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Single-Cell MRD Assay Helps Characterize Cells Surviving Acute Myeloid Leukemia Therapy

NEW YORK – A team led by investigators at Memorial Sloan Kettering Cancer Center has developed a measurable residual disease (MRD) assay for identifying and studying cells that survive after treatment for acute myeloid leukemia (AML).

The single-cell approach was described in a paper appearing in Science Advances on Wednesday.

There, investigators described a multiplex single-cell MRD assay that relies on flow cytometry-based enrichment of AML blasts and precursor cells, followed by single-cell sequencing of 31 AML-related genes and "immunophenotyping based on a panel of cell surface proteins."

"The potential implications of understanding clones at single cell levels are to pinpoint exact clones responsible for relapse and to provide a way to study therapeutic vulnerability on these high-risk clones," co-senior and co-corresponding author Wenbin Xiao, a hematopathologist at MSK, said in an email, noting that these features are not clear from conventional strategies for detecting MRD in AML patients.

Disease relapse is one of the most vexing features of AML, Xiao and his colleagues explained. While the condition — marked by immature myeloid blast cell expansions — tends to respond well to treatment, investigators are searching for strategies to accurately detect post-treatment disease remnants that may flare up again with time.

"Understanding the biology enabling MRD clones to resist therapy is necessary to guide the development of more effective curative treatments," the authors explained. "Discriminating between residual leukemic clones, preleukemic clones, and normal precursors remains a challenge with current MRD tools."

After assessing the sensitivity of their scMRD assay in sets of diluted AML blast and normal bone marrow samples, the investigators applied it to 30 previously frozen samples from 29 individuals with AML, including seven samples collected from six AML patients after allogeneic hematopoietic stem cell transplant treatments.

In those individuals, the assay showed sensitivity and specificity that was comparable to that of conventional bulk next-generation sequencing-based MRD assays, while providing additional clonal architecture clues.

The team saw similar MRD status with both bulk sequencing and the scMRD assay in more than three-quarters of the AML cases, identifying 44 overlapping mutations in a broader set of 76 mutations found by either method.

"Among the mutations detected by both scMRD and bulk NGS, variant allelic frequencies (VAFs) were significantly higher by scMRD," the authors reported, noting that scMRD "identified 17 mutations that were missed/unreported by bulk NGS."

They also conducted experiments showing that the scMRD assay can be used in a multiplexed manner to make it more cost-effective. In addition, the researchers used single-cell mutation and immunophenotyping clues found with the scMRD assay to tease out the clonal features found in residual disease.

In particular, the scMRD strategy "readily resolves clonal architecture and has the potential to distinguish between single-mutant [clonal hematopoiesis]/preleukemic versus leukemic clones with multiple co-occurring mutations," the authors wrote.

"As [clonal hematopoiesis]/preleukemic clones do not invariably portend relapse," they explained, "this distinction may be critical for clinical decision-making."