Fig. 2

Comparative analysis of mixed phenotype acute leukemia with other acute leukemias. A Split UMAP of leukemic and canonical cell types (n = 156,489 cells), separated based on leukemia type/subtype (i.e., AML, B-ALL, T-ALL, B/My MPAL, and T/My MPAL) and healthy samples. B Density plot showing stemness index distribution of the different blast cells from different acute leukemias including B/My MPAL and T/My MPAL, progenitor cells, and normal immune cells. The stemness index was calculated as the first principal component value of each cell after performing principal component analysis with the expression of the genes in a stem cell signature (Additional file 1: Table S3). C Heatmap with the top overexpressed markers for mixed phenotype acute leukemia (MPAL) and subtypes (i.e., B/My MPAL and T/My MPAL). The heatmap also shows the expression of MPAL marker genes in other acute leukemias (i.e., AML, B-ALL, T-ALL), (BM) and healthy immune cells. These markers were filtered to only include genes with low expression in healthy bone marrow cells. Overexpressed genes were identified for MPAL subtypes by comparing the profile of MPAL blast cells versus blast cells from other acute pediatric leukemias (i.e., AML, B-ALL, T-ALL) and healthy BM samples. The MPAL subtype significantly overexpressed genes (average log2FC > 0.25, adjusted p-value < 0.05, and pct. expressed > 50%) were further refined by selecting genes with low expression in healthy BM cells from HCA (avg. expression < 0.5). Finally, the top genes for the heatmap were chosen based on their highest average log2FC values. D Dot plots showing the expression of two canonical immune cell markers (CD79A and CD3D) and two MPAL blast cell markers (CD81 and LMO2), to show that these MPAL blast cells markers have low expression in various normal BM cell types and healthy hematopoietic stem cells. The size of the dots refers to the percentage of cells in each cell type cluster expressing the gene and the color represents averaged scaled gene expression level; cyan: low, red: high. X-axis is the cell type, and Y-axis is the genes. The expression of MPAL markers is marked with lasso. E Expression of MPAL blast markers in AML, T-ALL, and MPAL bulk RNA-seq data. The Y-axis shows the scaled values of the log2 of the normalized expression plus one, and the X-axis shows different subtypes for the bulk RNA-seq samples. Wilcoxon rank tests were performed to test the difference in expression between MPAL and AML, and MPAL and T-ALL for the three genes shown (*** for p-value < 0.001, ** for p-value < 0.01, and * for p-value < 0.05). F The top significantly enriched pathways of the filtered B/My MPAL blast cell marker genes. Each bar represents a significantly enriched pathway as determined using the P value (shown on the primary X-axis). The bar plot is sorted by the negative log of the hypergeometric distribution-based p-values of the results. The analysis for canonical pathways was performed using the MetaCore platform from Clarivate Inc. G The top significantly enriched pathways of the filtered T/My MPAL blast cells marker genes. H Kaplan–Meier curves-based survival association analysis of B/My MPAL marker gene, MTRNR2L12 in B/My MPAL TARGET samples (top) and T/My MPAL marker, PTEN in T/My MPAL TARGET samples (bottom). Survival association analysis was performed using the Cox Proportional Hazards Regression Model, with MTRNR2L12 expression having a hazard ratio of 4.80 (p = 0.059) and PTEN expression having a hazard ratio of 4.50 (p = 0.04), high expression of both genes indicated an association with poor survival