A Leukemic Stem Cell Expression Signature is Associated with Clinical Outcomes in Acute Myeloid Leukemia. Homo sapiens

Context: In many cancers, specific subpopulations of cells appear to be uniquely capable of initiating and maintaining tumors. The strongest support for this cancer stem cell model comes from transplantation assays in immune-deficient mice indicating that human acute myeloid leukemia (AML) is organized as a cellular hierarchy driven by self-renewing leukemia stem cells (LSC). This model has significant implications for the development of novel therapies, but its clinical significance remains unclear. Objective: To measure associations between a leukemic stem cell expression signature and clinical outcomes in AML. Design, Setting, and Patients: We defined a gene expression signature of LSC-enriched subpopulations from primary AML patient samples and xenografts, based on a functional definition in transplantation assays. Using previously published gene expression data of bulk AML from four independent cohorts totaling 1047 patients, we performed a retrospective cohort study, defining an LSC score and evaluating it for associations with known predictors of risk including cytogenetic subtype and molecular mutations, and as an independent prognostic factor. Main Outcome Measures: Reproducible associations between a leukemic stem cell signature and overall, event-free, and relapse-free survival. Results: The LSC score was similar across most AML subtypes, but was lower in promyelocytic leukemia, and prognostically favorable cases harboring NPM1 or CEBPA mutations. Strikingly, high scores associated with inferior overall (OS), event-free (EFS), and relapse-free survival (RFS) in these independent cohorts, whether considering patients with a normal karyotype [hazard ratio (HR) range for OS 1.13-1.18, p<0.012 in all cases], or those with cytogenetic anomalies (HR range for OS 1.07-1.15, p<0.01 in all cases). In multivariate analysis, the LSC score was associated with poor outcomes independently of age, FLT3 or NPM1 mutations, and cytogenetic risk group, and added to their prognostic value. Conclusions: High expression of a leukemic stem cell gene expression signature is independently associated with adverse outcomes in AML Overall design: Cellular fractionation and expression profiling of normal and leukemic subsets: Human samples were obtained at the Stanford University Medical Center according to an approved protocol of the Institutional Review Board after informed consent. Normal human bone marrow mononuclear cells were purchased from AllCells Inc. (Emeryville, CA) and human cord blood was obtained from Stanford University. For AML specimens, peripheral blood and/or bone marrow was obtained, and gene expression microarray data were generated using Affymetrix U133 Plus 2.0 microarrays from the following populations purified by fluorescence-activated cell sorting: AML LSC (Lin-CD34+CD38-CD90-, n=7), AML LPC (Lin-CD34+CD38+, n=7), AML Blasts (Lin-CD34-), normal hematopoietic stem cells (HSC, Lin-CD34+CD38-CD90+CD45RA-; bone marrow and cord blood, n=7), multipotent progenitors (Lin-CD34+CD38-CD90-CD45RA-; bone marrow and cord blood, n=7), common myeloid progenitors (Lin-CD34+CD38+CD123+CD45RA-; bone marrow, n=4), granulocyte-monocyte progenitors (Lin-CD34+CD38+CD123+CD45RA+; bone marrow, n=4), and megakaryocyte-erthythrocyte progenitors (Lin-CD34+CD38+CD123-CD45RA-; bone marrow, n=4). Raw data were deposited at the National Center for Biotechnology Information Gene Expression Omnibus (GEO, accession GSE24006). Detailed methods for purification of cellular subsets and clinical features of the corresponding AML patients have been reported previously. Microarray analysis and definition of LSC signature: Fourteen paired LSC and LPC samples from 7 patients described above were combined with 16 paired samples (8 LSC and 8 LPC) from an independent study to produce one dataset for analysis. Individual genes differentially expressed between paired LSC and LPC were identified using Significance Analysis of Microarrays, employing a paired metric (false discovery rate<10%). The ‘LSC signature’ in a given dataset was defined as the first principal component of these genes (the linear weighted sum of gene expression values that summarizes the maximum possible proportion of their total variance) across samples from that dataset. The LSC signature was evaluated across all purified subpopulations described above. To identify biological themes distinguishing LSC from LPC, all genes on microarrays were ranked by their geometric mean difference in expression between paired LSC/LPC samples, and evaluated using Gene Set Enrichment Analysis. Raw microarray data were obtained as Affymetrix CEL files for four publicly available bulk AML gene expression studies from NCBI GEO (GSE12417, n=163 normal-karyotype AML only, with OS outcomes; GSE10358, n=184, OS and EFS; GSE14468, n=527, OS, EFS and RFS) and the National Cancer Institute caArray database (accession willm-00119, n=170 non-FAB M3, OS only). Matrices of re-analyzed data linked below as supplementary files. Ingenuity Pathways Analysis was used to identify interaction networks of genes.

