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Introduction CD3/CD19 bi-specific T-cell engager blinatumomab provided excellent response in patients with relapsed/refractory (r/r) and MRD-positive B-cell precursor ALL (BCP-ALL). However, a subset of patients still relapses or does not respond to therapy. Loss of CD19 on leukemic blasts could be one of the immune escape mechanisms leading to CD19-negative ALL relapse. In addition to CD19 loss, expression of other markers can vary, seriously hamper flow cytometric MRD monitoring. The frequency of these changes is still unclear. We summarized our data on the flow cytometric monitoring of MRD and relapse diagnostics in patients treated with blinatumomab focusing on immunophenotypic changes. Methods We studied bone marrow blasts immunophenotype of 69 pediatric patients with r/r ВCP-ALL who received blinatumomab between December 2015 to May 2019. Cytogenetic aberrations have been identified in 58 (84.1%) cases: the most common abnormalities were KMT2A gene rearrangements (n=11), ETV6-RUNX1 fusion (n=6) and complex chromosomal aberrations (n=9). 51 patients underwent allo-HSCT. MRD monitoring was performed by 12-color flow cytometry. CD22 and CD24 were added to conventional antibodies’ combinations to avoid false-negative results if CD19 lacked. Results Sixteen patients were excluded from analysis as they never were MRD-positive after blinatumomab course. Twelve patients were resistant to blinatumomab therapy, and stable CD19 expression was found in all 7 of them with available immunophenotypic data. Twenty-five patients relapsed: in 13 cases tumor cells at relapse were CD19-positive, in 9 – CD19-negative; 1 patients experienced consequent CD19-negative and CD19-positive relapses. Two children developed relapse through «lineage switch» to AML. 16 patients, who achieved remission, had MRD at least once – blasts cell were either CD19-positive (n=8) or CD19-negative (n=7) or CD19-negative and positive at different time-points. Analyzing frequencies of changes in marker expression among different groups following findings were observed. In non-responders CD19 and CD38 tend to be the most stable antigens. Blast cells at CD19-negative relapses were more immunophenotypically stable comparing to CD19-positive cases. Up- and downmodulation of CD10 and CD20 expression were the most frequent shift types in CD19-positive relapses. At CD19-negative relapses we observed only in a few cases downmodulation of CD20 and CD45 and upmodulation of CD10 were observed. Conclusion We demonstrated that due to the changes in expression of all markers, not only CD19, more sophisticated algorithms of flow cytometric MRD monitoring should be applied.