Immunophenotypically defined stem cell subsets in paediatric AML are highly heterogeneous and demonstrate differences in BCL‐2 expression by cytogenetic subgroups

Abstract

In adult acute myeloid leukaemia (AML), immunophenotypic differences enable discrimination of leukaemic stem cells (LSCs) from healthy haematopoietic stem cells (HSCs). However, immunophenotypic stem cell characteristics are less explored in paediatric AML. Employing a 15‐colour flow cytometry assay, we analysed the expression of eight aberrant surface markers together with BCL‐2 on CD34+CD38− bone marrow stem cells from 38 paediatric AML patients and seven non‐leukaemic, age‐matched controls. Furthermore, clonality was investigated by genetic analyses of sorted immunophenotypically abnormal stem cells from six patients. A total of 50 aberrant marker positive (non‐HSC‐like) subsets with 41 different immunophenotypic profiles were detected. CD123, CLEC12A, and IL1RAP were the most frequently expressed markers. IL1RAP, CD93, and CD25 expression were not restricted to stem cells harbouring leukaemia‐associated mutations. Differential BCL‐2 expression was found among defined cytogenetic subgroups. Interestingly, only immunophenotypically abnormal non‐HSC‐like subsets demonstrated BCL‐2 overexpression. Collectively, we observed pronounced immunophenotypic heterogeneity within the stem cell compartment of paediatric AML patients. Additionally, certain aberrant markers used in adults seemed to be ineligible for detection of leukaemia‐representing stem cells in paediatric patients implying that inference from adult studies must be done with caution.

Proteomic Profiling Identifies Specific Leukemic Stem Cell-Associated Protein Expression Patterns in Pediatric AML Patients

“…Clearly, the AML-SCs from pAML23 and pAML29 were distributed differently from all other samples, while the AML-SCs from pAML17, pAML20, and pAML21 were situated parallel to the HSCs and HPCs ( Figure 2 a). As specified in Table 1 , cytogenetic analyses of the purified AML-SCs from pAML17 and pAML21 were negative for the KMT2A rearrangement by FISH, otherwise characterizing the leukemia [ 39 ]. Interestingly, in the PCA, the AML-SCs from these two particular cases were mapped near the HSCs, whereas the AML-SCs from pAML20, pAML23, and pAML29 were separate from this healthy counterpart.…”

“…Clinical data were obtained from the AML database administered by the Nordic Society of Pediatric Oncology and Hematology (NOPHO) ( Table 1 ). Flow cytometry data from the five pediatric AML patients, and the mutational status of the AML-SC compartment in three of the patient samples were previously determined [ 39 ]. In addition, cryopreserved MNCs from five hematologically healthy pediatric patients (aged 8 to 12 years, three females and two males) were included as controls.…”

Emerging and Future Targeted Therapies for Pediatric Acute Myeloid Leukemia: Targeting the Leukemia Stem Cells

“…Chavez-Gonzalez et al demonstrated increased expression of both CD123 and CD96, but not CD117 or CD90, in putative LSCs versus normal HSCs [57]. Petersen et al likewise confirmed increased expression of CD123, as well as CLL and IL1RAP, in primitive populations from AML patients versus healthy bone marrow donors, but noted that IL1RAP, CD93, and CD25 expression was not restricted to populations harboring AML-associated genetic mutations [58]. In retrospective analyses, both CD123 and CD200 expression have demonstrated a correlation with inferior survival and other poor prognostic features (high-risk genetics, persistent MRD) [60,63].…”

“…Much of what has been published about pediatric AML stem cell biology borrows heavily from certain foundational tenets of adult AML stem cells, which can at times create limitations in the data. For example, many of the pediatric LSC studies base their investigation of pediatric LSCs on CD34+CD38-sorted populations of cells, which in most cases is enriching for LSCs in the context of adult AML but may miss the LSC heterogeneity that we now know is present [57][58][59][60][61][62]. With those caveats in mind, the findings from these studies could still prove to be therapeutically and prognostically interesting.…”

Imaging Flow Cytometry and Convolutional Neural Network-Based Classification Enable Discrimination of Hematopoietic and Leukemic Stem Cells in Acute Myeloid Leukemia

“…Importantly, considerable immunophenotypic heterogeneity exists, and no single marker identifies all LSCs across AML samples [ 5 , 28 ]. For example, Petersen et al detected 50 LSC subsets showing 41 different immunophenotypic profiles within BM samples from 38 pediatric AML patients [ 29 , 30 ]. Also, the absence of an LSC marker on AML stem cells does not necessarily equal absence of disease, as shown by Bill et al demonstrating disease-related aberrancies within CLEC12A − stem cell subsets from AML cases [ 31 ].…”