B-cell Precursor Acute Lymphoblastic Leukemia Research Articles

Asparagine transport through SLC1A5/ASCT2 and SLC38A5/SNAT5 is essential for BCP-ALL cell survival and a potential therapeutic target.

B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) blasts strictly depend on the transport of extra-cellular asparagine (Asn), yielding a rationale for L-asparaginase (ASNase) therapy. However, the carriers used by ALL blasts for Asn transport have not been identified yet. Exploiting RS4;11 cells as BCP-ALL model, we have found that cell Asn is lowered by either silencing or inhibition of the transporters ASCT2 or SNAT5. The inhibitors V-9302 (for ASCT2) and GluγHA (for SNAT5) markedly lower cell proliferation and, when used together, suppress mTOR activity, induce autophagy and cause a severe nutritional stress, leading to a proliferative arrest and a massive cell death in both the ASNase-sensitive RS4;11 cells and the relatively ASNase-insensitive NALM-6 cells. The cytotoxic effect is not prevented by coculturing leukaemic cells with primary mesenchymal stromal cells. Leukaemic blasts of paediatric ALL patients express ASCT2 and SNAT5 at diagnosis and undergo marked cytotoxicity when exposed to the inhibitors. ASCT2 expression is positively correlated with the minimal residual disease at the end of the induction therapy. In conclusion, ASCT2 and SNAT5 are the carriers exploited by ALL cells to transport Asn, and ASCT2 expression is associated with a lower therapeutic response. ASCT2 may thus represent a novel therapeutic target in BCP-ALL.

Constitutional and acquired genetic variants in ARID5B in pediatric B-cell precursor acute lymphoblastic leukemia.

Constitutional polymorphisms in ARID5B are associated with an increased risk of developing high hyperdiploid (HeH; 51-67 chromosomes) pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL). Here, we investigated constitutional and somatic ARID5B variants in 1335 BCP ALL cases from five different cohorts, with a particular focus on HeH cases. In 353 HeH ALL that were heterozygous for risk alleles and trisomic for chromosome 10, where ARID5B is located, a significantly higher proportion of risk allele duplication was seen for the SNPs rs7090445 (p = 0.009), rs7089424 (p = 0.005), rs7073837 (p = 0.03), and rs10740055 (p = 0.04). Somatic ARID5B deletions were seen in 16/1335 cases (1.2%), being more common in HeH than in other genetic subtypes (2.2% vs. 0.4%; p = 0.002). The expression of ARID5B in HeH cases with genomic deletions was reduced, consistent with a functional role in leukemogenesis. Whole-genome sequencing and RNA-sequencing in HeH revealed additional somatic events involving ARID5B, resulting in a total frequency of 3.6% of HeH cases displaying a somatic ARID5B aberration. Overall, our results show that both constitutional and somatic events in ARID5B are involved in the leukemogenesis of pediatric BCP ALL, particularly in the HeH subtype.

The Evolving Landscape of Flowcytometric Minimal Residual Disease Monitoring in B-Cell Precursor Acute Lymphoblastic Leukemia.

Detection of minimal residual disease (MRD) is a major independent prognostic marker in the clinical management of pediatric and adult B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL), and risk stratification nowadays heavily relies on MRD diagnostics. MRD can be detected using flow cytometry based on aberrant expression of markers (antigens) during malignant B-cell maturation. Recent advances highlight the significance of novel markers (e.g., CD58, CD81, CD304, CD73, CD66c, and CD123), improving MRD identification. Second and next-generation flow cytometry, such as the EuroFlow consortium's eight-color protocol, can achieve sensitivities down to 10-5 (comparable with the PCR-based method) if sufficient cells are acquired. The introduction of targeted therapies (especially those targeting CD19, such as blinatumomab or CAR-T19) introduces several challenges for flow cytometric MRD analysis, such as the occurrence of CD19-negative relapses. Therefore, innovative flow cytometry panels, including alternative B-cell markers (e.g., CD22 and CD24), have been designed. (Semi-)automated MRD assessment, employing machine learning algorithms and clustering tools, shows promise but does not yet allow robust and sensitive automated analysis of MRD. Future directions involve integrating artificial intelligence, further automation, and exploring multicolor spectral flow cytometry to standardize MRD assessment and enhance diagnostic and prognostic robustness of MRD diagnostics in BCP-ALL.

