The bispecific T cell-binding antibody blinatumomab (CD19/CD3) has emerged as one of the most commonly used immunotherapeutic agents in pediatric oncology.1 It is approved for children with a recurrent or refractory (R/R) B-cell precursor acute lymphoblastic leukemia (BCP-ALL).2 3 In these patients, blinatumomab is often used as bridge therapy before hematopoietic stem cell transplantation (HSCT) to achieve a minimal residual disease (MRD) negativity before HSCT.1 Remarkable success was also achieved using immunotherapy in children with primary BCP-ALL.4 5 Mostly, it is used as an additional treatment element for patients with a slow response to therapy5 6 or with clinical high-risk characteristics (eg, infants with KMT2A gene rearrangements) with or without subsequent HSCT.4 7 8 Another approach to targeting CD19 in first-line therapy is to reduce treatment intensity and toxicity by replacing intensive chemotherapy with immunotherapy, for example, in patients with reduced tolerance to the usual therapy (eg, in children with Down syndrome).4 From this point of view, it makes sense to use immunotherapy also in children with first-line ALL to reduce toxicity as well as acute and long-term side effects. Here we report the efficacy of a single course of blinatumomab instead of consolidation chemotherapy in eliminating MRD in children with BCP-ALL.

Between February 2020 and November 2022, 177 children with BCP-ALL were enrolled in the pilot study ALL-MB 2019 (NCT04723342). The main aim of the study was to optimize the therapy of patients with primary BCP-ALL by including monoclonal bispecific antibodies in post-induction treatment with simultaneous reduction of chemotherapy without increase of relapse rate and decrease of event-free and overall survival. The data collected was analyzed as of December 2023. Children and adolescents with a white blood cell count less than 100×109 /L without central nervous system (CNS) leukemia (CNS-3 status) at diagnosis without translocation t(4;11) (q21;q23)/KMT2A::AFF1 who had achieved a complete remission at the end of induction therapy (EOI) received blinatumomab for 4 weeks, followed by 12 months of maintenance therapy (online supplemental figure S1). Initial patient characteristics are shown in table 1. As already described, the patients were divided into standard risk (SR) and intermediate risk (ImR) (online supplemental figure S1)9 10 and received the normal risk-adapted induction therapy in accordance with the ALL-MB protocol 2015 (figure 1).11 Before treatment, a centralized and standardized diagnosis including cytomorphology, immunophenotyping12 and cyto- and molecular genetics13 were routinely carried out in all patients. Using multicolor flow cytometry (MFC), MRD was measured at EOI, immediately after the treatment with blinatumomab and then four times at 3-month intervals during maintenance therapy (figure 1). The subsequent therapy was adapted to the risk group (figure 1, online supplemental figure S1).

Patient characteristics

ALL-MB 2019 Pilot protocol treatment overview. (A) Shows standard risk group, (B) intermediate risk group 1, (C) intermediate risk group 2, and (D) group F. DNR, daunorubicin; MRD, minimal residual disease; PEG-asparaginase, pegylated L-asparaginase.

MFC-MRD was examined using a 10-color antibody combination, which was specially adapted to the possible loss of CD19 after a CD19-oriented immunotherapy.14 Either commercially available (Beckman Coulter, BC, Indianapolis, Indiana, USA)15 or custom designed (Beckton Dickinson, BD, San Jose, California, USA) seven-color premanufactured tube containing CD19, CD10, CD34, CD20, CD45, CD58 and CD38 was used extended with additional antibodies to CD22, CD24 and intracellular (1) CD79a (all from BD) and DNA stain SYTO41 (Thermo Fisher Scientific, Waltham, Massachusetts, USA) as described previously.14 At least 1,000,000 nucleated cells (NC) were acquired with a 3-laser flow cytometer CytoFLEX (BC). EuroFlow guidelines for machine performance monitoring were used.16 CytoFLEX Daily QC Fluorospheres (BC) were used for daily cytometer optimization. MFC data was analyzed with Kaluza V.2.1 software (BC) according to a previously described algorithm.14 17 0.001% of definitive leukemia cells among all NCs were set as a threshold for the MFC-MRD positive.

