Supplementary Material for: Antigen Expression Varies Significantly between Molecular Subgroups of Acute Myeloid Leukemia Patients: Clinical Applicability Is Hampered by Establishment of Relevant Cutoffs

<b><i>Introduction:</i></b> In this single-center study of 268 acute myeloid leukemia (AML) patients, we have tested if a subset of 4 routinely employed immunophenotypic stem cell-associated markers correlated with the presence of recurrently mutated genes and if the markers were predictive for mutational status. <b><i>Methods:</i></b> Immunophenotypic data from 268 diagnostic AML samples obtained in 2009–2018 were analyzed retrospectively for the antigens CD34, CD117, CD123, and CLEC12A. Correlation between immunophenotypes and mutations was analyzed by Fischer’s exact test. Clinical applicability of the markers for predicting mutational status was evaluated by receiver operating characteristics analyses, where an area under the curve (AUC) of at least 0.85 was accepted as clinically relevant. <b><i>Results:</i></b> For a number of genes, the antigen expression differed significantly between mutated and wild-type gene expression. Despite low AUCs, CD123 and CLEC12A correlated with <i>FLT3</i>+<i>NPM1−</i> and <i>FLT3</i>+<i>NPM1</i>+. Three subsets met the AUC requirements (CD34+, CD34+CD117+, and CD34−CD117+) for predicting <i>FLT3−NPM1</i>+ or <i>FLT3</i>+<i>NPM1</i>+. <b><i>Conclusion:</i></b> The value of immunophenotypes as surrogate markers for mutational status in AML seems limited when employing CD123 and CLEC12A in combination with CD34 and CD117. Defining relevant cutoffs for given markers is challenging and hampered by variation between laboratories and patient groups.

**引言：** 本项针对268例急性髓系白血病（acute myeloid leukemia, AML）患者的单中心研究中，我们验证了4种常规应用的免疫表型干细胞相关标志物组成的亚群是否与复发性突变基因的存在相关，同时评估了这些标志物对基因突变状态的预测效能。
**方法：** 本研究对2009年至2018年间获取的268例初诊急性髓系白血病样本的免疫表型数据进行回顾性分析，检测的靶抗原包括CD34、CD117、CD123及CLEC12A。采用费希尔精确检验（Fischer’s exact test）分析免疫表型与基因突变的相关性。通过受试者工作特征（receiver operating characteristics, ROC）曲线分析评估上述标志物预测突变状态的临床应用价值，将曲线下面积（area under the curve, AUC）≥0.85认定为具有临床相关性。
**结果：** 对于部分基因而言，突变型与野生型对应的抗原表达水平存在显著差异。尽管整体曲线下面积数值偏低，但CD123与CLEC12A的表达分别与FLT3+NPM1−及FLT3+NPM1+基因型存在显著相关性。另有3种标志物亚群（CD34+、CD34+CD117+及CD34−CD117+）满足预测FLT3−NPM1+或FLT3+NPM1+基因型的曲线下面积阈值要求。
**结论：** 将CD123、CLEC12A与CD34、CD117联合使用时，免疫表型作为急性髓系白血病突变状态替代标志物的临床价值较为有限。针对特定标志物确定合理的临界值颇具挑战性，且会受到不同实验室及患者群体间异质性的限制。