Mass Cytometric Functional Profiling of Acute Myeloid Leukemia Defines Cell-Cycle and Immunophenotypic Properties That Correlate with Known Responses to Therapy

Abstract

Acute myeloid leukemia (AML) is characterized by a high relapse rate that has been attributed to the quiescence of leukemia stem cells (LSC), which renders them resistant to chemotherapy. However, this hypothesis is largely supported by indirect evidence and fails to explain the large differences in relapse rates across AML subtypes. To address this, bone marrow aspirates from 41 AML patients and five healthy donors were analyzed by high-dimensional mass cytometry. All patients displayed immunophenotypic and intracellular signaling abnormalities within CD34(+)CD38(lo) populations, and several karyotype- and genotype-specific surface marker patterns were identified. The immunophenotypic stem and early progenitor cell populations from patients with clinically favorable core-binding factor AML demonstrated a 5-fold higher fraction of cells in S-phase compared with other AML samples. Conversely, LSCs in less clinically favorable FLT3-ITD AML exhibited dramatic reductions in S-phase fraction. Mass cytometry also allowed direct observation of the in vivo effects of cytotoxic chemotherapy.

Significance: The mechanisms underlying differences in relapse rates across AML subtypes are poorly understood. This study suggests that known chemotherapy sensitivities of common AML subsets are mediated by cell-cycle differences among LSCs and provides a basis for using in vivo functional characterization of AML cells to inform therapy selection.

Figure 1

SPADE plots of normal bone marrow sample #6. SPADE clustering was performed on all samples (normal and AML) simultaneously to generate a single tree structure for all samples. All of the cell events from each sample were then mapped to the common tree structure. Each node of the SPADE tree is colored for the median expression of the indicated markers from low (blue) to high (red). The size of each node is correlated to the fraction of cells mapping to the node; however, a minimum size was enforced for most nodes to allow visualization of node color. Immunophenotypic grouping of nodes was performed manually on the basis of the median marker expression level of each node, and based on analysis of the relevant biaxial plots (e.g., CD38 vs. CD34).

Figure 2

Different AML samples demonstrate karyotype and genotype-specific patterns of cell distribution across developmental populations. A) SPADE tree colored for the fraction of total cells in each node from lowest (blue) to highest (red). The size of each node is correlated to the fraction of cells mapping to the node; however, a minimum size was enforced for most nodes to allow visualization of node color. B) Heat map demonstrating the relative frequency of cells in each of the indicated manually-gated immunophenotypic subsets averaged across the patients in the indicated karyotypic and genotypic groups. C) The frequency of cells in the indicated (manually-gated) stem and progenitor cell compartments for patients of each AML subtype. Error bars indicate standard errors of the means. *The 14 replicate normal samples came from 5 donors.

Figure 2

Different AML samples demonstrate karyotype and genotype-specific patterns of cell distribution across developmental populations. A) SPADE tree colored for the fraction of total cells in each node from lowest (blue) to highest (red). The size of each node is correlated to the fraction of cells mapping to the node; however, a minimum size was enforced for most nodes to allow visualization of node color. B) Heat map demonstrating the relative frequency of cells in each of the indicated manually-gated immunophenotypic subsets averaged across the patients in the indicated karyotypic and genotypic groups. C) The frequency of cells in the indicated (manually-gated) stem and progenitor cell compartments for patients of each AML subtype. Error bars indicate standard errors of the means. *The 14 replicate normal samples came from 5 donors.

Figure 2

Different AML samples demonstrate karyotype and genotype-specific patterns of cell distribution across developmental populations. A) SPADE tree colored for the fraction of total cells in each node from lowest (blue) to highest (red). The size of each node is correlated to the fraction of cells mapping to the node; however, a minimum size was enforced for most nodes to allow visualization of node color. B) Heat map demonstrating the relative frequency of cells in each of the indicated manually-gated immunophenotypic subsets averaged across the patients in the indicated karyotypic and genotypic groups. C) The frequency of cells in the indicated (manually-gated) stem and progenitor cell compartments for patients of each AML subtype. Error bars indicate standard errors of the means. *The 14 replicate normal samples came from 5 donors.

