MRD in AML Detected by Deep Sequencing CD34+ Cells

For patients with hematological malignancies such as AML or high-risk myelodysplastic syndrome (MDS), allogeneic hematopoietic stem cell transplantation (allo-HSCT) often remains the only curative treatment option. Nevertheless, relapse after allo-HSCT occurs in 30% to 70% of patients with AML and is the major cause of treatment failure, with dismal prognosis and a 2-year survival of less than 20%.

Medical Scientists at the University Hospital Carl Gustav Carus (Dresden, Germany) evaluated the feasibility of a novel approach for MRD detection in peripheral blood (PB), which combines immunomagnetic pre-enrichment and fluorescence-activated cell sorting (FACS) for isolation of CD34+ cells with error-reduced targeted next-generation sequencing (NGS). For clinical validation, they retrospectively analyzed 429 PB and 55 bone marrow (BM) samples of 40 patients with AML or high-risk MDS, with/without molecular relapse based on CD34+ donor chimerism (DC), in complete remission after allogeneic stem cell transplantation.

The scientists ensured consistent enrichment of CD34+/CD117+ cell populations to high purity (i.e. >90%). CD34+/CD117+ cells were enriched by using a combination of magnetic cell separation (MACS) and FACS for all samples. Mononuclear cells (MNCs) were extracted by density gradient centrifugation and CD34+/CD117+ cells were isolated from MNCs using MACS by positive selection with the CD34+ or CD117+ Microbead Kit (Miltenyi Biotec, Bergisch-Gladbach, Germany). Sorting of the CD34+/CD117+ cells was then conducted on a BD FACS Aria II cell sorter (BD Biosciences, San Jose, CA, USA), aiming for 5,000 to 10,000 CD34+/CD117+ cells and a purity of >90%. Sequencing of genomic DNA from sorted CD34+/CD117+ cells and unsorted material was performed according to an optimized protocol for error-reduced NGS-based detection of low-level, single-nucleotide variants.

The investigators reported that enrichment of CD34+ cells for NGS increased the detection of mutant alleles in PB ∼1,000-fold (median variant allele frequency, 1.27% versus 0.0046% in unsorted PB). Although a strong correlation was observed for the parallel analysis of CD34+ PB cells with NGS and DC the combination of FACS and NGS improved sensitivity for MRD detection in dilution experiments ∼10-fold to levels of 10−6. In both assays, MRD detection was superior using PB versus BM for CD34+ enrichment. Importantly, next-generation sequencing (NGS) on CD34+ PB cells enabled prediction of molecular relapse with high sensitivity (100%) and specificity (91%), and significantly earlier (median, 48 days; range, 0-281) than by CD34+ DC or NGS of unsorted PB, providing additional time for therapeutic intervention. Moreover, panel sequencing in CD34+ cells allowed for the early assessment of clonal trajectories in hematological complete remission.

The authors proposed a novel, easily accessible, robust method for ultrasensitive MRD detection in PB that is applicable to most patients with AML. Initial results demonstrated the feasibility of targeted deep sequencing of CD34+ cells for early relapse prediction in clinical settings, with superior sensitivity and specificity, as compared with chimerism-based MRD assessment or the use of unsorted PB for NGS. The study was published on June 2, 2022 in the journal Blood Advances.

Related Links:
University Hospital Carl Gustav Carus 
Miltenyi Biotec 
BD Biosciences