Acute myeloid leukemia (AML) is the most frequent myeloid malignancy in adults, causing death of about 90% of elderly patients. Cytogenetic and molecular abnormalities are used to categorize AML in favorable, intermediate and adverse molecular risk groups. Classification in one of these groups will directly affect clinical decision-making. Although molecular criteria have significantly improved prognostication and thus AML patient stratification and risk-based treatment, they still do not allow full risk prediction.
Using a xenotransplantation model of human AML samples into NOD/SCID/IL2Rgnull(NSG) mice, we have previously shown that favorable risk AML engraft NSG mice after longer latency when compared to intermediate or adverse risk AML (Paczulla et al., 2017). This suggests that this mouse model accurately depicts important features of human AML. To further investigate the relationship between in vivo leukemia latency in NSG mice and AML outcome in patients, we have expanded our analyses to a cohort of n=23 favorable AML patient samples with diverse cytogenetic and molecular features. We hypothesize that the time-to-leukemia induction in mice correlates with the capacity of homing to the bone marrow (BM) as well as with the outcome in patients.
AML cells were derived from peripheral blood mononuclear cells of patients, and depleted for CD19-positive B- as well as CD3-positive T-cells by MACS. Patient-derived AML blasts were freshly thawed before each transplantation and intra-femorally transplanted into previously irradiated 6-8 week-old female NSG (500.000 cells per mouse). Transplanted mice were monitored for engraftment at two time points post-transplantation (16 weeks and 6 months) via BM biopsy and detection of human hematopoietic markers (CD33 or CD34, depending on patients features) using flow cytometry. At signs of disease or when more than 80% leukemic blasts were detected in the BM biopsy, mice were euthanized and leukemic infiltration of BM, peripheral blood (PB) and organs investigated using multi-color flow cytometry. Homing capacity was investigated by intravenous transplantation of 1.000.000 CFSE-labeled leukemic blasts in non-irradiated 6-8 week-old male NSG mice. 16 hours later, mice were euthanized and BM and PB analyzed for detection of fluorescent leukemic cells.
Interestingly, all transplanted favorable AML samples were able to engraft NSG mice within 54 weeks after transplantations, with very different time-to-leukemia induction between patients. Survival proportion at week 16 was 81.5%, 78.1% at week 26 (6 months) and dropped to 27.3 % at week 40 (9 months).
From the BM punctures performed at week 16 (n=65 mice, n=23 AML patients), 44.62% showed more than 1%, 76.92% more than 0.1% and 84.62% more than 0.01 % human leukemic cells. Notably, although some mice showed very low engraftment at week 16, they nevertheless developed leukemia at a later time point. Interestingly, results from BM punctures seem to nicely correlate with long-term survival. Indeed, mice for which more than 1% engraftment was detected at the 16-week time-point showed a 50% survival rate at week 26, 42% at week 26 and 27% at week 40 (in comparison to respectively 92%, 80% and 61% survival for mice with <1% leukemic cells at the same time-point).
Furthermore, homing capacity of leukemic cells also seems to correlate with long-term engraftment (n=56 mice, n=20 AML patients). In fact, we could observe a tendency for increased homing to the BM for the samples that resulted in more than 1% engraftment at week 16 in comparison to those with less than 1% (median homing 0.0049% vs. 0.0029%).
Taken together, our recent work demonstrates in a large set of patient samples that - in contrast to several previous publications - human AML cells of favorable molecular risk indeed routinely engraft NSG mice. Furthermore this work points out to correlations between homing capacity, BM engraftment at week 16 and long-term survival and time-to-leukemia in NSG mice. Comparative studies using AML cells of intermediate and adverse molecular risk are underway. These findings provide proof-of-principle data for a prospective clinical study analyzing correlations between AML cell homing capacity in mice and outcome of disease in patients.
Disclosures
No relevant conflicts of interest to declare.
Piperaquine Pharmacodynamics and Parasite Viability in a Murine Malaria Model