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.

The Promise of Single-cell Technology in Providing New Insights Into the Molecular Heterogeneity and Management of Acute Lymphoblastic Leukemia

“…For example, targeted scDNA-seq combined with high-throughput droplet-based microfluidics technology accurately identified SNVs and small insertion-deletion mutations in 305 genomic regions in over 100,000 cells from both T-ALL 30 and B-ALL patients. 72 Both studies provided a comprehensive insight into the clonal diversity of pediatric ALL at diagnosis. Interestingly, whereas the majority of T-ALL patients accumulated subclonal mutations, 30 clonal heterogeneity in B-ALL appeared to be more subtype-specific, where patients with high hyperdiploidy or PAX5 alterations had a higher number of subclones.…”

“…Interestingly, whereas the majority of T-ALL patients accumulated subclonal mutations, 30 clonal heterogeneity in B-ALL appeared to be more subtype-specific, where patients with high hyperdiploidy or PAX5 alterations had a higher number of subclones. 72 Additionally, Alberti-Servera et al highlighted a high degree of heterogeneity of NOTCH1 mutations in T-ALL. NOTCH1 was the most frequently mutated gene, with 33 variants identified across 8 patients.…”

“…The limitation of low cell number is now in part overcome, with more recent studies taking advantage of new technology, which utilize high‐throughput methods to capture thousands of cells per sample. For example, targeted scDNA‐seq combined with high‐throughput droplet‐based microfluidics technology accurately identified SNVs and small insertion‐deletion mutations in 305 genomic regions in over 100,000 cells from both T‐ALL 30 and B‐ALL patients 72 . Both studies provided a comprehensive insight into the clonal diversity of pediatric ALL at diagnosis.…”

XBP1 promotes NRASG12D pre‐B acute lymphoblastic leukaemia through IL‐7 receptor signalling and provides a therapeutic vulnerability for oncogenic RAS

“…However, in B‐ALL, activating RAS mutations, namely in NRAS and KRAS , are frequently found upon relapse and are known to confer resistance to chemotherapy 1,3–5 by aberrant activation of the downstream signalling cascade via RAF/MEK/ERK with a connection to PI3K/AKT/mTOR signalling 6–8 . The prevalence of RAS pathway mutations has been found to differ significantly depending on the selection of the study population and varies from around 10% in a study with no preselection of the study population to 50% in a population encompassing high‐risk common ALL 7,9–11 . Activation of RAS–ERK signalling results in the expression of MAPK negative regulators dual specificity phosphatase 6 (DUSP6), ETS variant 5 (ETV5) and sprouty RTK signalling antagonist 2 (SPRY2), which regulate RAS–ERK signalling via a negative feedback loop 12–15 .…”

“…[6][7][8] The prevalence of RAS pathway mutations has been found to differ significantly depending on the selection of the study population and varies from around 10% in a study with no preselection of the study population to 50% in a population encompassing high-risk common ALL. 7,[9][10][11] Activation of RAS-ERK signalling results in the expression of MAPK negative regulators dual specificity phosphatase 6 (DUSP6), ETS variant 5 (ETV5) and sprouty RTK signalling antagonist 2 (SPRY2), which regulate RAS-ERK signalling via a negative feedback loop. [12][13][14][15] Interleukin-7 (IL-7) is a critical cytokine for survival, homeostasis and development of B and T cells.…”