All 177 patients successfully completed induction therapy and achieved complete remission (CR). In 174 of them, MFC-MRD was measured at EOI; no samples were sent to the central laboratory from three children. MFC-MRD was negative in 143 patients (82.2%) (figure 2A), and the remaining 31 patients had varying degrees of MFC-MRD positivity (figure 2B). Two-thirds of the MFC MRD positive patients had values below 0.1%, and values >1% were found in only four children (figure 2B). The proportion of MFC-MRD-negative children was similar in the SR and ImR groups: 93 of 113 (82.3%) compared with 55 of 66 (83.3%). Quantitative MFC-MRD values were different in SR and ImR patients: an MRD value greater than 0.1% was seen in only 4 of 20 (20.0%) SR patients, compared with 6 of 11 (54.5%) ImR patients (figure 2B).

Results of MFC-MRD monitoring in children with B-lineage ALL treated with the ALL-MB 2019 Pilot protocol. (A) Overall results, (B) distribution of MF-MRD levels at the end of induction (day 36, EOI) according to the risk group; (C) relation of MFC-MRD data before (day 36) and after the blinatumomab course in patients who were MFC-MRD-positive at EOI. ALL, acute lymphoblastic leukemia; EOI, end of induction; ImR, intermediate risk; MFC, multicolor flow cytometry; MRD, minimal residual disease; ND, no data; SR, standard risk.

MFC-MRD was studied in all 176 patients who completed the blinatumomab course. In one ImR patient (MFC-MRD negative at EOI), blinatumomab treatment had to be discontinued due to severe neurotoxicity, and the child was switched to the conventional ImR treatment plan of the ALL-MB 2015 protocol. All but one patient achieved MFC-MRD negativity after blinatumomab, regardless of MFC-MRD score on EOI (figure 2C, figure 3A–B). One adolescent girl with high MFC-MRD positivity at EOI (more than 1%) remained MFC-MRD positive (0.007%, figure 3C). She was enrolled in the high-risk (HR) group of the ALL-MB 2015 protocol, became MFC-MRD negative after the first HR block, and subsequently received HSCT.

Representative examples of MRD elimination after the blinatumomab course. (A) shows MRD elimination in a case of high EOI leukemic burden; (B) shows an example of low EOI-MRD elimination. (C) Shows MRD persistence after the blinatumomab course in a patient who had a high level of MRD at EOI. (D) Shows MRD reappearance in the middle of maintenance therapy in a patient who had MRD-negativity before the blinatumomab course. CD22+iCD79a+cells are displayed. Blue, mature B cells; red, leukemic cells; green, normal B-cell precursors. EOI, end of induction; MRD, minimal residual disease.

Of 175 patients who had completed 6 months of maintenance therapy, MFC-MRD data was available for 156 children. For the remaining 18 children, no MRD samples had been received by the laboratory. Of these 156 patients, 155 (99.4%) remained MFC-MRD negative. Only one boy with t(12;21) (p13;q22)/ETV6::RUNX1 became MFC-MRD positive again (figure 3D). The fusion gene transcript was identified by PCR, and FISH was positive in leukemia cells sorted by flow analysis. The patient was switched to the ALL-MB 2015 ImR group regimen but later suffered a relapse with the change of the dominant leukemic clone. Therefore, 174 children had completed all therapy, including 12-month maintenance therapy. Of them, MFC-MRD was tested in 154 children (MRD samples were not available in the remaining 20 patients). Among them, all except one remained MFC-MRD negative. A girl with hypodiploid BCP-ALL showed MFC-MRD recurrence (partially CD19-negative) with subsequent complete CD19-positive relapse.

