Eleven‐marker 10‐color flow cytometric assessment of measurable residual disease for T‐cell acute lymphoblastic leukemia using an approach of exclusion

Measurable/minimal residual disease (MRD) status has been suggested as a powerful indicator of clinical‐outcome in T‐cell lymphoblastic leukemia/lymphoma (T‐ALL). Multicolor flow cytometric (MFC)‐based T‐ALL MRD reports are limited and traditionally based on the utilization of markers‐of‐immaturity like TdT and CD99. Moreover, studies demonstrating the multicolor flow cytometric (MFC) approach for the assessment of T‐ALL MRD are sparse. Herein, we describe an 11‐marker, 10‐color MFC‐based T‐ALL MRD method using an “approach of exclusion.” Methods The study included 269 childhood T‐ALL patients treated with a modified‐MCP841 protocol. An 11‐marker, 10‐color MFC‐based MRD was performed in bone marrow (BM) samples at the end‐of‐induction (EOI) and end‐of‐consolidation (EOC) time‐points using Kaluza‐version‐1.3 software. Results We studied EOI‐MRD in 269 and EOC‐MRD in 105 childhood T‐ALL patients. EOI‐MRD was detectable in 125 (46.5%) samples (median, 0.3%; range, 0.0007–66.3%), and EOC‐MRD was detectable in 34/105 (32.4%) samples (median, 0.055%; range, 0.0008–27.6%). Leukemia‐associated immunophenotypes (LAIPs) found useful for MRD assessment were dual‐negative CD4/CD8 (40.9%), dual‐positive CD4/CD8 (23.3%) and only CD4 or CD8 expression (35.8%); dim/subset/dim‐negative surface‐CD3 (39%), dim/subset/dim‐negative/negative CD5 (28.3%), dim/dim‐negative/negative/heterogeneous CD45 (44.7%) and co‐expression of CD5/CD56 (7.5%). EOI‐MRD‐positive status was found to be the most‐relevant independent factor in the prediction of inferior relapse‐free and overall survival. Conclusion We described an 11‐marker 10‐color MFC‐based highly sensitive MRD assay in T‐ALL using an approach of exclusion. The addition of CD4 and CD8 to the pan‐T‐cell markers in a 10‐color assay is highly useful in T‐ALL MRD assessment and extends its applicability to almost all T‐ALL patients.