Nodality, Inc, South San Francisco, California, United States of America.
PLoS One. 2010 Oct 27;5(10):e13543. doi: 10.1371/journal.pone.0013543.
Molecular characterization of the FMS-like tyrosine kinase 3 receptor (FLT3) in cytogenetically normal acute myeloid leukemia (AML) has recently been incorporated into clinical guidelines based on correlations between FLT3 internal tandem duplications (FLT3-ITD) and decreased disease-free and overall survival. These mutations result in constitutive activation of FLT3, and FLT3 inhibitors are currently undergoing trials in AML patients selected on FLT3 molecular status. However, the transient and partial responses observed suggest that FLT3 mutational status alone does not provide complete information on FLT3 biological activity at the individual patient level. Examination of variation in cellular responsiveness to signaling modulation may be more informative.
METHODOLOGY/PRINCIPAL FINDINGS: Using single cell network profiling (SCNP), cells were treated with extracellular modulators and their functional responses were quantified by multiparametric flow cytometry. Intracellular signaling responses were compared between healthy bone marrow myeloblasts (BMMb) and AML leukemic blasts characterized as FLT3 wild type (FLT3-WT) or FLT3-ITD. Compared to healthy BMMb, FLT3-WT leukemic blasts demonstrated a wide range of signaling responses to FLT3 ligand (FLT3L), including elevated and sustained PI3K and Ras/Raf/Erk signaling. Distinct signaling and apoptosis profiles were observed in FLT3-WT and FLT3-ITD AML samples, with more uniform signaling observed in FLT3-ITD AML samples. Specifically, increased basal p-Stat5 levels, decreased FLT3L induced activation of the PI3K and Ras/Raf/Erk pathways, decreased IL-27 induced activation of the Jak/Stat pathway, and heightened apoptotic responses to agents inducing DNA damage were observed in FLT3-ITD AML samples. Preliminary analysis correlating these findings with clinical outcomes suggests that classification of patient samples based on signaling profiles may more accurately reflect FLT3 signaling deregulation and provide additional information for disease characterization and management.
CONCLUSIONS/SIGNIFICANCE: These studies show the feasibility of SCNP to assess modulated intracellular signaling pathways and characterize the biology of individual AML samples in the context of genetic alterations.
在细胞遗传学正常的急性髓细胞白血病（AML）中，对 FMS 样酪氨酸激酶 3 受体（FLT3）的分子特征分析最近已被纳入临床指南，这是基于 FLT3 内部串联重复（FLT3-ITD）与无病生存和总生存时间减少之间的相关性。这些突变导致 FLT3 的组成性激活，目前正在对根据 FLT3 分子状态选择的 AML 患者进行 FLT3 抑制剂试验。然而，观察到的短暂和部分反应表明，FLT3 突变状态本身并不能为个体患者水平的 FLT3 生物学活性提供完整信息。检查细胞对信号调节的反应变化可能更具信息量。
方法/主要发现：使用单细胞网络分析（SCNP），用细胞外调节剂处理细胞，并通过多参数流式细胞术定量其功能反应。比较了健康骨髓髓样前体细胞（BMMb）和 FLT3 野生型（FLT3-WT）或 FLT3-ITD 的 AML 白血病细胞的细胞内信号反应。与健康 BMMb 相比，FLT3-WT 白血病细胞对 FLT3 配体（FLT3L）表现出广泛的信号反应范围，包括升高和持续的 PI3K 和 Ras/Raf/Erk 信号。在 FLT3-WT 和 FLT3-ITD AML 样本中观察到不同的信号和凋亡谱，在 FLT3-ITD AML 样本中观察到更均匀的信号。具体而言，在 FLT3-ITD AML 样本中观察到增加的基础 p-Stat5 水平、FLT3L 诱导的 PI3K 和 Ras/Raf/Erk 途径激活减少、IL-27 诱导的 Jak/Stat 途径激活减少以及诱导 DNA 损伤的药物引起的凋亡反应增加。对这些发现与临床结果进行初步分析表明，基于信号谱对患者样本进行分类可能更准确地反映 FLT3 信号失调，并为疾病特征描述和管理提供更多信息。
结论/意义：这些研究表明，SCNP 可用于评估调节后的细胞内信号通路，并在遗传改变的背景下描述个体 AML 样本的生物学特性。
