Baseline biomarkers associated with blast reduction in BM and PB. (A) BM blast reduction-associated immunophenotype clusters identified in BM after UMAP dimensionality reduction and FlowSOM clustering of high-dimensional cellular immunophenotyping data at baseline (P value < .05; t test; n = 13). Cluster frequencies of significant CD4⁺ cluster 4 (CD4_C4) and CD8⁺ cluster 1 (CD8_C1) are shown (as % of parent) on logit-scaled axes; the mean is indicated by the line. Cluster composition based on manually gated immune cell subsets is shown within the plot as a colored stacked bar chart. (B) PB blast reduction–associated immunophenotype clusters identified in PB after UMAP dimensionality reduction and FlowSOM clustering of high-dimensional cellular immunophenotyping data at baseline (P value < .05; t test; n = 43). Cluster frequencies of significant CD8⁺ clusters 16, 6, and 1 (CD8_C16, CD8_C6, CD8_C1) are shown (as % of parent) on logit-scaled axes; the mean is indicated by the line. Cluster composition based on manually gated immune cell subsets is shown within the plot as a colored stacked bar chart. (C) Significantly expressed immune cell markers in each significant cluster from BM (panel A) and PB (panel B). The median marker intensity was tested for the respective cluster vs all other clusters (adjusted P value < .05; Wilcoxon rank sum test) and arrows indicate significant higher ↑ or lower ↓ intensity. Markers are ranked by significance; exhaustion markers are indicated in bold. (D) Baseline CD8 T-cell states in BM measured by scRNA-seq of BMMC samples (n = 14). Enrichment scores were calculated per cell and averaged per sample using previously described gene sets.²⁹ (E) WT1 messenger RNA target expression in PB shown as (normalized copy number) NCN measured by quantitative reverse transcription polymerase chain reaction; the line indicates the median (n = 30). Samples with WT1 below the level of quantification (BLQ) are shown in red. The dashed line indicates the value above which WT1 is considered to be overexpressed in normal PB (NCN = 50).³⁰ (F) Intracellular WT1 and cell surface HLA-A2 protein expression measured by flow cytometry in PB (left, n = 40) and BM (right, n = 27). Levels are shown as frequency (%) of blasts in PB or BM; the mean is indicated by the line and P values were determined after FDR adjustment (t test on logit-transformed values). (G) On-treatment dynamics of WT1+ (left) and HLA-A2+ (right) percentage of AML cells in BM measured by scRNA-seq of BMMC samples (n = 10). WT1+ / HLA-A+ cells defined by unique molecular identifier (UMI) >0; the color code indicates the clinical response at the time of sampling. (H) Distribution of %WT1+ cells within different AML subpopulations in BM at baseline as evaluated by scRNA-seq of BMMC samples (n = 14). WT1+ cells were defined by a UMI >0. (I) Change in AML subpopulations in BM at the time of progression as evaluated by scRNA-seq of BMMC samples (n = 9). Expansion defined as >5% increase relative to baseline; shrinkage defined as >5% decrease relative to baseline. (J) Percentage of WT1+/PSMB9+, WT1+/PSMB9−, and WT1− cells in expanding mono-like AML subpopulation at screening and the time point of disease progression as evaluated by scRNA-seq of BMMC samples (n = 9). WT1+ / PSMB9+ cells defined by a unique molecular identifier (UMI) >0. AML, acute myeloid leukemia; BMMC, bone marrow mononuclear cells; C, cluster; cDC, conventional dendritic cell; CM, central memory; CR, complete remission; CxDx, Cycle x Day x; EM, effector memory; FDR, false discovery rate; GMP, granulocyte-monocyte progenitors; hem., hematologic; LSPC, leukemia stem and progenitor cells; Mono, monocyte; NCN, normalized copy number; ProMono, pro-monocyte; PD, progressive disease; PSMB9, Proteasome 20S Subunit Beta 9; SCRN, screening; scRNA-seq, single-cell RNA sequencing; SD, stable disease; TEMRA, terminally differentiated effector memory cells re-expressing CD45RA; UMAP, Uniform Manifold Approximation and Projection; WT1, Wilms Tumor Protein 1.