Intention-to-treat outcomes utilising a stringent event definition in children and young people treated with tisagenlecleucel for r/r ALL through a national access scheme.

CAR T-cell therapy has transformed relapsed/refractory (r/r) B-cell precursor acute lymphoblastic leukaemia (B-ALL) management and outcomes, but following CAR T infusion, interventions are often needed. In a UK multicentre study, we retrospectively evaluated tisagenlecleucel outcomes in all eligible patients, analysing overall survival (OS) and event-free survival (EFS) with standard and stringent definitions, the latter including measurable residual disease (MRD) emergence and further anti-leukaemic therapy. Both intention-to-treat and infused cohorts were considered. We collected data on feasibility of delivery, manufacture, toxicity, cause of therapy failure and followed patients until death from any cause. Of 142 eligible patients, 125 received tisagenlecleucel, 115/125 (92%) achieved complete remission (CR/CRi). Severe cytokine release syndrome and neurotoxicity occurred in 16/123 (13%) and 10/123 (8.1%), procedural mortality was 3/126 (2.4%). The 2-year intent to treat OS and EFS were 65.2% (95%CI 57.2-74.2%) and 46.5% (95%CI 37.6-57.6%), 2-year intent to treat stringent EFS was 35.6% (95%CI 28.1-44.9%). Median OS was not reached. Sixty-two responding patients experienced CAR T failure by the stringent event definition. Post failure, 1-year OS and standard EFS were 61.2% (95%CI 49.3-75.8) and 55.3% (95%CI 43.6-70.2). Investigation of CAR T-cell therapy for B-ALL delivered on a country-wide basis, including following patients beyond therapy failure, provides clinicians with robust outcome measures. Previously, outcomes post CAR T-cell therapy failure were under-reported. Our data show that patients can be successfully salvaged in this context with good short-term survival.

Mutational and transcriptional landscape of pediatric B-cell precursor lymphoblastic lymphoma

Pediatric B-cell precursor (BCP) lymphoblastic malignancies are neoplasms with manifestation either in bone marrow/blood (BCP acute lymphoblastic leukemia, BCP-ALL) or less common in extramedullary tissue (BCP lymphoblastic lymphoma, BCP-LBL). Although both presentations are similar in morphology and immunophenotype, molecular studies are virtually restricted to BCP-ALL so far. The lack of molecular studies on BCP-LBL is due to its rarity and the restriction to small, mostly formalin-fixed paraffin embedded (FFPE) tissues. Here we present the first comprehensive mutational and transcriptional analysis of what we consider the largest BCP-LBL cohort described to date (n=97). Whole exome sequencing indicates a mutational spectrum of BCP-LBL strikingly similar to that found in BCP-ALL. However, epigenetic modifiers were more frequently mutated in BCP-LBL, whereas BCP-ALL was more frequently affected by mutation in genes involved in B-cell development. Integrating copy number alterations, somatic mutations and gene expression by RNA-sequencing revealed that virtually all molecular subtypes originally defined in BCP-ALL are present in BCP-LBL too, with only 7% of lymphomas that were not assigned to a subtype. Similar to BCP-ALL, the most frequent subtypes of BCP-LBL were high hyperdiploidy and ETV6::RUNX1. Tyrosine kinase/cytokine-receptor rearrangements were detected in 7% of BCP-LBL. These results indicate that genetic subtypes can be identified in BCP-LBL using next-generation sequencing, even on FFPE tissue, and may be relevant to guide treatment.

CD9 shapes glucocorticoid sensitivity in pediatric B-cell precursor acute lymphoblastic leukemia.

