Real-World Performance of 2022 Revised World Health Organization (WHO) Criteria for Mixed Phenotype Acute Leukemia (MPAL) B/Myeloid Defined Only By Isolated MPO Expression (isoMPO)

Mixed phenotype acute leukemia (MPAL) is biologically and immunophenotypically heterogeneous, and optimal diagnostic criteria remain unclear. The two current pathologic classifications [2022 World Health Organization (WHO) 5^th Ed. and International Consensus Classification (ICC)] group MPAL by cell lineages and, when present, cytogenetic drivers. In both, myeloperoxidase (MPO) is a key determinant of myeloid lineage. However, the diagnosis of MPAL has been inconsistently applied to cases of acute leukemia expressing MPO alone to define myeloid lineage. To address this, the 2022 WHO revised prior cell lineage definitions to emphasize antigen intensity (≥50%) relative to a positive control population, attempting to standardize lineage assignment across flow cytometer settings and pathologist interpreters. Distinction between pediatric B-lymphoblastic leukemia (B-ALL) and MPAL B/Myeloid with isolated MPO is crucial for therapeutic decision-making. For example, on Children’s Oncology Group (COG) regimens, patients with MPAL B/Myeloid receive an anthracycline during induction and intensified chemotherapy post-induction, whereas standard-risk B-ALL patients do not. We hypothesized that the 2022 WHO criteria applied to MPO expression in children with acute leukemia would identify leukemic subtypes (B-ALL vs. MPAL B/Myeloid with isolated MPO) with differences in clinical characteristics and/or outcome.

We applied 2022 WHO criteria to flow cytometry data from a multicenter pediatric cohort of leukemias originally classified as MPAL B/Myeloid or B-ALL (Oberley AJCP 2017; Raikar Blood 2018; Oberley Leukemia 2020). Maximal antigen intensity on the leukemic population was measured in 0.5 log increments and normalized to maximal intensity of a control (neutrophil) population. MPO expression was scored as none, low (<50% intensity of mature neutrophils, the typical level in B-ALL with isolated MPO expression) or high (≥50% intensity of neutrophils, meeting 2022 WHO criteria for myeloid lineage assignment). Since the current study focused on MPAL B/Myeloid cases with isolated MPO expression, cases of MPAL T/Myeloid and of MPAL B/Myeloid with myeloid lineage assignment based on monocytic markers were excluded. Clinicopathologic data were compared with MPO expression scores by univariate and multivariate analyses. Primary endpoint was categorical difference in presenting features. Secondary endpoints were end of induction minimal residual disease (EOI MRD) ≥0.01%, event-free survival (EFS) and overall survival (OS). All analyses were two-sided; p<0.05 was the threshold for statistical significance.

Among all patients at diagnosis (n=176), median age was 6.14 years (range 0.3-21), 68% were <10 years old, 59% were male, 63% were Hispanic, 76% had initial white blood cell (WBC) count <50K/µL, and 81% were CNS1. MPO was absent, low (<50%) or high (≥50%) in 48%, 29% and 23% of the cohort, respectively. Prevalence of favorable cytogenetics (ETV6::RUNX1 or double trisomy) was inversely proportional to MPO intensity [no MPO (50%), low MPO (27%), high MPO (17%); p<0.001). MPO expression (low or high) was more prevalent in patients >10 years of age (p<0.001). There was no difference in MPO by presenting WBC count (p=0.40). Among patients treated with ALL induction with known EOI MRD results (n=166), MPO intensity was not significantly associated with EOI MRD positivity, EFS or OS (multivariate p>0.40 for all three endpoints after accounting for presenting features and initial therapy).

Our study demonstrates that 2022 WHO criteria for interpreting MPO intensity can be consistently applied to flow cytometry data from a variety of laboratories. Based on our analysis, only 44% of previous MPAL B/Myeloid cases with isolated MPO were now classified as MPAL. However, the clinical significance of this distinction remains unclear. After accounting for differences in presenting features, MPO expression was not additionally predictive of MRD positivity, EFS or OS. Further investigation is required to support the proposed threshold of 50% MPO intensity of a neutrophil control population as clinically relevant to assign myeloid lineage. Prospective standardized application of 2022 WHO cell lineage assignment criteria across centers will facilitate detailed understanding of the biological underpinnings and optimal therapeutic strategies for this heterogeneous disease.