Combinatory Flowcytometric Approach in Pediatric Acute Lymphoid Leukemia Identifies Surrogate Minimal Residual Disease Markers

Abstract

Background/Objectives: Minimal residual disease (MRD) refers to the resistant clonal population of leukemia cells that survive induction chemotherapy, serving as a critical indicator of treatment response in pediatric Acute Lymphoid Leukemia (ALL). While flow cytometry (FCM) and molecular methods are standard for MRD detection, novel leukemia-associated immunophenotype (LAIP) markers are needed when conventional markers are insufficient. Methods: MRD was assessed in 218 pediatric B-ALL patients using a combinatory approach of Different-from-Normal (DfN) and LAIP strategies. An eight-color flow cytometry panel included routine MRD markers (e.g., CD10, CD19, and CD20) and less commonly used markers (e.g., CD123, CD73, CD86). Cytogenetic and molecular profiling were integrated to evaluate the association between genetic abnormalities and MRD positivity. Results: The combined DfN and LAIP approach enhanced MRD detection sensitivity compared to individual methods. CD7 showed a significant association with MRD positivity (p = 0.003), whereas CD73 (p = 0.000) and CD86 (p = 0.002) correlated with MRD-negative status. CD123 exhibited the highest aberrancy among MRD-positive cases, while CD81 had the lowest. These findings highlight the prognostic potential of CD73 and CD86 for MRD-negative status, complementing the established utility of CD123. Conclusions: Incorporating novel markers (CD123, CD73, CD86, and CD81) into MRD panels enhances detection sensitivity and clinical applicability. These markers are compatible with standard flow cytometry, supporting their integration into routine practice for comprehensive MRD evaluation, ultimately improving therapeutic outcomes in pediatric B-ALL.

Keywords: DfN; LAIP; acute leukemia; flow cytometry; minimal residual disease (MRD).

Figure 1

This figure outlines the gating strategy and methodology used for MRD detection, utilizing an 8-colour, 3-laser flow cytometry analysis on a BD FACS CANTO^TM II cytometer, with data analyzed using BD FACS DIVA ^TM v 8.03 software. (A) Time Gate vs. FSC-H: All events were initially monitored to confirm data acquisition stability. (B) Singlet Gate: A gate was applied on FSC-H vs. FSC-A to exclude doublets. (C) Viable Cell Gate: Debris, platelet clumps, and artifacts were excluded based on FSC and SSC properties to isolate viable cells. (D) CD45 Plot: Representative cell populations were visualized on a CD45 dot plot. (E) CD19+ B Cell Gate: B cells were identified using CD19 vs. SSC plots. (F) CD45 vs. CD19 Plot: This step refined the isolation of CD19+ cells for further analysis. (G) CD10 vs. CD19 Plot: Expression patterns of various markers on B cells were examined. (H) CD34 vs. CD19 Plot: B cell subsets, including immature (CD19+CD34+) and mature populations, were analyzed. (I–L): Residual leukemic cells (MRD) were clearly identified within predefined gates. Statistics for MRD events are provided in the figure. (Flow graphs of the patients are given in Supplementary Images).