Data_Sheet_1_Reliability of CD44, CD24, and ALDH1A1 immunohistochemical staining: Pathologist assessment compared to quantitative image analysis.doc

BackgroundThe data on the expression of stem cell markers CD44, CD24, and ALDH1A1 in the breast tissue of cancer-free women is very limited and no previous studies have explored the agreement between pathologist and computational assessments of these markers. We compared the immunohistochemical (IHC) expression assessment for CD44, CD24, and ALDH1A1 by an expert pathologist with the automated image analysis results and assessed the homogeneity of the markers across multiple cores pertaining to each woman.
MethodsWe included 81 cancer-free women (399 cores) with biopsy-confirmed benign breast disease in the Nurses’ Health Study (NHS) and NHSII cohorts. IHC was conducted with commercial antibodies [CD44 (Dako, Santa Clara, CA, USA) 1:25 dilution; CD24 (Invitrogen, Waltham, MA, USA) 1:200 dilution and ALDH1A1 (Abcam, Cambridge, United Kingdom) 1:300 dilution]. For each core, the percent positivity was quantified by the pathologist and Definiens Tissue Studio. Correlations between pathologist and computational scores were evaluated with Spearman correlation (for categorical positivity: 0, >0–<1, 1–10, >10–50, and >50%) and sensitivity/specificity (for binary positivity defined with 1 and 10% cut-offs), using the pathologist scores as the gold standard. Expression homogeneity was examined with intra-class correlation (ICC). Analyses were stratified by core [normal terminal duct-lobular units (TDLUs), benign lesions] and tissue type (epithelium, stroma).
ResultsSpearman correlation between pathologist and Definiens ranged between 0.40–0.64 for stroma and 0.66–0.68 for epithelium in normal TDLUs cores and between 0.24–0.60 for stroma and 0.61–0.64 for epithelium in benign lesions. For stroma, sensitivity and specificity ranged between 0.92–0.95 and 0.24–0.60, respectively, with 1% cut-off and between 0.43–0.88 and 0.73–0.85, respectively, with 10% cut-off. For epithelium, 10% cut-off resulted in better estimates for both sensitivity and specificity. ICC between the cores was strongest for CD44 for both stroma and epithelium in normal TDLUs cores and benign lesions (range 0.74–0.80). ICC for CD24 and ALDH1A ranged between 0.42–0.63 and 0.44–0.55, respectively.
ConclusionOur findings show that computational assessments for CD44, CD24, and ALDH1A1 exhibit variable correlations with manual assessment. These findings support the use of computational platforms for IHC evaluation of stem cell markers in large-scale epidemiologic studies. Pilot studies maybe also needed to determine appropriate cut-offs for defining staining positivity.

研究背景
目前关于无癌女性乳腺组织中干细胞标志物CD44、CD24及ALDH1A1表达情况的数据十分有限，且尚无研究探讨病理学家与自动化计算方法对这些标志物评估结果的一致性。本研究对比了资深病理学家对CD44、CD24及ALDH1A1的免疫组化（immunohistochemical, IHC）表达评估结果与自动化图像分析结果，并评估了每名女性对应多个组织芯中标志物的表达均一性。

研究方法
本研究纳入护士健康研究（Nurses’ Health Study, NHS）及护士健康研究II（NHSII）队列中81名经活检证实患有良性乳腺疾病的无癌女性（共399个组织芯）。采用商品化抗体开展免疫组化染色：CD44（Dako，美国加利福尼亚州圣克拉拉，稀释比例1:25）、CD24（Invitrogen，美国马萨诸塞州沃尔瑟姆，稀释比例1:200）及ALDH1A1（Abcam，英国剑桥，稀释比例1:300）。针对每个组织芯，由病理学家及Definiens Tissue Studio软件量化阳性细胞百分比。以病理学家评分为金标准，采用斯皮尔曼相关分析（针对分类阳性等级：0、>0~<1、1~10、>10~50及>50%）及灵敏度/特异度（针对以1%和10%作为截断值界定的二元阳性）评估病理学家评分与自动化计算评分间的相关性。采用组内相关系数（intra-class correlation, ICC）分析标志物的表达均一性。分析按组织芯类型[正常末端导管小叶单位（terminal duct-lobular units, TDLUs）、良性病变]及组织类型（上皮组织、间质）进行分层。

研究结果
在正常TDLUs组织芯中，病理学家评分与Definiens软件分析结果的斯皮尔曼相关系数在间质中为0.40~0.64，在上皮组织中为0.66~0.68；在良性病变组织芯中，间质相关系数为0.24~0.60，上皮组织为0.61~0.64。针对间质组织，以1%作为截断值时，灵敏度与特异度分别为0.92~0.95和0.24~0.60；以10%作为截断值时，两者分别为0.43~0.88和0.73~0.85。针对上皮组织，采用10%截断值时，灵敏度与特异度的估算效果更优。在正常TDLUs组织芯及良性病变中，CD44在间质与上皮组织中的组内相关系数最高（范围0.74~0.80）；CD24与ALDH1A1的组内相关系数分别为0.42~0.63和0.44~0.55。

研究结论
本研究结果显示，CD44、CD24及ALDH1A1的自动化评估结果与人工评估结果间存在程度不一的相关性。上述结果支持在大规模流行病学研究中采用自动化分析平台开展干细胞标志物的免疫组化评估。此外，或需开展预试验以确定界定染色阳性的合适截断值。