Page 198 - Haematologica March 2020

H. Kuusanmäki et al.
ple t-tests P-values were adjusted using the Benjamin-Hochberg method (P<0.10 used to determine significance). The Kruskal- Wallis test was used when more than two groups were tested and significant comparisons were validated with post-hoc analysis (Dunn’s test). Statistical dependence between two variables was assessed by Spearman’s rank correlation.
Results
Analysis of the AML bone marrow compartment
To measure blast-specific drug responses in mononu- clear cell (MNC) enriched BM AML samples, we tested 34 AML samples collected at diagnosis or relapse with seven drugs. Following a 72-hour drug treatment we analyzed the samples by both FC and CTG-based cell viability assays (Figure 1A). With the CTG assay we measured the overall BM-MNC sensitivity, while with the FC analysis the number of viable cells in different cell populations was measured. We used four cell surface markers (CD45,
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CD34, CD33, CD14) to identify the major leukocyte pop- ulations present in the AML BM: leukemic blasts, imma- ture granulocytes, promonocytes/monocytes and lympho- cytes (Figure 1B). In the studied samples, the fraction of blasts out of CD45+ positive leukocytes varied between 17-92% and the lymphocyte population ranged from 1-49% (Online Supplementary Table S3). As expected, we observed high numbers of monocytic cells in FAB M4/5 samples, whereas M0/1 samples mainly consisted of blasts and lymphocytes (Figure 1C). After 72-hours in cul- ture, we observed monocytic maturation in several M5 samples,24 and in many samples the granulopoietic cell population diminished or was completely lost (Online Supplementary Figure S2).
FC versus homogeneous cell viability assay-based drug sensitivity profiling
In order to determine the correlation between drug sen- sitivity of the samples measured by FC or CTG-based methods, we converted the cell viability readouts from
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Figure 3. Maturation stage of acute myeloid leukemia (AML) cells affects drug sensitivities. (A) Drug sensitivity score (DSS) values for distinct cell populations in 33 AML samples (blue) and 2-3 healthy controls (orange). Cell population means were compared against blasts with Kruskal-Wallis test (Dunn’s test, *P<0.05, **P<0.001, ***P<0.0001). (B) Representative flow cyometric (FC) scatter plots of the effects of venetoclax and trametinib on blasts, monocytic cells (CD14+) and lymphocytes after 72h drug treatment with the indicated concentrations. Absolute cell counts inside the gates were calculated after drug treatment and normalized to the cell counts in the DMSO-treatment wells (represented as percentages). (C) Inter- and intra-patient comparison of the DSS values in blasts and monocytic cell fraction calculated with Mann-Whitney U test. HSPC: healthy hematopoietic stem/progenitor cells.
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haematologica | 2020; 105(3)