Resistance to glucocorticoids (GCs), the common agents for remission induction in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), poses a significant therapeutic hurdle. Therefore, dissecting the mechanisms shaping GC resistance could lead to new treatment modalities. Here, we showed that CD9- BCP-ALL cells were preferentially resistant to prednisone and dexamethasone over other standard cytotoxic agents. Concordantly, we identified significantly more poor responders to the prednisone prephase among BCP-ALL patients with a CD9- phenotype, especially for those with adverse presenting features including older age, higher white cell count and BCR-ABL1. Furthermore, gain- and loss-of-function experiments dictated a definitive functional linkage between CD9 expression and GC susceptibility, as demonstrated by the reversal and acquisition of relative GC resistance in CD9low and CD9high BCP-ALL cells, respectively. Despite physical binding to the GC receptor NR3C1, CD9 did not alter its expression, phosphorylation or nuclear translocation but potentiated the induction of GC-responsive genes in GCresistant cells. Importantly, the MEK inhibitor trametinib exhibited higher synergy with GCs against CD9- than CD9+ lymphoblasts to reverse drug resistance in vitro and in vivo. Collectively, our results elucidate a previously unrecognized regulatory function of CD9 in GC sensitivity, and inform new strategies for management of children with resistant BCP-ALL.

Flow cytometric minimal residual disease measurement accounting for cytogenetics in children with non-high-risk acute lymphoblastic leukemia treated according to the ALL-MB 2008 protocol.

Quantitative measurement of minimal residual disease (MRD) is the "gold standard" for estimating the response to therapy in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Nevertheless, the speed of the MRD response differs for different cytogenetic subgroups. Here we present results of MRD measurement in children with BCP-ALL, in terms of genetic subgroups with relation to clinically defined risk groups. A total of 485 children with non-high-risk BCP-ALL with available cytogenetic data and MRD studied at the end-of-induction (EOI) by multicolor flow cytometry (MFC) were included. All patients were treated with standard-risk (SR) of intermediate-risk (ImR) regimens of "ALL-MB 2008" reduced-intensity protocol. Among all study group patients, 203 were found to have low-risk cytogenetics (ETV6::RUNX1 or high hyperdiploidy), while remaining 282 children were classified in intermediate cytogenetic risk group. For the patients with favorable and intermediate risk cytogenetics, the most significant thresholds for MFC-MRD values were different: 0.03% and 0.04% respectively. Nevertheless, the most meaningful thresholds were different for clinically defined SR and ImR groups. For the SR group, irrespective to presence/absence of favorable genetic lesions, MFC-MRD threshold of 0.1% was the most clinically valuable, although for ImR group the most informative thresholds were different in patients from low-(0.03%) and intermediate (0.01%) cytogenetic risk groups. Our data show that combining clinical risk factors with MFC-MRD measurement is the most useful tool for risk group stratification of children with BCP-ALL in the reduced-intensity protocols. However, this algorithm can be supplemented with cytogenetic data for part of the ImR group.

Central role of the mTORC1 pathway in glucocorticoid activity against B-ALL cells

Glucocorticoids (GCs), such as dexamethasone and prednisone, are crucial components of B-cell precursor acute lymphoblastic leukemia (B-ALL) therapies. However, the molecular basis of GC-induced cell death remains elusive. Here we show that GC suppresses mTORC1 (mechanistic target of rapamycin complex 1) signaling and that, conversely, oncogenic activation of mTORC1 confers resistance to GC. Our genome-wide CRISPR/Cas9 dropout screens reveal that depletion of components of either the GATOR1 (Gap Activity TOward Rags 1) or TSC (Tuberous Sclerosis Complex) complexes, both negative regulators of mTORC1 signaling, significantly attenuates B-ALL cell sensitivity to dexamethasone. Dexamethasone primarily induces B-ALL cell death by downregulating mTORC1 activity, thus promoting autophagy and impairing protein synthesis. Dexamethasone treatment failed to suppress mTORC1 activity in B-ALL cells expressing mutant glucocorticoid receptors lacking DNA-binding capacity, suggesting that dexamethasone transcriptionally represses mTORC1 activity. RNA-sequencing analysis identified multiple dexamethasone target genes that negatively regulate mTORC1 activity. Our findings suggest that GC sensitivity is significantly influenced by oncogenic stimuli and/or growth factors that activate the PI3K-AKT-mTORC1 pathway. This is consistent with the frequent GC resistance found in Ph and Ph-like ALLs.

