Identification of an abnormal immunophenotype by flow cytometric analysis is important for the diagnosis of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Several markers have been identified that are expressed in leukemic blasts but are usually absent in normal myeloid precursors including CD56, CD7 and CD11b. However, these markers are only expressed in approximately 20-30% of acute myeloid leukemias (Raspadori, D. 2001; Jen, W-Y. 2024; Juncà, J. 2014) and can be present in non-neoplastic conditions. Regardless, the presence of markers of aberrancy can aid diagnosis and, if present, can significantly improve detection of measurable residual disease at follow-up. We aim to demonstrate the utility of recently described markers, IL1RAP and CD366 in clinical flow cytometric analysis.

IL1RAP is a co-receptor required for IL-1 signaling that has been reproted to be expressed on the leukemic stem cells but absent on normal myeloid precursors (Weeda, V 2022). Additionally, it may be a viable therapy target with bispecific antibodies under investigation (Ågerstam, H. 2015; Askmyr, M. 2013; Meng, W. 2017). However, IL1RAP expression by flow cytometric analysis has essentially only been studied on cell line or leukemic stem cell models and not by clinical flow cytometric analysis on patients.

CD366 (TIM-3) is expressed on IFN-γ producing activated T cells, playing a role in immune tolerance. A few studies have described a significantly higher frequency of CD366 expression in AML by flow cytometry with CD366 being absent on normal hematopoietic stem cells (Roth, C. 2013; Zeijlemaker, W. 2016; Haubner, S. 2018).

We validated a 12-color flow cytometry assay for the evaluation of myeloid neoplasia that includes these markers, following CAP and CLSI guidelines (Wang, S. 2023). We retrospectively reviewed IL1RAP and CD366 expression in blasts in the setting of myeloid neoplasia versus expression in normal hematopoietic precursors, systematically, using the fold-over-background methods we have described elsewhere to classify cases as positive or negative for expression (Maggard, J. 2025). All cases were received for clinical flow cytometric analysis at our institution.

The myeloid neoplasia cohort consisted of 20 bone marrow and 7 blood specimens from 27 different patients with the diagnosis (N) of AML (15), MDS (4), MDS/AML (5), chronic myeloid leukemia (2), and clonal cytopenia of undetermined significance (1). The mean patient age was 68 years with a male:female ratio of 1.7. IL1RAP was positive in the neoplastic blast population in 6 cases (22.22%) while CD366 was positive in only 1 case (3.7%). The lone CD366 positive case also expressed IL1RAP and was an AML with NPM1 mutation. Among the IL1RAP positive cases, the average MFI (median fluorescence intensity) was 605.43 (interquartile range 308.3-947.29). The lone CD366 positive case had a CD366 MFI of 1761.75.

Maturing myeloid precursors in 16 normal bone marrows from different patients with no history of myeloid neoplasia showed an average MFI for IL1RAP of 204.42 (95.49-409.537, p=0.001 vs the myeloid neoplasia cohort's positive cases) and 655.5 (435.29-818.58) for CD366. Of these, 2 were classified as IL1RAP positive (12.5%) and 1 of these was also classified as CD366 positive (6.25%).

We are the first center to report on the aberrant expression of IL1RAP in myeloid neoplasia using clinical flow cytometric analysis of actual patient specimens. We were unable to reproduce the finding of increased CD366 expression in AML described by others, calling into question its use. While our sample size is limited, IL1RAP shows promise as a marker of aberrancy, with a similar frequency of expression as other markers like CD56 and should be considered for inclusion in clinical flow cytometry panels.

All authors contributed equally to this abstract.

No AI was used to generate this abstract.

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2025
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