Healthcare cost and utilization for chimeric antigen receptor (CAR) T-cell therapy in the treatment of pediatric acute lymphoblastic leukemia: A commercial insurance claims database analysis.

BACKGROUND: B-lineage acute lymphoblastic leukemia (B-ALL) is the most common malignancy of childhood. With the introduction of novel cellular therapies, cost of care is a critical component and the financial burden experienced by patients and society requires evaluation. AIMS: This study aims to assess the utilization and cost of care for chimeric antigen receptor T-cell (CAR-T) therapy for pediatric ALL patients with commercial insurance coverage in the United States. METHODS AND RESULTS: Using de-identified commercial insurance data from the OptumLabs® Data Warehouse, a cohort of 37 patients, aged 1-25 years, with B-ALL treated with CAR-T therapy between Oct 2016 and Dec 2021 in the United States was identified. Cost was evaluated for a 90 day period encompassing CAR-T infusion and by administration and complication characteristics. Among the 37 identified B-ALL patients that received a CAR-T product infusion, 14 patients were female, median age at administration was 13 years. The median 90-day total cost was $620,500 (Mean: $589,108). Inpatient cost accounted for approximately 71% of the total cost with an average of 28 inpatient days per patient. Although inpatient cost was slightly higher in the older age group (aged 10-25 years) and in patients with a code for cytokine release syndrome (CRS), these differences were not statistically significant. CONCLUSION: This real-world cost analysis shows for the first time the encompassing cost of CAR-T therapy for pediatric B-ALL patients in the US with commercial insurance. This study provides a valuable benchmark that can be used to analyze the financial implications of CAR-T therapy for pediatric B-ALL therapy on health systems.

15-color highly sensitive flow cytometry assay for post anti-CD19 targeted therapy (anti-CD19-CAR-T and blinatumomab) measurable residual disease assessment in B-lymphoblastic leukemia/lymphoma: Real-world applicability and challenges.

OBJECTIVES: Measurable residual disease (MRD) is the most relevant predictor of disease-free survival in B-cell acute lymphoblastic leukemia (B-ALL). We aimed to establish a highly sensitive flow cytometry (MFC)-based B-ALL-MRD (BMRD) assay for patients receiving anti-CD19 immunotherapy with an alternate gating approach and to document the prevalence and immunophenotype of recurrently occurring low-level mimics and confounding populations. METHODS: We standardized a 15-color highly-sensitive BMRD assay with an alternate CD19-free gating approach. The study included 137 MRD samples from 43 relapsed/refractory B-ALL patients considered for anti-CD19 immunotherapy. RESULTS: The 15-color BMRD assay with CD22/CD24/CD81/CD33-based gating approach was routinely applicable in 137 BM samples and could achieve a sensitivity of 0.0005%. MRD was detected in 29.9% (41/137) samples with 31.7% (13/41) of them showing <.01% MRD. Recurrently occurring low-level cells that showed immunophenotypic overlap with leukemic B-blasts included: (a) CD19+CD10+CD34+CD22+CD24+CD81+CD123+CD304+ plasmacytoid dendritic cells, (b) CD73bright/CD304bright/CD81bright mesenchymal stromal/stem cells (CD10+) and endothelial cells (CD34+CD24+), (c) CD22dim/CD34+/CD38dim/CD81dim/CD19-/CD10-/CD24- early lymphoid progenitor/precursor type-1 cells (ELP-1) and (d) CD22+/CD34+/CD10heterogeneous/CD38moderate/CD81moderate/CD19-/CD24- stage-0 B-cell precursors or ELP-2 cells. CONCLUSIONS: We standardized a highly sensitive 15-color BMRD assay with a non-CD19-based gating strategy for patients receiving anti-CD19 immunotherapy. We also described the immunophenotypes of recurrently occurring low-level populations that can be misinterpreted as MRD in real-world practice.

Genetic alterations and MRD refine risk assessment for KMT2A-rearranged B-cell precursor ALL in adults: a GRAALL study.