Involvement of BCR::ABL1 in laminin adhesion of Philadelphia chromosome-positive acute lymphoblastic leukemia through upregulation of integrin α6.

Adhesion of cancer cells to extracellular matrix laminin through the integrin superfamily reportedly induces drug resistance. Heterodimers of integrin α6 (CD49f) with integrin β1 (CD29) or β4 (CD104) are major functional receptors for laminin. Higher CD49f expression is reportedly associated with a poorer response to induction therapy in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Moreover, a xenograft mouse model transplanted with primary BCP-ALL cells revealed that neutralized antibody against CD49f improved survival after chemotherapy. Considering the poor outcomes in Philadelphia chromosome (Ph)-positive ALL treated with conventional chemotherapy without tyrosine kinase inhibitors, we sought to investigate an involvement of the laminin adhesion. Ph-positive ALL cell lines expressed the highest levels of CD49f among the BCP-ALL cell lines with representative translocations, while CD29 and CD104 were ubiquitously expressed in BCP-ALL cell lines. The association of Ph-positive ALL with high levels of CD49f gene expression was also confirmed in two databases of childhood ALL cohorts. Ph-positive ALL cell lines attached to laminin and their laminin-binding properties were disrupted by blocking antibodies against CD49f and CD29 but not CD104. The cell surface expression of CD49f, but not CD29 and CD104, was downregulated by imatinib treatment in Ph-positive ALL cell lines, but not in their T315I-acquired sublines. Consistently, the laminin-binding properties were disrupted by the imatinib pre-treatment in the Ph-positive ALL cell line, but not in its T315I-acquired subline. BCR::ABL1 plays an essential role in the laminin adhesion of Ph-positive ALL cells through upregulation of CD49f.

Evaluation of next-generation sequencing for measurable residual disease monitoring in three major fusion transcript subtypes of B-precursor acute lymphoblastic leukaemia

The use of next-generation sequencing (NGS) for monitoring measurable residual disease (MRD) in acute lymphoblastic leukaemia (ALL) has been gaining traction. This study aimed to investigate the utility of NGS in MRD monitoring for the three major fusion transcript (FT) subtypes of B-precursor ALL (B-ALL). The MRD results for 104 bone marrow samples from 56 patients were analysed through NGS and real time quantitative reverse transcription PCR (RT-qPCR) for the three major FTs: BCR::ABL1, TCF3::PBX1, and ETV6::RUNX1. To validate the NGS approach, NGS-MRD was initially compared with allele-specific oligonucleotide-qPCR-MRD, and the coefficient of determination was good (R2=0.8158). A subsequent comparison of NGS-MRD with FT-MRD yielded a good coefficient of determination (R2=0.7690), but the coefficient varied by subtype. Specifically, the R2 was excellent for TCF3::PBX1 ALL (R2=0.9157), good for ETV6::RUNX1 ALL (R2=0.8606), and subpar for BCR::ABL1 ALL (R2=0.5763). The overall concordance between the two methods was 83.7%, and an excellent concordance rate of 95.8% was achieved for TCF3::PBX1 ALL. Major discordance, which was defined as a >1 log difference between discordant NGS-MRD and FT-MRD, occurred in 6.7% of the samples, with all but one sample being BCR::ABL1 ALL. Among the four non-transplanted patients with BCR::ABL1-MRD (+)/NGS-MRD (-), three did not relapse after long-term follow-up. Our finding indicates that NGS-MRD has a better prognostic impact than RT-qPCR-MRD in ETV6::RUNX1 and BCR::ABL1 ALL, whereas in TCF3::PBX1 ALL, both methods exhibit comparable efficacy.

