Comprehensive Analysis of Target and Immune Checkpoint Markers in Pediatric AML

Introduction Pediatric acute myeloid leukemia (AML) is a rare hematologic malignancy in which first-line treatment leads to relapse or refractory disease in one-third of patients, often with a devastating prognosis. To date, immunotherapeutic approaches have not been successfully introduced in the treatment of AML, which is mainly due to the lack of suitable target antigens and functional immunoinhibitory checkpoint proteins (IC). Here, we investigate the expression of nine different IC and target antigens on AML blasts using multicolor flow cytometry (MFC) and correlate the most promising expression patterns with prognostic and therapeutic factors for the outcome of pediatric AML.

Methods Candidate target and IC antigens were analyzed in bone marrow or peripheral blood samples from 207 pediatric AML patients (0-18 years) at diagnosis within the AML-BFM study group (Germany) from July 2021 to May 2024. MFC analysis was performed using Beckman Coulter’s Kaluza Analysis Software 2.1. Positivity was defined for each marker using positive and/or negative reference populations with cut-off values of 10% (negative vs weak positive) and 50% (weak vs strong positive) according to the AIEOP-BFM Consensus Guidelines 2016 for Flow Cytometric Immunophenotyping of Pediatric ALL (Dworzak et al., 2017). Expression of each candidate antigen was assessed for CD34⁺ and CD34⁺CD38^- subsets of blasts. MFC results were correlated with clinical and molecular genetic data.

Results The highest percentages of positive blasts were observed for the myeloid marker CD33/SIGLEC3 (N=181, 97.3±7%), CD312/EMR2 (N=70, 92.3±16%), CD371/CLL1 (N=181, 86.7±24%) and CD123/IL3Rα (N=181, 59.5±28%). Among the immunoregulatory markers, CD47/IAP (N=179, 70.9±28%), CD85d/LILRB2 (N=46, 50.2±27%), CD120b/TNFR2 (N=105, 61.2±33%) and CD366/TIM3 (N=155, 35.5±26%) showed the highest positivity on pediatric AML blasts, while CD274/PD-L1 (N=71, 15.0±19%), Mesothelin (N=47, 10.3±18%), and VISTA (N=92, 6.5±10%) were only weakly expressed.

A relevant proportion of CD34⁺CD38^- cells was found among blasts positive for CD123 (12%), CD85d (11%), CD47 (10%) and CD312 (9%), with total CD34⁺-fractions ranging from 37% to 48%. CD123 and CD366 showed the most favorable off-target profiles, with only 4.8% and 7.8% positive cells among healthy lymphocytes and 21.2% and 29.8% on granulocytes, respectively.

In AML blasts, positivity for the immunoregulatory marker CD120b correlated with the expression of CD85d (r=0.6, p<.001) and CD70 (r=0.5, p<.001). The IC CD366 was most frequently co-expressed with CD371 (r=0.3, p<.001), which in turn was strongly co-expressed with the myeloid markers CD33 (r=0.3, p<.001) and CD312 (r=0.3, p<.001).

Percentage of positivity for CD120b was associated with KMT2A-rearranged AML (83.1 vs 52.5%, p<.001) and monocytic phenotype (AML FAB M5, 93.1 vs 50.9%, p<.001) compared to other classified AML. Positivity of CD371 and CD123 was increased within standard-risk (SR) rather than high-risk AML (93% vs 85%, p=.008 and 71% vs 56%, p=.001). This trend was also evident for double positivity of CD371/CD123, which was associated with SR AML (35 vs 11%, p=.002), and CBF-AML (25 vs 10%, p=.01) but negatively linked to complex karyotype (9 vs 25%, p=.006) compared to marker-negativity. Accordingly, the positivity of these two markers was related to favorable 2-year overall survival rates (CD123: 83.5±5% vs 58.1±2%, p=.01 and CD371: 89.6±4% vs 68.0±9%, p=.03).

Conclusion The results suggest favorable prognostic impact for CD123 and CD371 positivity at diagnosis in pediatric AML. Given the on-tumor and off-target profiles, we identified CD123 and the IC marker CD366 as the most promising target candidates in pediatric AML. Our results indicate that the combination of CD123/CD371 as well as CD85d/CD120b may provide promising combinatorial strategies for future therapeutic approaches. In general, this large-scale study provides fundamental data for a variety of therapeutic approaches for immunomodulation and targeted therapy strategies in pediatric AML.