KMT2A-rearranged (KMT2A-r) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is widely recognized as a high-risk leukemia in both children and adults. However, there is a paucity of data on adults treated in recent protocols, and the optimal treatment strategy for these patients is still a matter of debate. In this study, we set out to refine the prognosis of adult KMT2A-r BCP-ALL treated with modern chemotherapy regimen and investigate the prognostic impact of comutations and minimal residual disease (MRD). Of 1091 adult patients with Philadelphia-negative BCP-ALL enrolled in 3 consecutive trials from the Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL), 141 (12.9%) had KMT2A-r, with 5-year cumulative incidence of relapse (CIR) and overall survival (OS) rates of 40.7% and 53.3%, respectively. Molecular profiling highlighted a low mutational burden in this subtype, reminiscent of infant BCP-ALL. However, the presence of TP53 and/or IKZF1 alterations defined a subset of patients with significantly poorer CIR (69.3% vs 36.2%; P = .001) and OS (28.1% vs 60.7%; P = .006) rates. Next, we analyzed the prognostic implication of MRD measured after induction and first consolidation, using both immunoglobulin (IG) or T-cell receptor (TR) gene rearrangements and KMT2A genomic fusion as markers. In approximately one-third of patients, IG/TR rearrangements were absent or displayed clonal evolution during the disease course, compromising MRD monitoring. In contrast, KMT2A-based MRD was highly reliable and strongly associated with outcome, with early good responders having an excellent outcome (3-year CIR, 7.1%; OS, 92.9%). Altogether, our study reveals striking heterogeneity in outcomes within adults with KMT2A-r BCP-ALL and provides new biomarkers to guide risk-based therapeutic stratification.

Assessment of proxy-reported responses as predictors of motor and sensory peripheral neuropathy in children with B-lymphoblastic leukemia.

Chemotherapy-induced peripheral neuropathy (CIPN), a common condition in children with acute lymphoblastic leukemia, can be challenging to diagnose. Using data from Children's Oncology Group AALL0932 physical function study, we sought to determine if parent/guardian proxy-reported responses from the Pediatric Outcomes Data Collection Instrument could identify children with motor or sensory CIPN diagnosed by physical/occupational therapists (PT/OT). Four variables moderately discriminated between children with and without motor CIPN (c-index 0.76, 95% confidence interval [CI]: 0.64-0.84), but sensory and optimism-corrected models had weak discrimination (c-index sensory models 0.65, 95% CI: 0.54-0.74). New proxy-report measures are needed to identify children with PT/OT diagnosed CIPN.

PersonALL: a genetic scoring guide for personalized risk assessment in pediatric B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: Recurrent genetic lesions provide basis for risk assessment in pediatric acute lymphoblastic leukemia (ALL). However, current prognostic classifiers rely on a limited number of predefined sets of alterations. METHODS: Disease-relevant copy number aberrations (CNAs) were screened genome-wide in 260 children with B-cell precursor ALL. Results were integrated with cytogenetic data to improve risk assessment. RESULTS: CNAs were detected in 93.8% (n = 244) of the patients. First, cytogenetic profiles were combined with IKZF1 status (IKZF1normal, IKZF1del and IKZF1plus) and three prognostic subgroups were distinguished with significantly different 5-year event-free survival (EFS) rates, IKAROS-low (n = 215): 86.3%, IKAROS-medium (n = 27): 57.4% and IKAROS-high (n = 18): 37.5%. Second, contribution of genetic aberrations to the clinical outcome was assessed and an aberration-specific score was assigned to each prognostically relevant alteration. By aggregating the scores of aberrations emerging in individual patients, personalized cumulative values were calculated and used for defining four prognostic subgroups with distinct clinical outcomes. Two favorable subgroups included 60% of patients (n = 157) with a 5-year EFS of 96.3% (excellent risk, n = 105) and 87.2% (good risk, n = 52), respectively; while 40% of patients (n = 103) showed high (n = 74) or ultra-poor (n = 29) risk profile (5-year EFS: 67.4% and 39.0%, respectively). CONCLUSIONS: PersonALL, our conceptually novel prognostic classifier considers all combinations of co-segregating genetic alterations, providing a highly personalized patient stratification.