Long Non-coding RNA RP11-252C15.1 is a Potential Biomarker of Prognosis and Hallmark for Leukemogenesis in Children with B-cell Precursor Acute Lymphoblastic Leukemia.

Improved understanding of the prognostic biomarkers associated with childhood acute lymphoblastic leukemia (ALL) is needed for accurate risk group stratification. This study aimed to identify potential long-non coding RNA (lncRNA) markers and evaluate their prognostic value in children with ALL. We selected 50 children with newly diagnosed ALL and 20 age-matched patients with idiopathic immune thrombocytopenia (controls). RNA sequencing was performed to identify differentially expressed lncRNAs between the ALL and control groups. Correlation analysis was performed to determine the relationships between candidate lncRNAs, clinical features, and the risk of leukemogenesis. A total of 1,019 differentially expressed lncRNAs were identified between the ALL and control groups. Reverse-transcriptase (RT-qPCR) revealed that lncRNA RP11-252C15.1 and lncRNA RP11-701P16.2 were significantly upregulated in patients with ALL. Furthermore correlation analysis showed that lncRNA RP11-252C15.1 and lncRNA RP11-701P16.2 represent potential predictors of leukemogenesis; however, only lncRNA RP11-252C15.1 was associated with clinical features and outcome in children with B-cell precursor ALL (BCP-ALL). In vitro experiments confirmed that lncRNA RP11-252C15.1 was significantly overexpressed in BCP-ALL cell lines and promoted proliferation, and repressed apoptosis in MHH-CALL-3 cells. LncRNA RP11-252C15.1 is a potential oncogene in BCP-ALL pathogenesis and a prognostic biomarker in children with BCP-ALL.

Abstract 624: Ptpn1 deficiency collaborates with a NUP98::HOXD13 fusion gene to generate B cell precursor acute lymphoblastic leukemia

Abstract Background: Acute lymphoid leukemia is characterized by inherited or acquired mutations that effect critical differentiation and proliferation pathways. We have shown that Mcm2 deficient mice developed T cell acute lymphoblastic leukemia, due to copy number variations, most commonly interstitial deletions throughout the genome. When crossed with mice that expressed a NUP98::HOXD13 (NHD13) fusion gene, Mcm2−/−NHD13 mice developed B cell precursor ALL (BCP-ALL). A majority of these BCP-ALL had acquired homozygous deletions of Ptpn1, a protein tyrosine phosphatase, leading to the hypothesis that Ptpn1 deficiency combined with NHD13 expression leads to BCP ALL. Objective: To investigate the role of Ptpn1 deletion in BCP-ALL development using mouse models. Methods: Mice expressing an NHD13 fusion gene were crossed with Ptpn1 knockout mice, generating 6 possible genotypes. Mice were followed for 18 months. Mice with signs of leukemia were characterized by clinical evaluation, CBC, flow cytometry, and IHC. Primary BCP-ALL and derived BCP-ALL cell lines were also evaluated with RNA-seq and molecular pathway analysis. Results: NHD13+Ptpn1-/- mice developed BCP-ALL with 65% penetrance, characterized by hyperleukocytosis, anemia, thrombocytopenia, and invasion of non-hematopoietic tissues. Similar to human BCP-ALL, NHD13+Ptpn1-/- BCP-ALL had clonal IGH as well as clonal Tcrd gene rearrangements. Flow cytometry revealed CD19 and/or B220 expression. NHD13+Ptpn1+/- mice with BCP-ALL frequently lost the wild-type (WT) Ptpn1 allele in leukemic cells, reinforcing the hypothesis that Ptpn1 can function as a classic tumor suppressor gene in this context. Whole exome sequencing revealed acquired mutations in B-cell differentiation genes (Pax5 or Bcor) and activating mutations in tyrosine kinase genes (Jak1/3 and Flt3). Transcription signature analysis showed significant upregulation of Hoxa/b gene clusters, RNase12 and LncRNAs subsets. Conclusion: This study demonstrates that Ptpn1 loss combined with expression of NHD13 fusion gene leads to highly penetrant BCP-ALL in mice, suggesting a role for Ptpn1 in preventing malignant transformation. These findings present a collaborative model for BCP-ALL in which the NHD13 transgene leads to increased stem cell self-renewal, somatic Bcor or Pax5 mutations block normal B cell differentiation, and somatic signaling mutations (Jak1/3,Flt3) lead to hyperproliferation, which is potentiated by Ptpn1 deficiency. Citation Format: Nupur Nigam, Toshihiro Matsukawa, Ryan Bertoli, Michel L. Tremblay, Mianmian Yin, Peter D. Aplan. Ptpn1 deficiency collaborates with a NUP98::HOXD13 fusion gene to generate B cell precursor acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 624.

