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Scientific presentation

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


Sarah Meyers, PhD
Description

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 variants. We used the single-cell amplicon sequencing technology by Mission Bio to evaluate the clonal heterogeneity of ALL at diagnosis and during chemotherapy treatment.

We previously designed and optimized a custom DNA amplicon panel targeting mutational hotspot regions in ALL and validated this panel in a cohort of 8 T-ALL cases. In T-ALL, we typically observed a major clone at diagnosis accompanied by several minor clones. Half of the patients in our cohort had >2 mutations in NOTCH1, indicating a strong pressure to acquire NOTCH1 mutations in developing T-ALL cells. By analyzing longitudinal samples during treatment, we detected the presence and clonal nature of residual leukemic cells and found minor clones that evolved to clinically relevant major clones at later disease stages.

We later focused on B-ALL and sequenced bone marrow and blood samples of 12 patients with a median capture recovery of 7973 single cells per sample. Nine of the 12 cases showed at least 1 subclonal mutation, of which cases with PAX5 alterations or high hyperdiploidy showed a high number of subclones at diagnosis, defined by a variety of alterations in the JAK/STAT, RAS, or FLT3 signaling pathways. Cases with affected RAS pathway had multiple mutations in FLT3, NRAS, KRAS, or BRAF in various clones. For those patients where we detected multiple subclones in the diagnostic sample, we sequenced additional blood samples during the first weeks of chemotherapy treatment. During treatment, the leukemia clones disappeared with various kinetics, but we were able to detect remaining mutated cells even at low frequency. Our data illustrate that about half of the B-ALL cases show multiple subclones at diagnosis and that even very rare mutant cells can be detected by targeted single-cell DNA sequencing.



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