Significance of CEBPE Gene Promoter Polymorphism (Rs2239630 G > A ) Assessment in Childhood B-cell Acute Lymphoblastic Leukemia.

BACKGROUND: A significant association has been reported between CEBPE gene promoter polymorphisms (rs2239630 G > A ) and the incidence of B-cell acute lymphoblastic leukemia (B-ALL). However, no previous study on this issue has been included among the Egyptian cohort of pediatric patients with B-ALL. Therefore, this study was designed to address the associations between CEBPE polymorphisms and susceptibility to B-ALL, as well as its impact on the outcome of B-ALL Egyptian patients with B-ALL. PATIENTS AND METHODS: In the current study, we evaluated the rs2239630 polymorphism in 225 pediatric patients and 228 controls to assess the association of different rs2239630 genotypes with childhood susceptibility to B-ALL and the impact on the outcome of the patients. RESULTS: The frequency of the A allele was significantly higher in the cases of B-ALL compared with the control group ( P = 0.004). By analyzing different genotypes for the predictive value of disease development, the GA and AA genotypes have been identified to be the highest among multivariate factors with an odds ratio of 3.330 (95% CI: 1.105-10.035). Likewise, the A allele was significantly associated with the shortest overall survival. CONCLUSIONS: CEBPE gene promoter polymorphism (rs2239630 G > A ) AA is frequently associated with B-ALL; and has the worst overall survival among the 3 genotypes, followed by the GA and GG genotypes ( P < 0.001).

Switching from salvage chemotherapy to immunotherapy in adult B-cell acute lymphoblastic leukemia.

About one-half of adults with acute B-cell lymphoblastic leukemia (B-ALL) who do not achieve molecular complete remission or who subsequently relapse are not cured by current chemo- or targeted therapies. Previously, the sole therapeutic option for such persons was a hematopoietic stem cell transplant. Recently, several immune therapies including monoclonal antibodies, bispecific T-cell engagers (BiTEs), antibody-drug conjugates (ADCs), and chimeric antigen receptor T-cells (CARs) have been shown safe and effective in this setting. In this manuscript, we summarize data on US FDA-approved immune therapies of advanced adult B-ALL including rituximab, blinatumomab, inotuzumab ozogamicin, tisagenlecleucel and brexucabtagene autoleucel. We consider the results of clinical trials focusing on efficacy, safety, and quality of life (QoL). Real-world evidence is presented as well. We also briefly discuss pharmacodynamics, pharmacokinetics, and pharmacoeconomics followed by risk-benefit analyses. Lastly, we present future directions of immune therapies for advanced B-ALL in adults.

Flow cytometric assessment for minimal/measurable residual disease in B lymphoblastic leukemia/lymphoma in the era of immunotherapy.

Minimal/measurable residual disease (MRD) is the most important independent prognostic factor for patients with B-lymphoblastic leukemia (B-LL). MRD post therapy has been incorporated into risk stratification and clinical management, resulting in substantially improved outcomes in pediatric and adult patients. Currently, MRD in B-ALL is most commonly assessed by multiparametric flow cytometry and molecular (polymerase chain reaction or high-throughput sequencing based) methods. The detection of MRD by flow cytometry in B-ALL often begins with B cell antigen-based gating strategies. Over the past several years, targeted immunotherapy directed against B cell markers has been introduced in patients with relapsed or refractory B-ALL and has demonstrated encouraging results. However, targeted therapies have significant impact on the immunophenotype of leukemic blasts, in particular, downregulation or loss of targeted antigens on blasts and normal B cell precursors, posing challenges for MRD detection using standard gating strategies. Novel flow cytometric approaches, using alternative strategies for population identification, sometimes including alternative gating reagents, have been developed and implemented to monitor MRD in the setting of post targeted therapy.

United we stand, divided we fall. Multicentre standardization of measurable residual disease assessment in acute leukaemia is the way forward.