Raman spectroscopy can recognize the KMT2A rearrangement as a distinct subtype of leukemia

Acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) are the two most common hematologic malignancies, challenging to treat and associated with high recurrence and mortality rates. This work aims to identify specific Raman biomarkers of ALL cells with the KMT2A gene rearrangement (KMT2A-r), representing a highly aggressive subtype of childhood leukemia with a poor prognosis. The proposed approach combines the sensitivity and specificity of Raman spectroscopy with machine learning and allows us to distinguish not only myelo- and lymphoblasts but also discriminate B-cell precursor (BCP) ALL with KMT2A-r from other blasts of BCP-ALL. We have found that KMT2A-r ALL cells fixed with 0.5% glutaraldehyde exhibit a unique spectroscopic profile that enables us to identify this subtype from other leukemias and normal cells. Therefore, a rapid and label-free method was developed to identify ALL blasts with KMT2A-r based on the ratio of the two Raman bands assigned to phenylalanine – 1040 and 1008 cm−1. This is the first time that a particular group of leukemic cells has been identified in a label-free way. The identified biomarker can be used as a screening method in diagnostic laboratories or non-reference medical centers.

The Implementation of Chimeric Antigen Receptor (CAR) T-cell Therapy in Pediatric Patients: Where Did We Come From, Where Are We Now, and Where are We Going?

CD19-directed Chimeric Antigen Receptor (CAR) T-cell therapy has revolutionized the treatment of patients with B-cell acute lymphoblastic leukemia (B-ALL). Somewhat uniquely among oncologic clinical trials, early clinical development occurred simultaneously in both children and adults. In subsequent years however, the larger number of adult patients with relapsed/refractory (r/r) malignancies has led to accelerated development of multiple CAR T-cell products that target a variety of malignancies, resulting in six currently FDA-approved for adult patients. By comparison, only a single CAR-T cell therapy is approved by the FDA for pediatric patients: tisagenlecleucel, which is approved for patients  25 years with refractory B-cell precursor ALL, or B-cell ALL in second or later relapse. Tisagenlecleucel is also under evaluation in pediatric patients with relapsed/refractory B-cell non-Hodgkin lymphoma, but is not yet been approved for this indication. All the other FDA-approved CD19-directed CAR-T cell therapies available for adult patients (axicabtagene ciloleucel, brexucabtagene autoleucel, and lisocabtagene maraleucel) are currently under investigations among children, with preliminary results available in some cases. As the volume and complexity of data continue to grow, so too does the necessity of rapid assimilation and implementation of those data. This is particularly true when considering “atypical” situations, e.g. those arising when patients do not precisely conform to the profile of those included in pivotal clinical trials, or when alternative treatment options (e.g. hematopoietic stem cell transplantation (HSCT) or bispecific T-cell engagers (BITEs)) are also available. We have therefore developed a relevant summary of the currently available literature pertaining to the use of CD19-directed CAR-T cell therapies in pediatric patients, and sought to provide guidance for clinicians seeking additional data about specific clinical situations.

