AML engraftment in the NOD/SCID assay reflects the outcome of AML: implications for our understanding of the heterogeneity of AML

Abstract

The nonobese diabetic/severe combined immunodeficient (NOD/SCID) assay is the current model for assessment of human normal and leukemic stem cells. We explored why 51% of 59 acute myeloid leukemia (AML) patients were unable to initiate leukemia in NOD/SCID mice. Increasing the cell dose, using more permissive recipients, and alternative tissue sources did not cause AML engraftment in most previously nonengrafting AML samples. Homing of AML cells to the marrow was the same between engrafters and nonengrafters. FLT3 internal tandem duplication (ITD) and nucleophosmin mutations occurred at a similar frequency in engrafters and nonengrafters. The only variable that was related to engraftment ability was the karyotypically defined risk stratification of individual AML cases. Of interest, follow-up of younger patients with intermediate-risk AML revealed a significant difference in overall survival between NOD/SCID engrafting and nonengrafting AMLs. Hence, the ability of AML to engraft in the NOD/SCID assay seems to be an inherent property of AML cells, independent of homing, conditioning, or cell frequency/source, which is directly related to prognosis. Our results suggest an important difference between leukemic initiating cells between engrafting and nonengrafting AML cases that correlates with treatment response.

Figure 1.

Confirmation of AML cell growth in NOD/SCID mice. Ten million cells were injected into NOD/SCID mice and marrows were analyzed for human myeloid cell content 6 weeks later. (A) Diagnostic peripheral blood smear from AML-M2 patient 10. Cells were stained with hematoxylin and eosin (H&E) before analysis at 400 × magnification via a 40 ×/0.75 objective lens. (B) Murine marrow that was injected with cells from the same AML-M2 patient as in panel A. Myeloblasts, featuring Auer rods (arrow), are present, indicating AML. Cells were stained with H&E before analysis at 1000 × magnification via a 100 ×/1.3 objective lens. (C) Dual fusion, dual-color fluorescent in situ hybridization of a relapsed t[8,21] AML-M2 sample. Cells positive for the rearrangement exhibit 1 green, 1 red, and 2 orange spots. (D-F) Examples of NOD/SCID engrafted, FACSorted CD33⁺/CD45⁺ cells exhibiting AML-M2 t[8,21] rearrangement. Pictures of blood smears were taken on a Leica DFC 300F camera.

Figure 2.

Gene expression analysis of engrafted AML cells. A dendrogram is shown from the unsupervised hierarchical cluster analysis of the 8 chips for the 2260 genes passing the variation filter. Independent of karyotype, AML patients were grouped between before and after engraftment. This means that the AML in the original patient is very much related to the AML that has grown in the mouse. The samples corresponding to before and after engraftment were always adjacent to each other, reflecting a very close relationship between them. Pt indicates patient. Height is an arbitrary unit of association; higher numbers indicate greater degree of similarity.

Figure 3.

Most cases of nonengrafting AML do not engraft in the B2m^-/- NOD/SCID model. Ten million mononuclear cells from 23 different AML patients were injected into both NOD/SCID () and B2m^-/- NOD/SCID (▪) mice in paired experiments. Six weeks later, bone marrow engraftment was assessed via flow cytometry. AML engraftment was recorded if human CD33⁺/CD45⁺ myeloid cells were present without an accompanying CD19⁺/CD45⁺ B-cell population. Ten of 12 AML cases that failed to engraft in the NOD/SCID assay did not engraft in the B2m^-/- NOD/SCID model.

Figure 4.

Calcium flux in cells from both NOD/SCID engrafting and nonengrafting AML cases. Samples were labeled with Indo-1 dye as described in “Calcium flux measurement.” The ratio of fluorescence due to dye bound to calcium over fluorescence due to unbound dye is displayed against time. Data were collected for 30 seconds, before addition of 100 ng/mL of SDF-1 and further analysis. All samples analyzed produced detectable intracellular calcium upon SDF-1 stimulation and no differences could be detected between NOD/SCID engrafting and nonengrafting AML cases.

Figure 5.

Overall survival and EFS of NOD/SCID engrafting and nonengrafting AML samples. (A) Overall survival data of NOD/SCID engrafting and nonengrafting AML samples. The overall and event-free survival data of 25 de novo intermediate-risk AML cases (< 60 years old) that received intensive multi-agent chemotherapy. Four cases were censored at allograft in first complete remission (2 in each group). NOD/SCID engrafting AML cases had a poor overall survival that was statistically lower than NOD/SCID nonengrafting AML cases. (B) Event-free data of NOD/SCID engrafting and nonengrafting AML samples. The event-free survival data of 25 de novo intermediate-risk AML cases (< 60 years old) that received intensive multi-agent chemotherapy. Four cases were censored at allograft in first complete remission (2 patients in each group). NOD/SCID engrafting AML cases had a poor event-free survival when compared with nonengrafting AML cases, though this did not reach statistical significance.