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Fig. 1 | Journal of Hematology & Oncology

Fig. 1

From: Adverse stem cell clones within a single patient’s tumor predict clinical outcome in AML patients

Fig. 1

Sequencing divided 12 PDX AML single stem cell clones according to first and second relapse. A Primary AML cells from a 52-year-old female patient at time of initial diagnosis (ID), first (REL1) and second relapse (REL2) were transplanted into NSG mice. REL1 and REL2, but not ID, allowed engraftment. B Primary tumor (n = 1), REL1 PDX (n = 9) and REL2 PDX (n = 3) cells were analyzed by targeted sequencing. Variant allele frequency (VAF) is depicted. C–H Generation and characterization of single PDX AML stem cell clones. C Experimental procedure; passage-1 bulk REL1 or REL2 PDX cells were transduced with a genetic barcode and marker+ cells injected into mice in limiting dilutions (REL1: 1100–33,000 cells, n = 18; REL2: 100–10,000 cells, n = 11). At advanced leukemia, PDX cells were re-isolated and barcodes quantified. D Numbers of barcodes within REL1 or REL2 populations; one dot represents one mouse. PDX populations consisting of a single barcode were defined as single stem cell clones (red box). E NRASQ61K was determined in PDX clones and compared to proportion of NRASQ61K cells within bulk REL1 and REL2 PDX cells (mean ± SD, see B). F Leukemia initiating cell (LIC) frequency of clone 4 (NRASQ61K) and clone 8 (NRASwt); cells were injected into mice in limiting dilutions and positive engraftment analyzed. Frequency of LIC and statistical significance was calculated using the ELDA software. Mean (solid line) ± 95% CI (dashed line) is depicted. G Gene expression profile was analysed via prime-seq from 3–4 biological replicates per clone and a t-distributed stochastic neighbor embedding (t-SNE) plot built by unsupervised clustering. H 424 single nucleotide variants (SNVs) were identified from exome sequencing and used to calculate a phylogenetic tree; the length of each branch correlates to number of SNV changes (grey boxes). 50 SNVs of the trunk refer to the complete remission control. Depicted are major chromosomal changes and AML related mutations at each intersection (black), numbers of individual clones (colored boxes), and name of clusters (colored letters)

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