Assessment of measurable residual disease (MRD) using multiparameter flow cytometry in precursor B-cell acute lymphoblastic leukaemia (ALL) is routine. However, studies on the harmonization of laboratory techniques as well as on the interpretation of results are limited. Here, Ikoma-Colturato and colleagues from Brazil demonstrate multicentric standardization of B-ALL MRD using EuroFlow protocols. Commentary on: Ikoma-Colturato et al., Multicentric standardization of minimal/measurable residual disease in B-cell precursor acute lymphoblastic leukaemia using next-generation flow cytometry in a low/middle-level income country. Br J Haematol 2023;200:381-384.

A prospective, open-label, randomised, parallel design study of 4 generic formulations of intramuscular L-asparaginase in childhood precursor B-cell acute lymphoblastic leukaemia (ALL).

AIMS: L-asparaginase is an essential medicine for childhood ALL. The quality of generic L-asparaginase available in India is a matter of concern. We compared four commonly used generic formulations of L-asparaginase in India. MATERIALS AND METHODS: We conducted a prospective, open-label, randomised trial of four generic formulations of asparaginase for the treatment of patients with newly diagnosed intermediate-risk B-ALL. Patients were randomly assigned in a 1:1:1:1 ratio to receive generic asparaginase at a dose of at 10,000 IU/m 2 on days 9, 12, 15, and 18 of a 35-day cycle (Induction treatment). The primary end points were to determine the difference in the asparaginase activity and asparagine depletion. Historical patients who received L-asparaginase Medac (innovator) served as controls. RESULTS: A total of 48 patients underwent randomization; 12 patients each in the four arms. Failure to achieve predefined activity threshold of 100 IU/L was observed in 9/40 samples of Generic A (22·5%), 23/40 of Generic B (57·5%), and 43/44 (98%) each of Generic C and D. All 27 samples from seven historical patients who were administered Medac had activity > 100 IU/L. The average activity was significantly higher for Genericm A, 154 (70·3, 285·4) IU/L followed by Generic B 84·5(44·2, 289·1) IU/L, Generic C 45(14·4, 58·4) IU/L, and Generic D 20·4(13, 35) IU/L. Only 6 patients had asparaginase activity > 100 IU/L on each of the four occasions (Generic A = 5; Generic B = 1), and none of them developed Anti-Asparaginase Antibodies (AAA). On the other hand, AAA was observed in 12/36 patients who had at least one level < 100 IU/L (P < 0·05): Generic A 3/5, Generic B = 3/9, Generic D (4/11), and Generic C (5/11). CONCLUSION: Generic A and B had better trough asparaginase activity compared to Generic D and C. Overall, generic formulations had lower asparaginase activity which raises serious clinical concerns regarding their quality. Until strict regulatory enforcement improves the quality of these generics, dose adaptive approaches coupled with therapeutic drug monitoring need to be considered.

Flow cytometric minimal residual disease assessment in B-cell precursor acute lymphoblastic leukaemia patients treated with CD19-targeted therapies - a EuroFlow study.

The standardized EuroFlow protocol, including CD19 as primary B-cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients treated with chemotherapy. We developed and validated an alternative gating strategy allowing reliable MRD analysis in BCP-ALL patients treated with CD19-targeting therapies. Concordant data were obtained in 92% of targeted therapy patients who remained CD19-positive, whereas this was 81% in patients that became (partially) CD19-negative. Nevertheless, in both groups median MRD values showed excellent correlation with the original MRD data, indicating that, despite higher interlaboratory variation, the overall MRD analysis was correct.

Critical evaluation of the utility of pre- and post-therapy immunophenotypes in assessment of measurable residual disease in B-ALL.