Faster clinical decisions in B-cell acute lymphoblastic leukaemia: A single flow cytometric 12-colour tube improves diagnosis and minimal residual disease follow-up.

Assessing minimal residual disease (MRD) in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) is essential for adjusting therapeutic strategies and predicting relapse. Quantitative polymerase chain reaction (qPCR) is the gold standard for MRD. Alternatively, flow cytometry is a quicker and cost-effective method that typically uses leukaemia-associated immunophenotype (LAIP) or different-from-normal (DFN) approaches for MRD assessment. This study describes an optimized 12-colour flow cytometry antibody panel designed for BCP-ALL diagnosis and MRD monitoring in a single tube. This method robustly differentiated hematogones and BCP-ALL cells using two specific markers: CD43 and CD81. These and other markers (e.g. CD73, CD66c and CD49f) enhanced the specificity of BCP-ALL cell detection. This innovative approach, based on a dual DFN/LAIP strategy with a principal component analysis method, can be used for all patients and enables MRD analysis even in the absence of a diagnostic sample. The robustness of our method for MRD monitoring was confirmed by the strong correlation (r = 0.87) with the qPCR results. Moreover, it simplifies and accelerates the preanalytical process through the use of a stain/lysis/wash method within a single tube (<2 h). Our flow cytometry-based methodology improves the BCP-ALL diagnosis efficiency and MRD management, offering a complementary method with considerable benefits for clinical laboratories.

Reduced-dose chemotherapy followed by blinatumomab in induction therapy for newly diagnosed B-cell acute lymphoblastic leukemia.

Blinatumomab early-line treatment in B-cell precursor acute lymphoblastic leukemia (B-ALL) might improve clinical outcomes. We conducted a retrospective real-world cohort analysis in 20 newly diagnosed B-ALL patients who received reduced-dose chemotherapy (idarubicin, vindesine, and dexamethasone) for 1-3 weeks, followed by blinatumomab for 1-4 weeks as an induction therapy. At the end of the induction therapy, a complete remission rate of 100% was achieved; 17 (85%) patients were minimal residual disease (MRD) negative (<1 × 10-4 ). Adverse events (AEs) were reported in 12 (60%) patients-43.8% were grade 1-2 and 56.2% were grade 3-4. No incidence of neurotoxicity or grade ≥3 cytokine release syndrome was reported. Blinatumomab demonstrated a significant improvement in clinical outcomes in patients with newly diagnosed B-ALL irrespective of their poor-risk factor status and the pretreatment blast burden.

B-cell precursor acute lymphoblastic leukemia elicits an interferon-α/β response in bone marrow-derived mesenchymal stroma.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) can hijack the normal bone marrow microenvironment to create a leukemic niche which facilitates blast cell survival and promotes drug resistance. Bone marrow-derived mesenchymal stromal cells (MSCs) mimic this protective environment in ex vivo co-cultures with leukemic cells obtained from children with newly diagnosed BCP-ALL. We examined the potential mechanisms of this protection by RNA sequencing of flowsorted MSCs after co-culture with BCP-ALL cells. Leukemic cells induced an interferon (IFN)-related gene signature in MSCs, which was partially dependent on direct cell-cell signaling. The signature was selectively induced by BCP-ALL cells, most profoundly by ETV6-RUNX1 positive ALL cells, as coculture of MSCs with healthy immune cells did not provoke a similar IFN signature. Leukemic cells and MSCs both secreted IFNα and IFNβ, but no IFNγ. In line, the IFN-gene signature was sensitive to blockade of IFNα/β signaling, but less to that of IFNγ. The viability of leukemic cells and level of resistance to three chemotherapeutic agents was not affected by interference with IFN signaling using selective IFNα/β inhibitors or silencing of IFN-related genes. Taken together, our data suggest that the leukemia-induced expression of IFNα/β-related genes by MSCs does not support survival of BCPALL cells but may serve a different role in the pathobiology of BCP-ALL.