Based on promising results in the treatment of R/R BCP-ALL,2 3 blinatumomab immunotherapy is currently used in protocols for primary B-lineage ALL.4 5 In most trials, blinatumomab is used to escalate treatment, sometimes as a replacement for intensified chemotherapy in poorly responding children, both with and without subsequent HSCT.4–6 For the same purpose, blinatumomab is used in infants with KMT2A-r ALL,7 8 sometimes regardless of their MRD response.7

Here, we present the results of MFC-MRD monitoring in a protocol using immunotherapy to significantly reduce chemotherapy in a first-line study. In fact, all three consolidation blocks are replaced by a single 28-day treatment with blinatumomab, and maintenance therapy is significantly shortened. The rationale behind this protocol was the assumption that CD19-targeted immunotherapy could achieve an equivalent or even deeper response (much lower than MRD detection thresholds) and reduce the leukemic burden after induction more effectively than ALL-MB chemotherapy, even in patients with negative MFC-MRD at the time of EOI. This is consistent with the philosophy of the MB group, which is to significantly reduce treatment for the majority of non-HR patients.9 10 18 We have previously shown that with careful consideration of clinical risk factors in combination with MFC-MRD response, half of children with BCP-ALL can be successfully treated with low-intensity therapy9 and another quarter with moderate-intensity therapy.10 However, further de-escalation of treatment with conventional drugs may not be possible without compromising cure rates. Therefore, the innovative design of the ALL-MB 2019 pilot protocol includes only one blinatumomab course instead of all three consolidations; the duration of maintenance therapy has also been shortened.

Of course, such a de-intensified therapy regimen requires constant monitoring of the disease course during treatment and safety measures in case patients become MRD-positive again after immunotherapy. Here, the protocol envisaged switching patients to therapy according to the conventional ALL-MB 2015 protocol, which simultaneously ran in parallel as a quasi “control arm”. For this reason, in contrast to our single-point approach,18 MFC-MRD monitoring at multiple time points after immunotherapy was implemented in the treatment protocol.

The results of MFC-MRD monitoring show that almost all children (99.2%) become MFC-MRD negative after treatment with blinatumomab. This high level of MFC-MRD elimination is achieved regardless of initial risk parameters and EOI-MFC-MRD response. The proportion is even higher when compared with the incidence of MFC-MRD negativity after initial conventional consolidation (end of consolidation, EOC) in ALL-MB protocols19 or other more intensive treatment regimens.20–22 Moreover, in the conventional ALL-MB protocol, one in four patients who were MFC-MRD positive at EOI maintained their MFC-MRD positivity at EOC.19 For the more intensive Children Oncology Group (COG) protocol, this proportion was significantly lower (6.8%22), as expected, but also higher than for ALL-MB 2019 Pilot (3.2%). Moreover, almost all patients studied had persistent MFC-MRD-negative CR at the end of therapy, although the consolidation phase did not consist of chemotherapy and maintenance therapy was significantly reduced. Only one adolescent patient experienced only a reduction in leukemia burden after immunotherapy and not complete MFC-MRD negativity. Two patients were diagnosed with MFC-MRD recurrence during surveillance, and both suffered subsequent relapses, although one patient was switched to conventional chemotherapy according to safety guidelines.

Unfortunately, scheduled MRD measurements were missed in some patients. Mostly this was due to technical and/or logistical reasons on the part of the parents. One of these patients suffered a relapse after 22 months and is alive and well in the second CR. All others who missed MRD values are alive, well and in continued first CR.

This report addresses only the results of MFC-MRD monitoring, as it is obviously too early to assess even the preliminary results of the entire study. Nevertheless, we can conclude that the 4-week blinatumomab course instead of the three blocks of consolidation chemotherapy leads to a deep MFC-MRD response, at least during the monitoring period, after significantly de-escalated chemotherapy.

Data are available upon reasonable request. The data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Consent obtained from parent(s)/guardian(s).

This study involves human participants and was approved by the Ethics Committee of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology (Decision ID 59-2). Participants gave informed consent to participate in the study before taking part.

The authors thank all doctors, nurses and laboratory personnel in participating institutions, who were involved in patients’ diagnostics, management and monitoring.