Immunophenotypical profiling of myeloid neoplasms with erythroid predominance using mass cytometry (CyTOF)

Abstract

Acute erythroid leukemia (AEL) is a disease continuum between Myelodysplastic syndrome (MDS) and Acute myeloid leukemia (AML) with the cellular hallmark of uncontrolled proliferation and impaired differentiation of erythroid progenitor cells. First described by Giovanni di Guglielmo in 1917 AEL accounts for less than 5% of all de novo AML cases. There have been efforts to characterize AEL at a molecular level, describing recurrent alterations in TP53, NPM1 and FLT3 genes. A genomic analysis of AEL cases confirmed its complexity. Despite these advances, the biology underlying erythroid proliferations remains unclear and the prognosis is dismal with a median survival of only 3 months for pure erythroid leukemia (PEL). Marker combinations suitable for the identification and characterization of leukemic stem cell (LSC) candidates, monitoring measurable residual disease (MRD) during chemotherapy treatment and the development of innovative targeted therapies are missing. Here, we developed a mass cytometry panel for an in-depth characterization of erythroid and myeloid blast cell populations from human AEL bone marrow samples in comparison to other AML subtypes and healthy counterparts. A total of 8 AEL samples were analyzed and compared to 28 AML samples from different molecular subtypes, healthy bone marrow counterparts (n = 5) and umbilical cord blood (n = 6) using highdimensional mass cytometry. Identification of erythroid and myeloid blast populations in high-dimensional mass cytometry data enabled a refined view on erythroblast differentiation stages present in AEL erythroid blasts and revealed immunophenotypical profiles specific to AEL. Profiling of phenotypic LSCs revealed aberrant erythroid marker expression in the CD34 + CD38 À stem cell compartment. In addition, the identification of novel candidate surface marker combinations and aberrancies might enhance clinical diagnostics of AEL. We present a high-parameter mass cytometry approach feasible for immunophenotypical analysis of blast and stem cell populations in myeloid neoplasms with erythroid predominance laying the foundation for more precise experimental approaches in the future.

Monitoring Measurable/Minimal Residual Disease in Acute Myeloid Leukemia: Multiparametric Flow Cytometry-Based Approach

“…The availability of new technologies such as CyTOF mass spectrometry and full-spectrum flow cytometry (FSFC) has demonstrated the capacity to utilize many markers (up to 27 colors) for AML MRD. 84 85 Additionally, by increasing the number of events from 1 million to 10 million, the sensitivity of MRD assay can easily be reached to as high as 2 × 10 −6 (0.0002%) MRD detection. 63 Thus, the recent availability of new flow cytometry technologies and newer dyes has improved the assay sensitivity and specificity for MRD assessment by flow cytometry.…”

Erythroblastic islands: A fertile ground for CD47 expression in myelodysplastic syndrome and acute erythroid leukaemia

“…Although it has not been demonstrated yet, a comparable mechanism may occur in neoplastic erythroblastic islands. As illustrated in this case, MDS and AEL are recognised as part of a disease continuum and increased CD47 expression has been observed in both myeloblasts and erythroblasts [2]. Therefore, anti‐CD47 immunotherapy should be a therapeutic option to consider in cases of AEL and MDS with increased dysplastic erythroid lineage.…”

Measurable residual disease monitoring in patients with acute myeloid leukemia treated with lower‐intensity therapy: Roadmap from an ELN‐DAVID expert panel

“…The feasibility, applicability, and prognostic effect of MFC‐MRD testing in lower‐intensity regimens have already been demonstrated ( Recommendation 1 ) 12,13 . However, the development of novel assays such NGS, digital droplet PCR, single‐cell sequencing, and highly multiplexed immunophenotyping (e.g., mass cytometry and Cytof) 28 is providing us with new avenues for detection and monitoring MRD as well as enabling us to better understand the biology of MRD. For the time being, technical recommendations provided in the ELN‐MRD guideline for MRD assessment by MFC‐MRD and molecular MRD apply also to patients treated with lower‐intensity regimens ( Recommendation 2 ) 24 .…”

“…25 The feasibility, applicability, and prognostic effect of MFC-MRD testing in lower-intensity regimens have already been demonstrated (Recommendation 1). 12,13 However, the development of novel assays such NGS, digital droplet PCR, single-cell sequencing, and highly multiplexed immunophenotyping (e.g., mass cytometry and Cytof) 28 aberrations. This has already been used extensively in intensive regimens but may also be applicable to patients treated with lowerintensity regimens.…”