MICSSS). The AUC of the automatically quantified Ki67-index based on double nuclear staining was 1.0 (95% CI: 1.0;1.0), whereas the AUC of conventional Ki67 single-stains was 0.87 (95% CI: 0.71;1.00).
CONCLUSIONS: The novel Ki67/SOX10 double nuclear stain highly improved the diagnostic precision of Ki67 interpretation. Both mIHC and mIF were useful methods for Ki67/SOX10 double nuclear staining, whereas the MICSSS method had challenges in the current setting. The Ki67/SOX10 double nuclear stain shows potential as a valuable diagnostic aid for melanocytic lesions.
摘要：
目的：病理学家在黑素细胞病变的诊断评估中经常使用增殖标志物Ki67的免疫组织化学染色。然而,Ki67的解释可能具有挑战性。我们提出了一种新的工作流程来提高Ki67指数的诊断效用。在此工作流中，Ki67在Ki67/SOX10双核染色中与黑素细胞肿瘤细胞标志物SOX10结合。然后使用数字图像分析(DIA)自动量化Ki67指数。这项研究的目的是优化和测试Ki67/SOX10双核染色的三种不同的多路复用方法。
方法：多重免疫荧光（mIF），多重免疫组织化学（mIHC），和多重免疫组织化学连续染色在单个载玻片(MICSSS)上被优化用于Ki67/SOX10双核染色。DIA应用被设计用于Ki67指数的自动定量。该方法在良性和恶性黑素细胞病变的先导病例对照队列中进行了测试（n=23）。
结果：使用Ki67/SOX10双核染色，恶性黑素细胞病变可以通过Ki67指数与良性病变完全区分.Ki67指数截止值分别为1.8%(mIF)和1.5%(mIHC和MICSSS)。基于双核染色的自动定量Ki67指数的AUC为1.0（95％CI：1.0;1.0），而常规Ki67单一染色的AUC为0.87(95%CI:0.71;1.00)。
结论：新的Ki67/SOX10双核染色法大大提高了Ki67解释的诊断精度。mIHC和mIF都是Ki67/SOX10双核染色的有用方法，而MICSSS方法在当前设置中存在挑战。Ki67/SOX10双核染色显示出作为黑素细胞病变的有价值的诊断辅助工具的潜力。
方法：多重免疫荧光（mIF），多重免疫组织化学（mIHC），和多重免疫组织化学连续染色在单个载玻片(MICSSS)上被优化用于Ki67/SOX10双核染色。DIA应用被设计用于Ki67指数的自动定量。该方法在良性和恶性黑素细胞病变的先导病例对照队列中进行了测试（n=23）。
结果：使用Ki67/SOX10双核染色，恶性黑素细胞病变可以通过Ki67指数与良性病变完全区分.Ki67指数截止值分别为1.8%(mIF)和1.5%(mIHC和MICSSS)。基于双核染色的自动定量Ki67指数的AUC为1.0（95％CI：1.0;1.0），而常规Ki67单一染色的AUC为0.87(95%CI:0.71;1.00)。
结论：新的Ki67/SOX10双核染色法大大提高了Ki67解释的诊断精度。mIHC和mIF都是Ki67/SOX10双核染色的有用方法，而MICSSS方法在当前设置中存在挑战。Ki67/SOX10双核染色显示出作为黑素细胞病变的有价值的诊断辅助工具的潜力。
