High frequency of heat shock protein 27 overexpression is a highly effective, high‐coverage marker for minimal residual disease detection in children with B‐cell acute lymphoblastic leukemia

Background Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. Minimal residual disease (MRD) detection is the most powerful prognostic tool for monitoring treatment efficacy and predicting clinical outcomes. We aimed to identify key leukemia‐associated markers, the proportions of differential expression in patients, and the most effective marker combination for MRD detection by flow cytometry. Methods Bone marrow samples were collected from 132 pediatric patients with newly diagnosed (n = 115) or relapsed (n = 17) B‐cell precursor (BCP)‐ALL. We used CD19, CD10, CD34, CD45 as backbone markers to identify immature B cells and analyzed the differential expression of 18 leukemia‐associated markers using seven‐color multiparameter flow cytometry. Results Leukemic cells in all 132 patients expressed leukemia‐associated markers. The most commonly overexpressed marker was heat shock protein 27 (Hsp27) (108 patients, 81%), followed by CD73 (102 patients, 77%) and CD123 (80 patients, 60%). CD38 was underexpressed in 64 patients (48%). Hsp27 overexpression persisted in 50 out of 57 follow‐up MRD bone marrow samples (87%) and was associated with older age at diagnosis. Hsp27 overexpression was not associated with MRD levels or genetic abnormalities including hyperdiploidy, t(12;21)/ETV6‐RUNX1, t(1;19)/TCF3‐PBX1, t(9;22)/BCR‐ABL1, or 11q23/KMT2A rearrangements. Four remaining leukemia‐associated markers (Hsp27, CD73, CD58, CD24) after in silico deletion from the original panel could collectively detect leukemia‐associated cell profiles in 100% of cases in this cohort and 98% of cases in a validation cohort. Conclusion Hsp27 combined with CD73, CD58, CD24, and backbone markers allows monitoring MRD in virtually all patients with BCP‐ALL.