We report incidence and deep molecular characteristics of lineage switch in 182 pediatric patients affected by B-cell precursor acute lymphoblastic leukemia (BCP-ALL), who were treated with blinatumomab. We documented six cases of lineage switch that occurred after or during blinatumomab exposure. Therefore, lineage conversion was found in 17.4% of all resistance cases (4/27) and 3.2% of relapses (2/63). Half of patients switched completely from BCP-ALL to CD19-negative acute myeloid leukemia, others retained CD19-positive B-blasts and acquired an additional CD19-negative blast population: myeloid or unclassifiable. Five patients had KMT2A gene rearrangements; one had TCF3::ZNF384 translocation. The presented cases showed consistency of gene rearrangements and fusion transcripts across initially diagnosed leukemia and lineage switch. In two of six patients, the clonal architecture assessed by IG/TR gene rearrangements was stable, while in others, loss of clones or gain of new clones was noted. KMT2A-r patients demonstrated very few additional mutations, while in the TCF3::ZNF384 case, lineage switch was accompanied by a large set of additional mutations. The immunophenotype of an existing leukemia sometimes changes via different mechanisms and with different additional molecular changes. Careful investigation of all BM compartments together with all molecular –minimal residual disease studies can lead to reliable identification of lineage switch.

We aimed to develop an antibody panel and data analysis algorithm for multicolor flow cytometry (MFC), which is a reliable method for minimal residual disease (MRD) detection in patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treated with CD19-directed therapy. The development of the approach, which was adapted for the case of possible CD19 loss, was based on the additional B-lineage marker expression data obtained from a study of primary BCP-ALL patients, an analysis of the immunophenotypic changes that occur during blinatumomab or CAR-T therapy, and an analysis of very early CD19-negative normal BCPs. We have developed a single-tube 11-color panel for MFC-MRD detection. CD22- and iCD79a-based primary B-lineage gating (preferably consecutive) was recommended. Based on patterns of antigen expression changes and the relative expansion of normal CD19-negative BCPs, guidelines for MFC data analysis and interpretation were established. The suggested approach was tested in comparison with the molecular techniques: IG/TR gene rearrangement detection by next-generation sequencing (NGS) and RQ-PCR for fusion-gene transcripts (FGTs). Qualitative concordance rates of 82.8% and 89.8% were obtained for NGS-MRD and FGT-MRD results, respectively. We have developed a sensitive and reliable approach that allows MFC-MRD monitoring after CD19-directed treatment, even in the case of possible CD19 loss.

The bispecific T cell-binding antibody blinatumomab (CD19/CD3) is widely and successfully used for the treatment of children with relapsed or refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Here, we report the efficacy of a single course of blinatumomab instead of consolidation chemotherapy to eliminate minimal residual disease (MRD) and maintain stable MRD-negativity in children with primary BCP-ALL. Between February 2020 and November 2022, 177 children with non-high-risk BCP-ALL were enrolled in the ALL-MB 2019 pilot study (NCT04723342). Patients received the usual risk-adapted induction therapy according to the ALL-MB 2015 protocol. Those who achieved a complete remission at the end of induction (EOI) received treatment with blinatumomab immediately after induction at a dose of 5 μg/m²/day for 7 days and 21 days at a dose of 15 μg/m²/day, followed by 12 months of maintenance therapy. MRD was measured using multicolor flow cytometry (MFC) at the EOI, then immediately after blinatumomab treatment, and then four times during maintenance therapy at 3-month intervals. All 177 patients successfully completed induction therapy and achieved a complete hematological remission. In 174 of these, MFC-MRD was measured at the EOI. 143 patients (82.2%) were MFC-MRD negative and the remaining 31 patients had varying degrees of MFC-MRD positivity. MFC-MRD was assessed in all 176 patients who completed the blinatumomab course. With one exception, all patients achieved MFC-MRD negativity after blinatumomab, regardless of the MFC-MRD score at EOI. One adolescent girl with high MFC-MRD positivity at EOI remained MFC-MRD positive. Of 175 patients who had completed 6 months of maintenance therapy, MFC-MRD data were available for 156 children. Of these, 155 (99.4%) were MFC-MRD negative. Only one boy with t(12;21) (p13;q22)/ETV6::RUNX1 became MFC-MRD positive again. The remaining 174 children had completed the entire therapy. MFC-MRD was examined in 154 of them, and 153 were MFC-MRD negative. A girl with hypodiploid BCP-ALL showed a reappearance of MFC-MRD with subsequent relapse. In summary, a single 28-day course of blinatumomab immediately after induction, followed by 12 months of maintenance therapy, is highly effective in achieving MRD-negativity in children with newly diagnosed non-high risk BCP-ALL and maintaining MRD-negative remission at least during the treatment period.

Multicolor flow cytometry (MFC) is a key method for assessing measurable residual disease (MRD) in acute lymphoblastic leukemia (ALL). However, very few approaches were developed for MRD in T-cell ALL (T-ALL). To identify MRD markers suitable for T-ALL, we analyzed the expression of CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD34, CD45, CD48, CD56, CD99, and HLA-DR in T-ALL patients at diagnosis. The median fluorescence intensities (MFIs) of surface CD3, CD4, CD5, CD7, CD8, CD45, CD48, CD99, and CD16+CD56 were also evaluated at Day 15 and the end-of-induction (EOI). The MFC data from 198 pediatric T-ALL patients were analyzed retrospectively. At diagnosis, the most common antigens were identified, and the MFI of T-lineage antigens in blasts was compared to that in T lymphocytes. At follow-up, the MFIs of the proposed MRD markers were compared to those observed at diagnosis. The most common T-ALL antigens were CD7 (100.0%), intracellular CD3 (100.0%), CD45 (98.5%), and CD5 (90.9%). The MFIs of T-lineage antigens in blasts differed significantly from those in T lymphocytes. By the EOI, a substantial modulation of sCD3, CD4, CD5, CD7, CD8, and CD45 was observed. CD48 and CD99 were the most stable markers. The proposed MRD markers (sCD3, CD4, CD5, CD7, CD8, CD45, CD48, CD99, CD16+CD56) enabled MFC-MRD monitoring in virtually all T-ALL patients.

Multicolor flow cytometry is now routinely used in laboratory practice for minimal residual disease (MRD) monitoring in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). This article describes the methodology of MRD detection in BCP-ALL using flow cytometry as recommended by the Russian-Belarusian multicenter group for pediatric acute leukemia studies. This wellharmonized approach includes recommendations for the choice of monoclonal antibodies, sample preparation, cytometer setup, flow cytometry data analysis and interpretation as well as for reporting. These guidelines allow application of multicolor flow cytometry for MRD monitoring in BCP-ALL in children and adults both in local laboratories and in multicenter settings in prospective clinical trials.