Tyrosine kinase inhibitor resistance in de novo BCR::ABL1–positive BCP-ALL beyond kinase domain mutations

AbstractA better understanding of ABL1 kinase domain mutation–independent causes of tyrosine kinase inhibitor (TKI) resistance is needed for BCR::ABL1–positive B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Although TKIs have dramatically improved outcomes, a subset of patients still experiences relapsed or refractory disease. We aimed to identify potential biomarkers of intrinsic TKI resistance at diagnosis in samples from 32 pediatric and 19 adult patients with BCR::ABL1–positive BCP-ALL. Reduced ex vivo imatinib sensitivity was observed in cells derived from newly diagnosed patients who relapsed after combined TKI and chemotherapy treatment compared with cells derived from patients who remained in continuous complete remission. We observed that ex vivo imatinib resistance was inversely correlated with the amount of (phosphorylated) BCR::ABL1/ABL1 protein present in samples that were taken at diagnosis without prior TKI exposure. This suggests an intrinsic cause of TKI resistance that is independent of functional BCR::ABL1 signaling. Simultaneous deletions of IKZF1 and CDKN2A/B and/or PAX5 (IKZF1plus), as well as deletions of PAX5 alone, were related to ex vivo imatinib resistance. In addition, somatic lesions involving ZEB2, SETD2, SH2B3, and CRLF2 were associated with reduced ex vivo imatinib sensitivity. Our data suggest that the poor prognostic value of IKZF1(plus) deletions is linked to intrinsic mechanisms of TKI resistance other than ABL1 kinase domain mutations in newly diagnosed pediatric and adult BCR::ABL1–positive BCP-ALL.

N-Acetylcysteine Alters Disease Progression and Increases Janus Kinase Mutation Frequency in a Mouse Model of Precursor B-Cell Acute Lymphoblastic Leukemia

N-Acetylcysteine Alters Disease Progression and Increases Janus Kinase Mutation Frequency in a Mouse Model of Precursor B-Cell Acute Lymphoblastic Leukemia

Enhanced potency of immunotherapy against B-cell precursor acute lymphoblastic leukemia by combination of an Fc-engineered CD19 antibody and CD47 blockade.

CD19-directed immunotherapy has become a cornerstone in the therapy of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). CD19-directed cellular and antibody-based therapeutics have entered therapy of primary and relapsed disease and contributed to improved outcomes in relapsed disease and lower therapy toxicity. However, efficacy remains limited in many cases due to a lack of therapy response, short remission phases, or antigen escape. Here, BCP-ALL cell lines, patient-derived xenograft (PDX) samples, human macrophages, and an in vivo transplantation model in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were used to examine the therapeutic potency of a CD19 antibody Fc-engineered for improved effector cell recruitment (CD19-DE) and antibody-dependent cellular phagocytosis (ADCP), in combination with a novel modified CD47 antibody (Hu5F9-IgG2σ). For the in vivo model, only samples refractory to CD19-DE monotherapy were chosen. Hu5F9-IgG2σ enhanced ADCP by CD19-DE in various BCP-ALL cell line models with varying CD19 surface expression and cytogenetic backgrounds, two of which contained the KMT2A-AFF1 fusion. Also, the antibody combination was efficient in inducing ADCP by human macrophages in pediatric PDX samples with and adult samples with and without KMT2A-rearrangement in vitro. In a randomized phase 2-like PDX trial using seven KMT2A-rearranged BCP-ALL samples in NSG mice, the CD19/CD47 antibody combination proved highly efficient. Our findings support that the efficacy of Fc-engineered CD19 antibodies may be substantially enhanced by a combination with CD47 blockade. This suggests that the combination may be a promising therapy option for BCP-ALL, especially in relapsed patients and/or patients refractory to CD19-directed therapy.