Monitoring of Leukemia Clones in B-cell Acute Lymphoblastic Leukemia at Diagnosis and During Treatment by Single-cell DNA Amplicon Sequencing

Abstract

Acute lymphoblastic leukemia (ALL) is characterized by the presence of chromosomal changes, including numerical changes, translocations, and deletions, which are often associated with additional single-nucleotide mutations. In this study, we used single cell-targeted DNA sequencing to evaluate the clonal heterogeneity of B-ALL at diagnosis and during chemotherapy treatment. We designed a custom DNA amplicon library targeting mutational hotspot regions (in 110 genes) present in ALL, and we measured the presence of mutations and small insertions/deletions (indels) in bone marrow or blood samples from 12 B-ALL patients, with a median of 7973 cells per sample. Nine of the 12 cases showed at least 1 subclonal mutation, of which cases with PAX5 alterations or high hyperdiploidy (with intermediate to good prognosis) showed a high number of subclones (1 to 7) at diagnosis, defined by a variety of mutations in the JAK/STAT, RAS, or FLT3 signaling pathways. Cases with RAS pathway mutations had multiple mutations in FLT3, NRAS, KRAS, or BRAF in various clones. For those cases where we detected multiple mutational clones at diagnosis, we also studied blood samples during the first weeks of chemotherapy treatment. The leukemia clones disappeared during treatment with various kinetics, and few cells with mutations were easily detectable, even at low frequency (<0.1%). Our data illustrate that about half of the B-ALL cases show >2 subclones at diagnosis and that even very rare mutant cells can be detected at diagnosis or during treatment by single cell-targeted DNA sequencing.

Figure 1.

Heatmap of genomic characteristics of all patient samples at diagnosis. Each column represents 1 of the 12 B-ALL patients and each row represents a specific genetic alteration. The upper part of the heatmap (orange gradient; number of variants per patient and gene) shows SNVs or indels identified with single-cell DNA sequencing, with the first column showing the number of variants in each gene and the top row (blue gradient) the number of variants per patient. The lower part of the heatmap (red gradient) displays the major chromosomal alterations that were found by molecular diagnostics, karyotyping, bulk RNA-sequencing, or Bionano Optical Genome Mapping. The top row (green gradient) shows the number of chromosomal alterations per patient.

Figure 2.

Quality metrics and amplicon coverage of B-ALL patient samples based on single-cell DNA amplicon sequencing. (A) Total number of sequenced cells (left panel), number of reads per amplicon per cell (middle panel), and mean allele dropout rates (right panel). Details on sample and sequencing metrics are in Suppl. Table S1 and ADO calculations in Suppl. Table S3. Each dot is one of the 23 sequenced samples of the 12 B-ALL patients and the horizontal line represents the median value. (B) Bar plot of average amplicon coverage per cell per sample. Each bar represents 1 of the 305 amplicons, and the height of the bar represents the average number of reads per cell. The 15 lowest performing amplicons with an average coverage below 10 reads (represented by horizontal dashed red line) are listed on the right. Detailed data of amplicon coverage per sample can be found in Suppl. Table S2.

Figure 3.

Clonal composition and mutational history based on single-cell sequencing data using the InfSCITE algoritm. (A) Heatmap of clonal architecture of diagnostic samples of patients XF109, XG111, XG115, and XG124. Because of potential allele dropout, zygosity information is not displayed (wild type [WT] = light gray, mutant [Mut] = dark gray). The heatmap shows the genotype consequence of each relevant somatic mutation and the colored horizontal bar at the top shows the clonal composition of the sample. (B) Phylogenetic trees showing the most likely order of variant acquisition during B-ALL development. Mutational history was inferred for diagnostic samples of patients XF109, XG111, XG115, and XG124 using the InfSCITE algorithm. Circles represent the clones and the outline color of the circles corresponds with the clone names in (A). The size of each circle represents the relative clone size in the sample, which is also indicated by the frequency next to the circles.

Figure 4.

In-frame alterations in FLT3 in case XG115. Overview of the alterations in the juxtamembrane domain of FLT3 in XG115. Alterations were identified based on inspection of sequencing reads in Integrative Genomics Viewer: yellow denotes deletions, purple denotes insertions, and green denotes mutations. All alterations identified were in-frame.

Figure 5.

Clonal evolution during chemotherapy treatment. Fish plots showing clonal evolution during chemotherapy treatment in patients XF109, XG111, XG115, and XG124. At each timepoint, the total number of cells passing the filtering parameters is indicated. The somatic mutations (which are covered by this amplicon panel) per clone and the number of cells and frequencies are shown in Suppl. Table S5.