Measurable residual disease (MRD) is an important parameter to predict outcome in B-cell acute lymphoblastic leukemia (B-ALL). Two different approaches have been used for the assessment of MRD by multiparametric flow cytometry that include the "Leukemia Associated Aberrant Immunophenotype (LAIP)" and "Difference from Normal (DFN)" approach. In this retrospective study, we analyzed 539 samples obtained from 281 patients of which 258 were paired samples and the remaining 23 samples were from post-induction time point only, to explore the utility of baseline immunophenotype (IPT) for MRD assessment. Single-tube 10-color panel was used both at diagnosis and MRD time points. Out of 281 patients, 31.67% (n = 89) were positive and 68.32% (n = 192) were negative for MRD. Among 258 paired diagnostic and follow-up samples, baseline IPT was required in only 9.31% (24/258) cases which included cases with hematogone pattern and isolated dim to negative CD10 expression patterns. Comparison of baseline IPT with post-induction MRD positive samples showed a change in expression of at least one antigen in 94.04% cases. Although the immunophenotypic change in expression of various antigens is frequent in post-induction samples of B-ALL, it does not adversely impact the MRD assessment. In conclusion, the baseline IPT is required in less than 10% of B-ALL, specifically those with hematogone pattern and/or dim to negative expression of CD10. Hence, a combination of DFN and LAIP approach is recommended for reliable MRD assessment.

Molecular classification improves risk assessment in adult BCR-ABL1-negative B-ALL.

Genomic classification has improved risk assignment of pediatric, but not adult B-lineage acute lymphoblastic leukemia (B-ALL). The international UKALLXII/ECOG-ACRIN E2993 (#NCT00002514) trial accrued 1229 adolescent/adult patients with BCR-ABL1- B-ALL (aged 14 to 65 years). Although 93% of patients achieved remission, 41% relapsed at a median of 13 months (range, 28 days to 12 years). Five-year overall survival (OS) was 42% (95% confidence interval, 39, 44). Transcriptome sequencing, gene expression profiling, cytogenetics, and fusion polymerase chain reaction enabled genomic subtyping of 282 patient samples, of which 264 were eligible for trial, accounting for 64.5% of E2993 patients. Among patients with outcome data, 29.5% with favorable outcomes (5-year OS 65% to 80%) were deemed standard risk (DUX4-rearranged [9.2%], ETV6-RUNX1/-like [2.3%], TCF3-PBX1 [6.9%], PAX5 P80R [4.1%], high-hyperdiploid [6.9%]); 50.2% had high-risk genotypes with 5-year OS of 0% to 27% (Ph-like [21.2%], KMT2A-AFF1 [12%], low-hypodiploid/near-haploid [14.3%], BCL2/MYC-rearranged [2.8%]); 20.3% had intermediate-risk genotypes with 5-year OS of 33% to 45% (PAX5alt [12.4%], ZNF384/-like [5.1%], MEF2D-rearranged [2.8%]). IKZF1 alterations occurred in 86% of Ph-like, and TP53 mutations in patients who were low-hypodiploid (54%) and BCL2/MYC-rearranged (33%) but were not independently associated with outcome. Of patients considered high risk based on presenting age and white blood cell count, 40% harbored subtype-defining genetic alterations associated with standard- or intermediate-risk outcomes. We identified distinct immunophenotypic features for DUX4-rearranged, PAX5 P80R, ZNF384-R/-like, and Ph-like genotypes. These data in a large adult B-ALL cohort treated with a non-risk-adapted approach on a single trial show the prognostic importance of genomic analyses, which may translate into future therapeutic benefits.

A single assessment of methotrexate levels at 42 hours permits safe administration and early discharge in children with lymphoblastic lymphoma and leukemia receiving high-dose methotrexate.

High-dose methotrexate (HDMTX) is an important component of treatment in pediatric acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LL). Optimal rescue therapy is essential for the safe administration of HDMTX. A cost-effective strategy that does not compromise safety is necessary for low- and middle-income countries. Consecutive admissions for HDMTX in children with ALL and LL over 12 months were analyzed. The dose of HDMTX was 3 g/m2 in B-ALL and B-LL and 5 g/m2 in T-ALL and T-LL. A methotrexate level was measured at 42 hours of starting HDMTX infusion (T42-MTX). Three doses of folinic acid at T42, T48, and T54 and alkalinized hydration till T54 were administered if T42-MTX <1 µM. A total of 282 cycles of HDMTX that were administered in 71 patients were analyzed. T42-MTX was <1 µM in 266 (94.3%) cycles. T42-MTX was ≥1 µM in 12% and 3% of cycles of HDMTX administered at a dose of 5 g/m2 and 3 g/m2, respectively (p = .074). The median duration of hospitalization for HDM was three days and did not differ with the dose of HDMTX administered (p = .427). Mucositis, delayed recovery of blood counts, and hospitalization for reversible toxicity occurred after 21 (7.4%), 28 (9.9%), and 19 (6.7%) cycles of HDMTX, respectively. Mucositis was greater following the administration of 5 g/m2 of HDMTX. A single T42-MTX measurement permits the safe administration of HDMTX and an expedited discharge from the hospital within three days in more than 90% of children with ALL/LL.