Figure 3

Systematic detection of multiple small aberrancies defines large karyotype and genotype-specific immunophenotypic changes across hematopoiesis in patients with AML. A) Method for defining aberrant marker expression. All immunophenotypic gates were defined on the basis of the normal samples and the same gates were applied to the each of the AML samples. Once each population was gated, the median expression of each of the 28 surface markers was extracted and compared to the median expression in the 14 samples from five healthy donors for each gated population. AML samples that were outside 2-fold the total variance of the normal samples were considered to be aberrant for that marker (CD45 is shown as an example). B) The total number of aberrant markers (of 28 measured markers) was summed for each population and each patient. Each box is colored for the number of the 28 markers that was aberrant for each patient (rows) in each gated immunophenotypic population (columns). The color scale ranges from green indicating no aberrant marker expression to the highest numbers of aberrancies colored red. The exact number of aberrant markers expressed (of the 28 tested) is printed in each box. The high rates of aberrancy observed in the pre-B cell population may be due in part to contamination of this gate with dimly CD19-positive malignant myeloid cells due to the limited number of markers defining this immunophenotypic subset (CD19 and CD10) and to the relatively dim staining of these antibodies in normal cells.

Figure 3

Systematic detection of multiple small aberrancies defines large karyotype and genotype-specific immunophenotypic changes across hematopoiesis in patients with AML. A) Method for defining aberrant marker expression. All immunophenotypic gates were defined on the basis of the normal samples and the same gates were applied to the each of the AML samples. Once each population was gated, the median expression of each of the 28 surface markers was extracted and compared to the median expression in the 14 samples from five healthy donors for each gated population. AML samples that were outside 2-fold the total variance of the normal samples were considered to be aberrant for that marker (CD45 is shown as an example). B) The total number of aberrant markers (of 28 measured markers) was summed for each population and each patient. Each box is colored for the number of the 28 markers that was aberrant for each patient (rows) in each gated immunophenotypic population (columns). The color scale ranges from green indicating no aberrant marker expression to the highest numbers of aberrancies colored red. The exact number of aberrant markers expressed (of the 28 tested) is printed in each box. The high rates of aberrancy observed in the pre-B cell population may be due in part to contamination of this gate with dimly CD19-positive malignant myeloid cells due to the limited number of markers defining this immunophenotypic subset (CD19 and CD10) and to the relatively dim staining of these antibodies in normal cells.

Figure 4

AML subtypes are characterized by specific marker aberrances in CD34⁺CD38^low cells. A) Median expression level of the indicated markers in the total CD34⁺CD38^low population, each data point represents the median expression level of one patient sample or one of the 14 sample aliquots from the five healthy donors. B) Upper panels: Biaxial plots of HLA-DR vs. CD33 in the gated CD34⁺CD38^low subset from three healthy donors. Lower panels: Biaxial plots of patient samples show that HLA-DR was either abnormally low (in APL samples) or abnormally high (AML sample #22) in immunophenotypically abnormal cells as compared to residual immunophenotypically normal cells (green circles).

Figure 4

AML subtypes are characterized by specific marker aberrances in CD34⁺CD38^low cells. A) Median expression level of the indicated markers in the total CD34⁺CD38^low population, each data point represents the median expression level of one patient sample or one of the 14 sample aliquots from the five healthy donors. B) Upper panels: Biaxial plots of HLA-DR vs. CD33 in the gated CD34⁺CD38^low subset from three healthy donors. Lower panels: Biaxial plots of patient samples show that HLA-DR was either abnormally low (in APL samples) or abnormally high (AML sample #22) in immunophenotypically abnormal cells as compared to residual immunophenotypically normal cells (green circles).

Figure 5

viSNE analysis of CD34⁺CD38^low subset reveals distinct karyotype and genotype-specific immunophenotypic patterns in high-dimensional space. Each sample was analyzed by viSNE (up to 5,000 sampled events per individual) using 19 dimensions (Supplementary Table 2). Two gates encompassing the vast majority of normal CD34⁺CD38^low events are shown for reference. The AML subtype is indicated for each sample. Each cell event is colored for it’s expression level of CD38 from blue (0 ion counts) to red (approximately 40 ion counts). Red cell events still fall within the CD34⁺CD38^low gate and demonstrated dim CD38 expression.