研究背景：在多种癌症中，特定的细胞亚群似乎是唯一能够启动并维持肿瘤生长的群体。癌症干细胞模型的最有力证据来自免疫缺陷小鼠的移植实验，该实验表明人类急性髓系白血病（acute myeloid leukemia, AML）呈现出由自我更新的白血病干细胞（leukemia stem cells, LSC）驱动的细胞层级结构。该模型对新型疗法的开发具有重要意义，但其临床意义仍不明确。
研究目标：探究白血病干细胞表达特征与AML患者临床结局之间的关联。
研究设计、研究场景与研究对象：本研究基于移植实验的功能定义，从原发性AML患者样本及异种移植模型中筛选出富集LSC的细胞亚群，并确定其基因表达特征。我们纳入四项独立队列的已发表AML整体基因表达数据（共计1047名患者），开展回顾性队列研究，构建LSC评分，并评估其与已知的风险预测因素（包括细胞遗传学亚型与分子突变）之间的关联，同时验证其作为独立预后因子的价值。
主要结局指标：白血病干细胞特征与总生存期（overall survival, OS）、无事件生存期（event-free survival, EFS）及无复发生存期（relapse-free survival, RFS）之间的可重复关联。
研究结果：LSC评分在大多数AML亚型中较为相似，但在急性早幼粒细胞白血病及携带NPM1或CEBPA突变的预后良好病例中评分较低。值得注意的是，在上述独立队列中，高LSC评分与较差的OS、EFS及RFS显著相关：无论是核型正常的患者[OS的风险比（hazard ratio, HR）范围为1.13~1.18，所有组均p<0.012]，还是存在细胞遗传学异常的患者[OS的HR范围为1.07~1.15，所有组均p<0.01]。多变量分析显示，LSC评分与不良结局的关联独立于年龄、FLT3或NPM1突变及细胞遗传学风险分组，且可提升上述因素的预后预测价值。
研究结论：白血病干细胞基因表达特征的高表达与AML患者的不良结局独立相关。
整体实验设计：正常与白血病细胞亚群的细胞分离与表达谱分析：本研究的人体样本获取自斯坦福大学医学中心，已获得机构审查委员会批准的研究方案及受试者的知情同意。正常人类骨髓单个核细胞购自AllCells公司（美国加利福尼亚州埃默里维尔市），人类脐带血样本获取自斯坦福大学。对于AML标本，采集外周血及/或骨髓样本，通过荧光激活细胞分选术纯化以下细胞群体，并使用Affymetrix U133 Plus 2.0芯片生成基因表达微阵列数据：AML LSC（Lin⁻CD34⁺CD38⁻CD90⁻，n=7）、AML LPC（Lin⁻CD34⁺CD38⁺，n=7）、AML母细胞（Lin⁻CD34⁻）、正常造血干细胞（hematopoietic stem cells, HSC，Lin⁻CD34⁺CD38⁻CD90⁺CD45RA⁻；骨髓及脐带血样本，n=7）、多能祖细胞（Lin⁻CD34⁺CD38⁻CD90⁻CD45RA⁻；骨髓及脐带血样本，n=7）、普通髓系祖细胞（Lin⁻CD34⁺CD38⁺CD123⁺CD45RA⁻；骨髓样本，n=4）、粒-单核细胞祖细胞（Lin⁻CD34⁺CD38⁺CD123⁺CD45RA⁺；骨髓样本，n=4）及巨核细胞-红细胞祖细胞（Lin⁻CD34⁺CD38⁺CD123⁻CD45RA⁻；骨髓样本，n=4）。原始数据已提交至美国国家生物技术信息中心基因表达综合数据库（National Center for Biotechnology Information Gene Expression Omnibus, GEO，登录号GSE24006）。细胞亚群纯化的详细方法及对应AML患者的临床特征已在既往研究中发表。
微阵列分析与LSC特征的定义：将上述7名患者的14对LSC与LPC样本，与另一项独立研究中的16对样本（8对LSC与8对LPC）合并，得到用于分析的数据集。采用微阵列显著性分析（Significance Analysis of Microarrays），以配对统计方法识别配对LSC与LPC之间差异表达的基因（错误发现率<10%）。给定数据集的‘LSC特征’定义为上述基因的第一主成分（即基因表达值的线性加权和，可最大化概括这些基因总方差的最大比例）。本研究评估了LSC特征在上述所有纯化细胞亚群中的表达情况。为识别区分LSC与LPC的生物学主题，本研究根据配对LSC/LPC样本间表达的几何均值差异对芯片上的所有基因进行排序，并通过基因集富集分析（Gene Set Enrichment Analysis）进行评估。本研究获取了四项公开的AML整体基因表达研究的Affymetrix CEL格式原始微阵列数据，数据来自NCBI GEO（GSE12417，仅纳入163名核型正常的AML患者，附带OS结局数据；GSE10358，184名患者，附带OS及EFS数据；GSE14468，527名患者，附带OS、EFS及RFS数据）及美国国家癌症研究所caArray数据库（登录号willm-00119，170名非FAB M3亚型患者，仅附带OS数据）。重新分析的数据矩阵以补充文件形式附于文末。本研究使用Ingenuity通路分析软件识别基因的相互作用网络。