Expression of CD304/neuropilin-1 in adult b-cell lymphoblastic leukemia/lymphoma and its utility for the measurable residual disease assessment.

INTRODUCTION: Many new markers are being evaluated to increase the sensitivity and applicability of multicolor flow cytometry (MFC)-based measurable residual disease (MRD) monitoring. However, most of the studies are limited to childhood B-cell lymphoblastic leukemia/lymphoma (B-ALL), and reports in adult B-ALL are extremely scarce and limited to small cohorts. We studied the expression of CD304/neuropilin-1 in a large cohort of adult B-ALL patients and evaluated its practical utility in MFC-based MRD analysis. METHODS: CD304 was studied in blasts from adult B-ALL patients and normal precursor B cells (NPBC) from non-B-ALL bone marrow samples using MFC. CD304 expression intensity and pattern were studied with normalized-mean fluorescent intensity (nMFI) and coefficient of variation of immunofluorescence (CVIF), respectively. MFC-based MRD was performed at end of induction (EOI; day-35), end of consolidation (EOC; day 78-80), and subsequent follow-up (SFU) time points. RESULTS: CD304 was positive in 120/214(56.07%) and was significantly associated with BCR-ABL1 fusion (P = .001). EOI-MRD and EOC-MRD were positive in 129/214(60.3%) and 50/81(61.72%), respectively. CD304 was positive in a significant percentage of EOI (48%, 62/129) and EOC (52%, 26/50) MRD-positive B-ALL samples. Its expression was retained, lost, and gained in 73.7%, 26.3%, and 11.3% of EOI-MRD and 85.7%, 14.3%, and none of EOC-MRD samples, respectively. Low-level MRD (<0.01%) was detectable in 34 of all (EOI + EOC + SFU = 189) MRD-positive samples, and CD304 was found useful in 50% of these samples. CONCLUSION: CD304 is commonly expressed in adult B-ALL and clearly distinguish B-ALL blasts from normal precursor B cells. It is a stable MRD marker and distinctly useful in the detection of MFC-based MRD monitoring, especially in high-sensitivity MRD assay.

Blast size-specific flowcytometric ploidy assessment using FxCycleTM Violet dye and its correlation with conventional cytogenetic ploidy in pediatric precursor B-lineage acute lymphoblastic leukemia patients.

INTRODUCTION: Numerical chromosomal abnormalities (aneuploidies), present in approximately 30%-50% of pediatric precursor B-lineage acute lymphoblastic leukemia (B-ALL) patients, are commonly identified through a laborious conventional cytogenetic (CG) technique. Flow cytometry (FCM) can identify both physical and fluorescent properties of cells together, and by using fluorescent nucleic-acid-binding dyes, FCM can identify variations in total nucleic-acid content of cells. FxCycleTM Violet dye (FxCV) is a selective DNA-binding dye which permits simultaneous multiparametric immunophenotyping and cell-cycle/ploidy assessment in a single assay. To date, only two studies have demonstrated the feasibility of FxCV-aided FCM-ploidy analysis in B-ALL patients and only one of these studies have compared their results with CG-ploidy. METHODOLOGY: Blast size-specific FCM-ploidy was prospectively analyzed using FxCV-dye in 109 pediatric B-ALL patients, and the results were compared with concurrent CG-ploidy status. RESULTS: FCM-ploidy categorization was feasible in 98% of samples tested and the results were 82% concordant with CG-ploidy status. We observed significant correlation between DNA content and blast size (r = .823, P < .001) and could demonstrate size differences between diploid vs low-hyperdiploid (P = .025), diploid vs high-hyperdiploid (P < .001) and low- vs high-hyperdiploid blasts (P = .007). CONCLUSION: FCM-ploidy assessment using FxCV dye is a reliable assay and the results closely concur with CG-based ploidy stratification and risk assessment. Using blast size-assisted DNA content analysis, the results of FCM-ploidy analysis can be further fine-tuned.

