MRD in AML Detected by Deep Sequencing CD34+ Cells

Monitoring of measurable residual disease (MRD) in patients with acute myeloid leukemia (AML) is predictive of disease recurrence and may identify patients who benefit from treatment intensification.

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 

Visit expo >

Automated ELISA-IFA-BLOT Processor AP 22

Gold Supplier

SARS-CoV-2 IgM ELISA Kit

SARS-CoV-2 (COVID-19) IgM ELISA

New

Gold Supplier

Influenza Type A & B Test

Influenza A+B DUO

New

Modular Sequencing Workstation

MGIFLP-L200