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Deep Sequencing of CD34+ Cells Detects Measurable Residual Disease in AML

By LabMedica International staff writers
Posted on 31 Mar 2022
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Image: The BD FACSAria III cell sorter is equipped with 4 lasers and the instrument enables multicolor analysis of up to 15 parameters (Photo courtesy of BD Biosciences)
Image: The BD FACSAria III cell sorter is equipped with 4 lasers and the instrument enables multicolor analysis of up to 15 parameters (Photo courtesy of BD Biosciences)

Monitoring of measurable residual disease (MRD) in patients with acute myeloid leukemia is predictive for disease recurrence and may identify patients who benefit from treatment intensification. Current MRD techniques rely on multicolor flow cytometry or molecular methods, but are limited in applicability or sensitivity.

For patients with hematological malignancies such as acute myeloid leukemia (AML) or high risk myelodysplastic syndrome (MDS), the application of allogeneic stem cell transplantation (alloHSCT) often remains the only curative treatment option. Nevertheless, relapse after SCT occurs in 30% to 70% of AML patients and is the major cause of treatment failure, with dismal prognosis and a two-year survival of <20%.

Hematologists at the University Hospital Carl Gustav Carus TU Dresden (Dresden, Germany) and their colleagues retrospectively analyzed 429 peripheral blood (PB) and 55 bone marrow (BM) samples of 40 AML and high-risk MDS patients, with/without molecular relapse based on CD34+ donor chimerism (DC), in complete remission after alloHSCT. The team evaluated the feasibility of a novel approach for MRD detection in 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).

The team isolated CD34+ / CD117+ cells from mononuclear cells (MNCs) using magnet activated cell sorting (MACS) by positive selection using the CD34+ or the CD117+ Microbead Kit (Miltenyi Biotec, Bergisch-Gladbach, Germany). For FACS-sorting of CD34+ / CD117+ cells, the CD34 or CD117 enriched fractions were incubated with the monoclonal antibodies CD45 FITC / CD34 PE (BD Biosciences, San Jose, CA, USA). Sorting of CD34+ / CD117+ cells was then conducted on a BD FACS Aria II cell sorter, aiming for 5,000-10,000 CD34+ / CD117+ cells and a purity of >90%. DNA extraction For DNA extraction, the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) or the “ZR 168 Viral DNA Kit” (Zymo Research, Orange, CA, USA), for CD34+ / CD117+ cell counts, was used.

The investigators reported that enrichment of CD34+ cells for NGS increased the detection of mutant alleles in PB ~1000-fold (median Variant Allele Frequency [VAF] 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 studies ~10-fold to levels of 10-6. In both assays, MRD detection was superior using PB versus BM for CD34+ enrichment. Importantly, 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 the early assessment of clonal trajectories in hematological complete remission.

The authors have proposed a novel, easily accessible and robust method for ultra-sensitive MRD detection in peripheral blood, which is applicable to the vast majority of AML patients. First results demonstrate the feasibility of targeted deep sequencing on CD34+ cells for early relapse prediction in clinical settings, with superior sensitivity and specificity as compared to chimerism-based MRD assessment or the use of unsorted PB for NGS. The study was published on March 23, 2022 in the journal Blood Advances.

Related Links:
University Hospital Carl Gustav Carus TU Dresden 
Miltenyi Biotec 
BD Biosciences 
Qiagen 
Zymo Research

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