Acute myeloid leukemia (AML) is a heterogenous disease with an emerging landscape of genetic and cytogenetic abnormalities. Natural killers (NK) cells recognize the tumor cells independent of MHC via cognate ligands of activating receptors such as DNAX accessory molecule-1(DNAM-1) and Natural Killer group 2, member D (NKG2D). Use of allogenic NK cells in a killer-cell immunoglobulin receptor (KIR)-mismatch setting has emerged as an encouraging treatment in patients diagnosed with AML. Knowledge about the expression of ligands of activating receptors on AML cells with various underlying genetic and cytogenetic abnormalities is important in the context of allogenic NK cell therapy. This study evaluates the expression of Nectin (CD112) and Poliovirus receptor (CD155) (which are ligands for DNAM-1) and MICA/BMHC class I polypeptide-related sequence A/B (MICA/B)(which are ligands for NKG2D) on CD34+CD38- leukemia- initiating cells (LICs) and CD38+CD13+ myeloid progenitor cells in AML cases harboring various genetic and cytogenetic abnormalities.

Patients with data on genetic and cytogenetic profile signed the informed consent and the study was conducted according to the declaration of Helsinki. The study was approved by the medical ethics committee of Aga Khan University (No.2024-8539-27830). Peripheral blood mononuclear cells were isolated from the whole blood of patients prior to the start of treatment. Cells were stained with CD45-FITC, CD34-APC, CD38-PE-Vio770, CD13-APC-Vio770 in three tubes followed by staining with CD112-PE, CD155-PE and MICA/B-PE in each tube. 7-AAD was added prior to the analysis by flow cytometry. Data was analyzed using FlowJo software using fluorescent minus one (FMO) control for gating. Live CD45+CD34+CD38- LICs and live CD45+CD38+CD13+ myeloid progenitor cells were analyzed for cell surface expression of CD112, CD155 and MICA/B by calculating relative mean fluorescence intensity (RMFI) using unstained control. Statistical analysis was done using GraphPad Prism and p value of <0.05 was considered statistically significant.

A total of 23 cases of AML were grouped as follows: APML with PML::RARĪ± fusion (9 cases), AML with RUNX1::RUNX1T1 fusion (5 cases) and AML with Nucleophosmin 1 (NPM-1) mutation (3 cases). Six cases of AML with defined cytogenetic abnormalities were grouped and included 2 cases of complex karyotype and one case each of 1q deletion, monosomy 7, trisomy 11, trisomy 21 (Khoury et.al. Leukemia 2022).

LICs harboring PML::RARĪ± fusion showed a statistically significant increase in the expression of CD112 (RFMI 572.3) compared to CD155 (RFMI 281.6) and MICA/B (RFMI 280.5). Similarly, progenitor cells from this group showed a statistically significant increase in the expression of CD112 (RFMI 515.9) compared to CD155 (RFMI 330.2) and MICA/B (RFMI 296.9). LICs in AML cases with RUNX1::RUNX1T1 fusion also showed statistically significant increase in the CD112 expression (RMFI 441.5) compared to CD155 (RMFI 223.1) and MICA/B (RMFI 176). Progenitors in this group showed statistically significant increase in CD112 expression (RMFI 514.9) compared to CD155 (RMFI 266.1) and MICA/B (RMFI 251.9). AML cases with NPM-1 mutation showed an increase in the expression of CD112 on LICs (RMFI 641) compared to CD155 (RMFI 470) and MICA/B (RMFI 461). Progenitors from this group showed a similar trend (RFMI of CD112: 963.7, RFMI of CD155: 597.3 and RFMI of MICA/B: 364.3). However, these data were not statistically significant. AML cases with defined cytogenetic abnormalities showed an increase in expression of CD112 (RFMI 365.3) and MICA/B (RFMI 260.5) compared to the expression of CD155 (RFMI 208.8) on LICs. These data were not found to be statistically significant. Progenitors from this group showed a statistically significant increased expression of CD112 (RFMI 960.4) compared to MICA/B (RFMI 284.3) but there was no significant difference between the expression of CD112 and CD155 (RFMI 382.4).

In conclusion, LICs and myeloid progenitors in AML cases with various genetic and cytogenetic abnormalities showed an increase in the expression of CD112 compared to CD155 and MICA/B. We suggest that the evaluation of expression of NK ligands on various populations of AML cells with different genetic and cytogenetic abnormalities will help in assessing the effectiveness of NK cell therapy in eradicating the LICs and rationalizing the use of NK therapy with strategies to enhance expression of specific ligands.

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2025
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