Tracking Response and Resistance in Acute Myeloid Leukemia through Single-Cell DNA Sequencing Helps Uncover New Therapeutic Targets

Abstract

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasia with a complex polyclonal architecture. Among driver lesions, those involving the FLT3 gene represent the most frequent mutations identified at diagnosis. The development of tyrosine kinase inhibitors (TKIs) has improved the clinical outcomes of FLT3-mutated patients (Pt). However, overcoming resistance to these drugs remains a challenge. To unravel the molecular mechanisms underlying therapy resistance and clonal selection, we conducted a longitudinal analysis using a single-cell DNA sequencing approach (MissionBioTapestri® platform, San Francisco, CA, USA) in two patients with FLT3-mutated AML. To this end, samples were collected at the time of diagnosis, during TKI therapy, and at relapse or complete remission. For Pt #1, disease resistance was associated with clonal expansion of minor clones, and 2nd line TKI therapy with gilteritinib provided a proliferative advantage to the clones carrying NRAS and KIT mutations, thereby responsible for relapse. In Pt #2, clonal architecture was less complex, and 1st line TKI therapy with midostaurin was able to eradicate the leukemic clones. Our results corroborate previous findings about clonal selection driven by TKIs, highlighting the importance of a deeper characterization of individual clonal architectures for choosing the best treatment plan for personalized approaches aimed at optimizing outcomes.

Keywords: FLT3 mutations; acute myeloid leukemia; clonal architecture; clonal evolution; tyrosine kinase inhibitors.

Figure 1

Bone marrow blasts (%) and treatments received at different time points for patient 1 (A) and patient 2 (B). The blue dotted lines indicate the time point relative to the percentage of blasts, and the boxes indicate the therapy administered. Abbreviations: 5-AZA = Azacitidine; Ven = venetoclax; Gilt = gilteritinib; Chemo = Chemotherapy; Mido = midostaurin; HD AraC = high-dose cytarabine; MDS = myelodysplastic syndrome; AML = acute myeloid leukemia.

Figure 2

Clonal and sub-clonal populations were identified by single-cell DNA sequencing in Pt #1 at different time points. (A) Number of identified mutated clones in each sample analyzed at the diagnosis of MDS, AML progression, treatment, and relapse. (B) Distribution of clonal populations with a given genotype identified across different time points (top panel). The heat map indicates the mutated genes and zygosity for each clone (bottom panel); gray boxes represent heterozygous mutations, black boxes represent homozygous mutations, and wild-type (WT) genotypes are shown with white boxes. (C) Heat map of the AML diagnosis sample shows chromosome ploidy (columns) of WT, C1, C2, and C3 clonal populations (rows). (D) Heat map of the AML diagnosis sample shows gene ploidy (columns) of the WT, C1, C2, and C3 clonal populations (rows). (E) Profile plot of C3 in the AML diagnosis sample shows ploidy (y-axis) for each amplicon ordered per chromosome (x-axis). Each dot is the median ploidy per amplicon. Abbreviations: C1 = TET2^N191Kfs*4 single-mutated clone; C2 = TET2^N191Kfs*4/RUNX1^R201Q_HET double-mutated clone; C3 = TET2^N191Kfs*4/RUNX1^R201Q_HOM double-mutated clone.

Figure 3

Clonal architecture and evolution shown by single-cell DNA sequencing. (A) Clonal and sub-clonal populations identified in Pt #1. Each column represents a specific time point, and the different colours represent the unique clonal populations identified. The total number of cells sequenced and the blast percentage for each sample are listed under the bar graph. (B) Patterns of clonal evolution during disease progression. Number of cells with the unique assigned genotype alongside the different time points analyzed. Legend: T1 = MDS diagnosis, T2 = AML diagnosis, T3 = during gilteritinib treatment, T4 = Relapse. (C) Fishplot visualizing patterns of clonal evolution according to variant allele frequencies at MDS diagnosis, AML progression, during gilteritinib treatment, and at relapse. Abbreviations: MDS = myelodysplastic syndrome; AML = acute myeloid leukemia; WT = wild type.

Figure 4

Clonal and sub-clonal populations identified by single-cell DNA sequencing in Pt #2 at different time points. (A) Number of mutated clones identified in each sample analyzed: at the diagnosis of AML, on treatment, and at complete remission (CR). (B) Distribution of clonal populations with a given genotype identified across the different time points (top panel). The heat map shows the mutated genes and zygosity of each clone (bottom panel). Gray boxes represent heterozygous mutations, whereas wild-type (WT) genotypes are shown with white boxes. Legend: T1 = AML diagnosis, T2 = intensive chemotherapy plus midostaurin, T3 = complete remission. (C) Clonal and sub-clonal populations identified in Pt #2. The bar graph refers to the baseline sample, and the different colors represent the unique clonal populations identified. (D) Patterns of clonal evolution during disease progression. Number of cells with the unique assigned genotype at the different analyzed time points. (E) Fish plot visualizing patterns of clonal evolution according to variant allele frequencies at AML diagnosis, during first-line treatment, and at complete remission (CR). Abbreviations: CR = complete remission; WT = wild type.

Figure 5

Representation of clonal evolution in each patient. Each line corresponds to an individual mutation and illustrates the presence of the mutation at each analyzed time point. Each circle corresponds to an individual cell clone defined by an identical set of mutations. Somatic mutations are represented by different symbols and colors. Cells with the “X” symbols represent wild-type cells without somatic mutations detectable by scDNAseq. The table indicates the variant allele frequencies (VAF, %) of the mutated genes in each analyzed sample (bottom panel). (A) Genetic evolution of Pt #1 with stable mutations and acquired mutations. Legend: T1 = myelodysplastic syndrome (MDS) diagnosis; T2 = acute myeloid leukemia (AML) diagnosis; T3 = on-treatment; T4 = relapse. (B) Genetic evolution of Pt #2 with lost mutations. Legend: T1 = acute myeloid leukemia (AML) diagnosis; T2 = on-treatment; T3 = complete remission. Abbreviations: VAF, variant allele frequency.