Association of a leukemic stem cell gene expression signature with clinical outcomes in acute myeloid leukemia

Abstract

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 immunodeficient mice, which indicate that human acute myeloid leukemia (AML) is driven by self-renewing leukemic stem cells (LSCs). This model has significant implications for the development of novel therapies, but its clinical relevance has yet to be determined.

Objective: To identify an LSC gene expression signature and test its association with clinical outcomes in AML.

Design, setting, and patients: Retrospective study of global gene expression (microarray) profiles of LSC-enriched subpopulations from primary AML and normal patient samples, which were obtained at a US medical center between April 2005 and July 2007, and validation data sets of global transcriptional profiles of AML tumors from 4 independent cohorts (n = 1047).

Main outcome measures: Identification of genes discriminating LSC-enriched populations from other subpopulations in AML tumors; and association of LSC-specific genes with overall, event-free, and relapse-free survival and with therapeutic response.

Results: Expression levels of 52 genes distinguished LSC-enriched populations from other subpopulations in cell-sorted AML samples. An LSC score summarizing expression of these genes in bulk primary AML tumor samples was associated with clinical outcomes in the 4 independent patient cohorts. High LSC scores were associated with worse overall, event-free, and relapse-free survival among patients with either normal karyotypes or chromosomal abnormalities. For the largest cohort of patients with normal karyotypes (n = 163), the LSC score was significantly associated with overall survival as a continuous variable (hazard ratio [HR], 1.15; 95% confidence interval [CI], 1.08-1.22; log-likelihood P <.001). The absolute risk of death by 3 years was 57% (95% CI, 43%-67%) for the low LSC score group compared with 78% (95% CI, 66%-86%) for the high LSC score group (HR, 1.9 [95% CI, 1.3-2.7]; log-rank P = .002). In another cohort with available data on event-free survival for 70 patients with normal karyotypes, the risk of an event by 3 years was 48% (95% CI, 27%-63%) in the low LSC score group vs 81% (95% CI, 60%-91%) in the high LSC score group (HR, 2.4 [95% CI, 1.3-4.5]; log-rank P = .006). In multivariate Cox regression including age, mutations in FLT3 and NPM1, and cytogenetic abnormalities, the HRs for LSC score in the 3 cohorts with data on all variables were 1.07 (95% CI, 1.01-1.13; P = .02), 1.10 (95% CI, 1.03-1.17; P = .005), and 1.17 (95% CI, 1.05-1.30; P = .005).

Conclusion: High expression of an LSC gene signature is independently associated with adverse outcomes in patients with AML.

Figure 1. LSC-Enriched Subpopulations Have a Distinct Gene Expression Signature That is Shared with Normal HSC

Genes distinguishing leukemic stem cells (LSC) from leukemic progenitor cells (LPC). (A) Gene expression heatmap, with each column representing the difference in expression between LSC/LPC-enriched subpopulations isolated from the same AML patient^-; ‘Hs’ denotes LSC/LPC profile purified from primary human patient specimen, and ‘Mm’ represents corresponding samples from mouse xenografts. 52 unique genes were identified as differentially expressed between LSC and LPC at 10% false discovery rate (eTable 1), with red indicating higher expression in LSC. (B) Enrichment analysis of relative expression between LSC and LPC of 17119 genes for the samples depicted in panel A (see eTable 2 for gene set definitions). Vertical bars in each of the six rows represent genes from each of the indicated gene sets. All nominal p-values were <0.001. NES: normalized enrichment score; FDR: false discovery rate. (C) Expression of the LSC signature across AML subpopulations (left) and normal hematopoietic stem and progenitor cell (HSPC) populations involved in myeloid differentiation (right), including AML leukemic stem cell (LSC), leukemic progenitor cell (LPC), and leukemic blast (BLAST) populations, as well as normal hematopoietic stem cell (HSC), multipotent progenitor (MPP), common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP), and megakaryocyte-erythrocyte progenitor (MEP). LSC and LPC samples are the same as those whose paired differences are depicted in panel A (Stanford cases). Boxes span the interquartile range, with median depicted by the thick horizontal bar. Each circle marks one sample. P-values were derived from Wilcoxon test comparing LSC to LPC/Blast, and for HSC/MPP compared to CMP/GMP/MEP.

Figure 2. Higher LSC Score is Associated with Worse Outcomes

Kaplan-Meier analysis of the association between the LSC score and survival outcomes in normal karyotype AML (NKAML). Excluding those with acute promyelocytic leukemia (APL), patients were split into high versus low LSC score groups according to the median value of the LSC score in the training cohort. Stratification of outcomes using this approach is depicted for OS of NKAML patients in the training set (A), in NKAML from one of the validation sets for OS (B), and for EFS (C). Vertical ticks on curves indicate censored events, and p-values shown are for the LSC score as a continuous predictor of survival (log-likelihood test; log-rank estimates provided in Table 3). Similar results were obtained in additional independent datasets (eFigure 5 and Table 3). (D) The LSC score was significantly associated with initial therapeutic response as determined by the ability to achieve clinical remission in two datasets for which this information was available^- (p-values derived from t-test). Boxes indicate the interquartile range, with median shown as the thick horizontal bar. Numbers within boxes indicate the sample sizes. OS=overall survival; EFS=event-free survival; CR=clinical remission.

Figure 3