Figure 6

Karyotype and genotype-specific patterns of S-phase fraction in AML. A) SPADE trees of representative patients of the indicated AML subtypes. Nodes are colored for the frequency of S-phase cells in each node from lowest (blue) to highest (red). B) AML cells exhibit a lower S-phase fraction than immunophenotypically similar normal cells. C) Stem and progenitor cell populations from patients with CBF-AML exhibit a higher S-phase fraction than immunophenotypically similar cells from patients with other AML subtypes (APL samples excluded from analysis). D) Stem and progenitor cell populations from patients with FLT3-ITD⁺ NK-AML exhibit a lower S-phase fraction than cell from patients with other AML subtypes (APL samples and patients with FLT3-TKD mutations excluded from analysis). Colored boxes group immunophenotypic populations: HSPC, hematopoietic stem and progenitor cells; B, blasts (immunophenotypic); Mono, monocyte lineage cells; Gran, granulocyte lineage; RBC, red blood cell lineage; B-Cell, B cell lineage. Error bars indicate standard errors.

Figure 6

Karyotype and genotype-specific patterns of S-phase fraction in AML. A) SPADE trees of representative patients of the indicated AML subtypes. Nodes are colored for the frequency of S-phase cells in each node from lowest (blue) to highest (red). B) AML cells exhibit a lower S-phase fraction than immunophenotypically similar normal cells. C) Stem and progenitor cell populations from patients with CBF-AML exhibit a higher S-phase fraction than immunophenotypically similar cells from patients with other AML subtypes (APL samples excluded from analysis). D) Stem and progenitor cell populations from patients with FLT3-ITD⁺ NK-AML exhibit a lower S-phase fraction than cell from patients with other AML subtypes (APL samples and patients with FLT3-TKD mutations excluded from analysis). Colored boxes group immunophenotypic populations: HSPC, hematopoietic stem and progenitor cells; B, blasts (immunophenotypic); Mono, monocyte lineage cells; Gran, granulocyte lineage; RBC, red blood cell lineage; B-Cell, B cell lineage. Error bars indicate standard errors.

Figure 6

Karyotype and genotype-specific patterns of S-phase fraction in AML. A) SPADE trees of representative patients of the indicated AML subtypes. Nodes are colored for the frequency of S-phase cells in each node from lowest (blue) to highest (red). B) AML cells exhibit a lower S-phase fraction than immunophenotypically similar normal cells. C) Stem and progenitor cell populations from patients with CBF-AML exhibit a higher S-phase fraction than immunophenotypically similar cells from patients with other AML subtypes (APL samples excluded from analysis). D) Stem and progenitor cell populations from patients with FLT3-ITD⁺ NK-AML exhibit a lower S-phase fraction than cell from patients with other AML subtypes (APL samples and patients with FLT3-TKD mutations excluded from analysis). Colored boxes group immunophenotypic populations: HSPC, hematopoietic stem and progenitor cells; B, blasts (immunophenotypic); Mono, monocyte lineage cells; Gran, granulocyte lineage; RBC, red blood cell lineage; B-Cell, B cell lineage. Error bars indicate standard errors.

Figure 6

Karyotype and genotype-specific patterns of S-phase fraction in AML. A) SPADE trees of representative patients of the indicated AML subtypes. Nodes are colored for the frequency of S-phase cells in each node from lowest (blue) to highest (red). B) AML cells exhibit a lower S-phase fraction than immunophenotypically similar normal cells. C) Stem and progenitor cell populations from patients with CBF-AML exhibit a higher S-phase fraction than immunophenotypically similar cells from patients with other AML subtypes (APL samples excluded from analysis). D) Stem and progenitor cell populations from patients with FLT3-ITD⁺ NK-AML exhibit a lower S-phase fraction than cell from patients with other AML subtypes (APL samples and patients with FLT3-TKD mutations excluded from analysis). Colored boxes group immunophenotypic populations: HSPC, hematopoietic stem and progenitor cells; B, blasts (immunophenotypic); Mono, monocyte lineage cells; Gran, granulocyte lineage; RBC, red blood cell lineage; B-Cell, B cell lineage. Error bars indicate standard errors.

