Specific biomarkers of AMKL. (A) Heat map of expression values in FPKM (RNAseq) of 7 top-ranked genes encoding cell surface proteins that are differentially expressed by a least 10-fold in leukemic BM cells derived from patients (pAMKL) or mouse xenograft models (xAMKL) in the middle panel, as compared with normal CB-CD34⁺ cells in the left panel, and expressed at low levels (≤5 FPKM) in CB-CD34⁺ cells. Genes with expression values of ≥5 FPKM in all N5A AMKL samples, fold change ≥10 compared with CB-CD34⁺ samples, and low expression levels (≤5 FPKM) in CB-CD34⁺ cells are listed in supplemental Table 8. Expression of the selected genes in leukemic BM cells derived from patients presenting other genetic subtypes of AMKL (non-N5A pAMKL) or non-AMKL leukemia subtype involving NUP98 rearrangement (NUPr pAML) are also indicated. Expression of ITGA2B/CD41, ITGB3/CD61, and NCAM1 is indicated in red for comparison. Expression of the selected genes in the validation cohort⁶  are shown in the right panel, represented as mean expression per indicated genetic group. (B) Distribution of selected gene expression values (FPKM) in BM-derived pediatric AML cells classified according to the FAB nomenclature (M0-M7); n = 284 pediatric AML cases from the National Cancer Institute (NCI) TARGET database. Horizontal lines represent median values. Pairwise gene expression comparisons between M7 and other FAB categories were performed with a Mann-Whitney rank sum test with the Benjamini-Hochberg correction (shown below graphs). M7 leukemia (n = 11) involved the following exclusive genetic lesions: NUP98-KDM5A (n = 1), CBFA2T3-GLIS2 (n = 4), KMT2A-MLLT10 (n = 1), and RBM15-MKL1 (n = 1). (C) Pairwise scatterplot representations showing correlative expression of the indicated genes in a pediatric AML (NCI, TARGET database). Representations were created with the bioinformatic tool MiSTIC.⁵¹  AML classified as FAB M7 or M6 are indicated in red and blue, respectively. (D) Selection of specimens expressing the highest levels of RHAG, NEO1, GP9, or ITGB3/CD61 combined with ITGA2B/CD41 significantly enriches for FAB M7 AML (eg, AMKL). M7: 8 of 9 selected P = 6.1e-11. See data set and bioinformatic tool in panel C. (E) SELP expression, as assessed by flow cytometry in freshly isolated CB-CD34⁺ cells and in AMKL BM cells from an N5A mouse xenograft model. (F) Expression of NEO1 detected by RT-PCR using RNA derived from the BM of leukemic xenograft models or from CB-CD34⁺ cells. KDM5B expression was used as the endogenous control. (G) Expression of NEO1 detected by RT-PCR using RNA derived from the BM of an infant with NUP98-BPTF AMKL. RNA was isolated at diagnosis (NUP98r pAMKL-3D) and after 2 cycles of chemotherapy when disease burden was ∼2% by cytology (NUP98r pAMKL-3 MRD). Human placental RNA was used as the nontumor control. ns/not significant P > .05; *P < .05; **P < .01; ***P < .001; ****P < .0001.