Immunophenotypic shift in the B-cell precursors from regenerating bone marrow samples: A critical consideration for measurable residual disease assessment in B-lymphoblastic leukemia.

Accurate knowledge of expression patterns/levels of commonly used MRD markers in regenerative normal-B-cell-precursors (BCP) is highly desirable to distinguish leukemic-blasts from regenerative-BCP for multicolor flow cytometry (MFC)-based measurable residual disease (MRD) assessment in B-lymphoblastic leukemia (B-ALL). However, the data highlighting therapy-related immunophenotypic-shift in regenerative-BCPs is scarce and limited to small cohort. Herein, we report the in-depth evaluation of immunophenotypic shift in regenerative-BCPs from a large cohort of BALL-MRD samples. Ten-color MFC-MRD analysis was performed in pediatric-BALL at the end-of-induction (EOI), end-of-consolidation (EOC), and subsequent-follow-up (SFU) time-points. We studied normalized-mean fluorescent intensity (nMFI) and coefficient-of-variation of immunofluorescence (CVIF) of CD10, CD19, CD20, CD34, CD38, and CD45 expression in regenerative-BCP (early, BCP1 and late, BCP2) from 200 BALL-MRD samples, and compared them with BCP from 15 regenerating control (RC) TALL-MRD samples and 20 treatment-naïve bone-marrow control (TNSC) samples. Regenerative-BCP1 showed downregulation in CD10 and CD34 expression with increased CVIF and reduced nMFI (p < 0.001), upregulation of CD20 with increased nMFI (p = 0.014) and heterogeneous CD45 expression with increased CVIF (p < 0.001). Immunophenotypic shift was less pronounced in the BCP2 compared to BCP1 compartment with increased CVIF in all but CD45 (p < 0.05) and reduced nMFI only in CD45 expression (p = 0.005). Downregulation of CD10/CD34 and upregulation of CD20 was higher at EOI than EOC and SFU time-points (p < 0.001). Regenerative-BCPs are characterized by the significant immunophenotypic shift in commonly used B-ALL-MRD markers, especially CD10 and CD34 expression, as compared to treatment-naïve BCPs. Therefore, the templates/database for BMRD analysis must be developed using regenerative-BCP.

Bispecific antibodies in acute lymphoblastic leukemia therapy.

INTRODUCTION: Blinatumomab, first in a class of bispecific T-cell engagers, revolutionized treatment paradigm of B-cell precursor relapsed/refractory or minimal residual disease positive acute lymphoblastic leukemia (ALL) in adults and children, inducing deep remissions in a proportion of patients. However, significant numbers of patients do not respond or eventually relapse. Strategies for improvement of treatment outcomes are required. AREAS COVERED: This review discusses the main structural and functional features of blinatumomab, and its place in the treatment of ALL. Furthermore, prospects to increase the efficacy of blinatumomab are addressed. The developments in the field of bispecific antibodies and their possible implications for treatment of ALL are reviewed. EXPERT OPINION: Better understanding the mechanisms of response and resistance to blinatumomab might help us to identify the group of patients benefiting most from treatment and to spare potentially toxic subsequent treatment strategies. Data emerging from ongoing clinical trials might change the treatment landscape of ALL and beyond. Early use of blinatumomab in frontline protocols with more advantageous treatment sequences and in combination with other targeted therapies might reduce the failure rates. Exponentially increasing number of novel treatment options and their possible combinations might complicate treatment decision-making without data from randomized trials.