Figure 7

In vivo response to hydroxyurea (HU) was readily detectable and distinct from the response of leukemia cell lines in vitro. A) IdU incorporation and Ki-67 staining of representative untreated and HU-treated AML samples demonstrates that treatment leads to a reduction in IdU incorporation with minimal change in S-phase fraction. B) IdU incorporation is significantly decreased in the S-phase cells of all immunophenotypic populations in samples from patients being treated with IdU at the time of their bone marrow biopsy. (HSC and MPP populations were combined for this analysis given the low numbers of S-phase cells in these rare cell populations.) C) HU treatment does not significantly decrease S-phase fraction in most immunophenotypic populations. D) Fraction of pRb⁺ cells does not decrease in response to HU treatment. Colored boxes group immunophenotypic populations: HSPC, hematopoietic stem and progenitor cells; B, blasts (immunophenotypic); Mono, monocyte lineage cells; Gran, granulocyte lineage; RBC, red blood cell lineage; B-Cell, B cell lineage. Error bars indicate standard errors.

Figure 7

In vivo response to hydroxyurea (HU) was readily detectable and distinct from the response of leukemia cell lines in vitro. A) IdU incorporation and Ki-67 staining of representative untreated and HU-treated AML samples demonstrates that treatment leads to a reduction in IdU incorporation with minimal change in S-phase fraction. B) IdU incorporation is significantly decreased in the S-phase cells of all immunophenotypic populations in samples from patients being treated with IdU at the time of their bone marrow biopsy. (HSC and MPP populations were combined for this analysis given the low numbers of S-phase cells in these rare cell populations.) C) HU treatment does not significantly decrease S-phase fraction in most immunophenotypic populations. D) Fraction of pRb⁺ cells does not decrease in response to HU treatment. Colored boxes group immunophenotypic populations: HSPC, hematopoietic stem and progenitor cells; B, blasts (immunophenotypic); Mono, monocyte lineage cells; Gran, granulocyte lineage; RBC, red blood cell lineage; B-Cell, B cell lineage. Error bars indicate standard errors.

Figure 7

In vivo response to hydroxyurea (HU) was readily detectable and distinct from the response of leukemia cell lines in vitro. A) IdU incorporation and Ki-67 staining of representative untreated and HU-treated AML samples demonstrates that treatment leads to a reduction in IdU incorporation with minimal change in S-phase fraction. B) IdU incorporation is significantly decreased in the S-phase cells of all immunophenotypic populations in samples from patients being treated with IdU at the time of their bone marrow biopsy. (HSC and MPP populations were combined for this analysis given the low numbers of S-phase cells in these rare cell populations.) C) HU treatment does not significantly decrease S-phase fraction in most immunophenotypic populations. D) Fraction of pRb⁺ cells does not decrease in response to HU treatment. Colored boxes group immunophenotypic populations: HSPC, hematopoietic stem and progenitor cells; B, blasts (immunophenotypic); Mono, monocyte lineage cells; Gran, granulocyte lineage; RBC, red blood cell lineage; B-Cell, B cell lineage. Error bars indicate standard errors.

Figure 7

In vivo response to hydroxyurea (HU) was readily detectable and distinct from the response of leukemia cell lines in vitro. A) IdU incorporation and Ki-67 staining of representative untreated and HU-treated AML samples demonstrates that treatment leads to a reduction in IdU incorporation with minimal change in S-phase fraction. B) IdU incorporation is significantly decreased in the S-phase cells of all immunophenotypic populations in samples from patients being treated with IdU at the time of their bone marrow biopsy. (HSC and MPP populations were combined for this analysis given the low numbers of S-phase cells in these rare cell populations.) C) HU treatment does not significantly decrease S-phase fraction in most immunophenotypic populations. D) Fraction of pRb⁺ cells does not decrease in response to HU treatment. Colored boxes group immunophenotypic populations: HSPC, hematopoietic stem and progenitor cells; B, blasts (immunophenotypic); Mono, monocyte lineage cells; Gran, granulocyte lineage; RBC, red blood cell lineage; B-Cell, B cell lineage. Error bars indicate standard errors.