In childhood acute lymphoblastic leukemia, blasts at different stages of immunophenotypic maturation have stem cell properties

Abstract

We examined the leukemic stem cell potential of blasts at different stages of maturation in childhood acute lymphoblastic leukemia (ALL). Human leukemic bone marrow was transplanted intrafemorally into NOD/scid mice. Cells sorted using the B precursor differentiation markers CD19, CD20, and CD34 were isolated from patient samples and engrafted mice before serial transplantation into primary or subsequent (up to quaternary) recipients. Surprisingly, blasts representative of all of the different maturational stages were able to reconstitute and reestablish the complete leukemic phenotype in vivo. Sorted blast populations mirrored normal B precursor cells with transcription of a number of stage-appropriate genes. These observations inform a model for leukemia-propagating stem cells in childhood ALL.

Figure 1. The morphology and immunophenotype of the leukemic blasts remain stable for four passages in the mice

(A) Original patient #14 blast cell morphology and immunophenotype. (B-E) Bone marrow samples derived from mice serially transplanted with CD34⁺CD19⁺ cells from the patient shown in A. Blast cell morphology and immunophenotype resemble the original patient sample in all four consecutive transplantations. (scale bar = 10μm)

Figure 2. Immunophenotypic composition of the leukemic grafts in the mice

(A) High-risk ALL. The composition of the human grafts in respect to the presence of CD34⁺CD19^- ■, CD34⁺CD19⁺ and CD34^-CD19⁺ cells resembles that of the original leukemias shown in C. All three subpopulations were able to reconstitute the complete phenotype of the original leukemia. However, mice transplanted with the more mature CD34^-CD19⁺ immunophenotype engrafted with a slightly lower level of CD34⁺CD19^- cells (0.04 vs 0.2 and 0.3%; p = 0.022) and higher levels of CD34^-CD19⁺ lymphoid cells (36 vs 6.3 and 7.4 %; p ≤ 0.001) as compared with mice transplanted with the other two cell fractions. (B) Standard-risk ALL. The composition of the graft was similar in mice that received CD34⁺CD19⁺ and CD34^-CD19⁺ cells. Two mice transplanted with CD34⁺CD19^- cells did not engraft. (C) Patient samples. Distribution of the three subpopulations in the original patient samples. Error bars show one SD (not calculated for 2 standard-risk patients).

Figure 3. All three transplanted subpopulations can self-renew and serially transfer the leukemia in the mice

Each dot plot (except for the patient sample) represents one mouse engrafted with human leukemic cells. Sorting gates and arrows indicate the subpopulation each mouse received from its predecessor. Mouse number and the exact cell dose transplanted into that mouse are provided in the heading of each dot plot.

Figure 4. Primary patient CD34^-CD19⁺ populations show significant upregulation of the small pre B cell specific transcription factor IRF4

Each bar represents pooled quantitative PCR data from four (E2A n=3) patient bone marrow specimens taken at diagnosis and sorted into CD34⁺CD19⁺ and CD34^-CD19⁺ blast populations. ΔΔCt values between CD34^-CD19⁺ and CD34⁺CD19⁺ populations have been transformed and normalized within a patient specimen against the B cell specific transcription factor PAX5 (error bars show 1 SD). Expression of IRF4 shows significant upregulation in the more mature CD34^-CD19⁺ population (p<0.01).

Figure 5. Model of the malleability and self-renewal seen in B precursor ALL blasts

(A) Normal hematopoiesis. The development from CD34⁺CD19^- early B progenitor, expressing transcription factors E2A and EBF1 through to CD34^-CD19⁺CD20^+/- small pre B cells expressing IRF4 and rearranging their immunoglobulin light chain loci. A period of clonal expansion in large pre B cells is shown. DJ_H – D_H-J_H segments rearranged, V_H-DJ_H – rearrangement of the V_H-D_H segment, VDJ_H – heavy chain allele rearranged. (B) ALL blast malleability and self-renewal. The blast compartment for High and Low risk ALL is shown. Within these relatively narrow compartments, blasts are able to up-regulate and down-regulate components of the normal developmental programme, including accessing the self-renewal programme at all stages. There is however, a block to differentiation beyond the B precursor stage. Genes demonstrated as being upregulated are shown below the schema.