Page 59 - Haematologica June

Cytokines increase AML but not MDS engraftment
engraftment levels were measured in female mice by bone mar- row aspiration of either the ipsilateral or contralateral femur at the time points indicated.
The methods are described in more detail in the Online Supplementary Appendix.
Results
Increased levels of acute myeloid leukemia engraft- ment in NSG-S mice
We first investigated the engraftment of 77 AML sam- ples in NSG mice, representing all French-American- British and prognostic groups (Table 1). We observed that only half of the samples (n=39, 51%) were able to engraft at a significant level (>0.5% human blasts in mouse bone marrow) (Figure 1A). In order to compare engraftment between the two mouse strains, 18 of the 39 NSG-engraft- ing AML samples were also injected in NSG-S mice. All 18 NSG-engrafting samples engrafted in NSG-S mice. Representative flow cytometry plots of human CD45+CD33+ cells in the bone marrow, spleen and periph- eral blood from 2 AML samples that engrafted in both strains are shown in Figure 1B. Importantly, for 44% (8 out of 18) of the NSG-engrafting AML samples, the use of NSG-S mice as recipients was associated with very rapid engraftment, excessive leukemic burden, anemia, weight loss and lethargy, leading to a significantly shorter overall survival (P≤0.005) (Figure 1C). Consequently, a quantita- tive comparison of engraftment at the same time point for these 8 patients’ samples was not feasible. The enhanced engraftment, and associated mortality, observed in 26% (8/31) of all NSG-S-engrafting samples should be taken into account when using this strain for pre-clinical studies. For 9 of the 18 patients’ samples tested in both NSG and NSG-S, we were able to sacrifice the mice at the same time (Figure 1D). We observed a significantly higher leukemia burden in bone marrow and spleen from NSG-S compared to NSG mice (32±23% vs. 21±22% of bone marrow blasts, P≤0.035; 8±14% vs. 6±9% splenic blasts, P≤0.0001) (Figure 1E and F, respectively). Interestingly, NSG-S mice showed a dramatic increase in peripheral blast count (2771±7208 vs. 137±166 blasts/mL peripheral blood, P≤0.034) (Figure 1G). This may represent an improvement over NSG mice because it extends the use- fulness of peripheral blood sampling to monitor engraft- ment and response to treatment in pre-clinical studies. Overall, these data demonstrate that AML engraftment in NSG-S mice is more rapid and yields a higher leukemic burden than in NSG mice.
NSG-S mice could support the engraftment of samples incapable of engrafting in NSG mice
To test whether NSG-S mice could support the engraft- ment of samples incapable of engrafting in NSG mice, we transplanted NSG-S mice with 21 of the 38 NSG non- engrafting samples (Figure 1A). Remarkably, 67% (14/21) of the non-engrafting samples did engraft in NSG-S mice (18±17.5% bone marrow blasts, 9.2±13.7% splenocytes, and 1799±4848 blasts/mL peripheral blood) (Figure 2A). Thus, overall our results show that 82% (32/39) of all test- ed AML samples engrafted in NSG-S mice, compared to 51% in NSG mice. The degree of engraftment observed in bone marrow and spleen for 11 representative patients is shown in Figure 2B. These results indicate that the pres-
ence of systemic human stem cell factor, granulocyte- macrophage colony-stimulating factor and interleukin-3 in NSG-S mice contribute to support leukemia-initiating cells for most AML samples. However, 7 out of 39 samples (18%) still failed to engraft in NSG-S mice, indicating that the bone marrow microenvironment in NSG-S mice may remain suboptimal for a minority of AML samples.
We investigated whether engraftment in NSG-S mice was correlated with surface expression of CD116 (granu- locyte-macrophage colony-stimulating factor receptor), CD117 (c-kit), and CD123 (interleukin-3 receptor α−chain) on leukemic cells. As shown in Figure 2C, we found no significant difference in the density of cytokine receptor expression or cytogenetic profiles, mutations, and prognosis between NSG-S engrafting and non- engrafting samples. These results indicate that, in a small minority of AML samples, leukemia-initiating cells have requirements beyond the combination of human granulo- cyte-macrophage colony-stimulating factor, interleukin-3 and stem cell factor capable of supporting the vast major- ity of primary AML samples in mice.
Inv(16) acute myeloid leukemia shows enhanced engraftment in NSG-S mice
Core binding factor (CBF), a heterodimeric transcription factor that plays an essential role in controlling and regu- lating normal and leukemic differentiation, is a frequent target of gene rearrangements and mutations in AML.25 CBF-AML patients represent 10-15% of all patients with AML and are characterized by two recurrent transloca- tions: t(8;21)(q22;q22) and inv(16)(p13.1; q22) or t(16;16)(p13.1;q22).26,27 These AML samples with favorable karyotypes are known to engraft poorly in NSG mice. We included 10 CBF-AML samples [2 with t(8;21) and 8 with inv(16)] in our strain comparison study, and were able to successfully engraft all 8 inv(16) AML samples in NSG-S mice (Figure 2D). Interestingly, the 2 t(8;21) samples did not show enhanced engraftment in the NSG-S mice sug- gesting perhaps a specific defect for the particular translo- cation. The presence of chromosomal abnormalities was confirmed in bone marrow blasts from engrafted NSG-S mice using a reverse transcriptase polymerase chain reac- tion to amplify the CBFβ-MYH11 fusion transcript and fluorescence in situ hybridization to detect the inv(16) breakpoint region (Figure 2E). Thus, NSG-S mice provide a permissive environment to support leukemia-initiating cells from low risk inv(16) patients. Whether this reflects a particular requirement of cytokines for inv(16) AML stem cells will require further studies.
Characterization of myelodysplastic syndrome cell engraftment in xenotransplantation models
We next turned to characterizing MDS cell engraftment in the two mouse strains. MDS engraftment in patient- derived xenotransplantation models is less well described than AML. Unlike AML, MDS should provide multi-lin- eage engraftment which requires further characterization. It has also been reported that MDS engraftment is enhanced by co-injection of MSC and we designed exper- iments to study these variables. We initially transplanted 7 MDS patient samples in NSG and/or NSG-S immuno- compromised mice (Figure 3A). The patients’ samples used were separately considered as high risk (MDS with excess blasts-1/2), or low risk (MDS/myeloproliferative neoplasm, unclassified low-risk MDS, therapy-related
haematologica | 2018; 103(6)
961