Predictive model for CAR-T cell therapy success in patients with relapsed/refractory B-cell acute lymphoblastic leukaemia.

Chimeric antigen receptor T-cell (CAR-T) therapy has demonstrated remarkable efficacy in treating relapsed/refractory acute B-cell lymphoblastic leukaemia (R/R B-ALL). However, a subset of patients does not benefit from CAR-T therapy. Our study aims to identify predictive indicators and establish a model to evaluate the feasibility of CAR-T therapy. Fifty-five R/R B-ALL patients and 22 healthy donors were enrolled. Peripheral blood lymphocyte subsets were analysed using flow cytometry. Sensitivity, specificity, accuracy, positive and negative predictive values and receiver operating characteristic (ROC) areas under the curve (AUC) were determined to evaluate the predictive values of the indicators. We identified B lymphocyte, regulatory T cell (Treg) and peripheral blood minimal residual leukaemia cells (B-MRD) as indicators for predicting the success of CAR-T cell preparation with AUC 0.936, 0.857 and 0.914. Furthermore, a model based on CD3+ T count, CD4+ T/CD8+ T ratio, Treg and extramedullary diseases (EMD) was used to predict the response to CAR-T therapy with AUC of 0.938. Notably, a model based on CD4+ T/CD8+ T ratio, B, Treg and EMD were used in predicting the success of CAR-T therapy with AUC 0.966 [0.908-1.000], with specificity (92.59%) and sensitivity (91.67%). In the validated group, the predictive model predicted the success of CAR-T therapy with specificity (90.91%) and sensitivity (100%). We have identified several predictive indicators for CAR-T cell therapy success and a model has demonstrated robust predictive capacity for the success of CAR-T therapy. These results show great potential for guiding informed clinical decisions in the field of CAR-T cell therapy.

[Clinical efficacy and safety of blinatumomab bridging CAR-T cell therapy in the treatment of patients with adult acute B-cell lymphoblastic leukemia].

Objective: Exploring the efficacy and safety of bridging blinatumomab (BiTE) in combination with chimeric antigen receptor T (CAR-T) cell therapy for the treatment of adult patients with acute B-cell lymphoblastic leukemia (B-ALL) . Methods: Clinical data from 36 adult B-ALL patients treated at the First Affiliated Hospital of Suzhou University from August 2018 to May 2023 were retrospectively analyzed. A total of 36 cases were included: 18 men and 18 women. The median age was 43.5 years (21-72 years). Moreover, 21 cases of Philadelphia chromosome-positive acute lymphoblastic leukemia were reported, and 16 of these cases were relapsed or refractory. Eighteen patients underwent blinatumomab bridging followed by CAR-T cell therapy, and 18 patients received CAR-T cell therapy. This study analyzed the efficacy and safety of treatment in two groups of patients. Results: In the BiTE bridge-to-CAR-T group, 16 patients achieved complete remission (CR) after BiTE immunotherapy, with a CR rate of 88.9%. One month after bridging CAR-T therapy, bone marrow examination showed a CR rate of 100.0%, and the minimal residual disease (MRD) negativity rate was higher than the nonbridging therapy group (94.4% vs. 61.1%, Fisher, P=0.041). The incidence of cytokine release syndrome and other adverse reactions in the BiTE bridge-to-CAR-T group was lower than that in the nonbridging therapy group (11.1% vs. 50.0%, Fisher, P=0.027). The follow-up reveals that 13 patients continued to maintain MRD negativity, and five patients experienced relapse 8.40 months (2.57-10.20 months) after treatment. Two of five patients with relapse achieved CR after receiving the second CAR-T cell therapy. In the nonbridging therapy group, 10 patients maintained continuous MRD negativity, 7 experienced relapse, and 6 died. The 1 year overall survival rate in the BiTE bridge-to-CAR-T group was higher than that in the nonbridging therapy group, with a statistically significant difference at the 0.1 level (88.9%±10.5% vs. 66.7%±10.9%, P=0.091) . Conclusion: BiTE bridging CAR-T cell therapy demonstrates excellent efficacy in adult B-ALL treatment, with a low recent recurrence rate and ongoing assessment of long-term efficacy during follow-up.

The Role of the JAK-STAT Pathway in Childhood B-Cell Acute Lymphoblastic Leukemia.

B-cell lymphoblastic leukemia is a hematologic neoplasm that poses a serious health concern in childhood. Genetic aberrations, such as mutations in the genes IL-7, IL7R, JAK1, JAK2, TLSP, CRLF2, and KTM2A or gene fusions involving BCR::ABL1, ETV6::RUNX1, and PAX5::JAK2, often correlate with the onset of this disease. These aberrations can lead to malfunction of the JAK-STAT signaling pathway, which is implicated in various important biological processes, including those related to immunology. Understanding the mechanisms underlying the malfunction of the JAK-STAT pathway holds potential for research on drugs targeting its components. Available drugs that interfere with the JAK-STAT pathway include fludarabine, ruxolitinib, and fedratinib.

ActivinA modulates B-acute lymphoblastic leukaemia cell communication and survival by inducing extracellular vesicles production.

Extracellular vesicles (EVs) are a new mechanism of cellular communication, by delivering their cargo into target cells to modulate molecular pathways. EV-mediated crosstalk contributes to tumor survival and resistance to cellular stress. However, the role of EVs in B-cell Acute Lymphoblastic Leukaemia (B-ALL) awaits to be thoroughly investigated. We recently published that ActivinA increases intracellular calcium levels and promotes actin polymerization in B-ALL cells. These biological processes guide cytoskeleton reorganization, which is a crucial event for EV secretion and internalization. Hence, we investigated the role of EVs in the context of B-ALL and the impact of ActivinA on this phenomenon. We demonstrated that leukemic cells release a higher number of EVs in response to ActivinA treatment, and they can actively uptake EVs released by other B-ALL cells. Under culture-induced stress conditions, EVs coculture promoted cell survival in B-ALL cells in a dose-dependent manner. Direct stimulation of B-ALL cells with ActivinA or with EVs isolated from ActivinA-stimulated cells was even more effective in preventing cell death. This effect can be possibly ascribed to the increase of vesiculation and modifications of EV-associated microRNAs induced by ActivinA. These data demonstrate that ActivinA boosts EV-mediated B-ALL crosstalk, improving leukemia survival in stress conditions.

[The safety and efficacy of blinatumomab in the treatment of B-cell acute lymphoblastic leukemia in children].

To explore the safety and efficacy of blinatumomab in the treatment of CD19 positive (CD19+) B-cell acute lymphoblastic leukemia (B-ALL) in children. A retrospective analysis was conducted on the clinical data of pediatric B-ALL patients who received blinatumomab treatment from Hematology & Blood Diseases Hospital of Chinese Academy of Medical Sciences from August 2021 to October 2023. Based on their disease status, the patients were divided into refractory/relapsed(RR) group, minimal residual disease clearance (MC) group, and chemotherapy intolerance (IC) group. Clinical data of the children were collected to evaluate the adverse drug reactions, therapeutic efficacy and survival of the children. In total, 35 patients were included, with 20 males and 15 females, aged from 0.6 to 16.4 (9.9±4.2) years old. There were 10 cases in the RR group, 20 cases in the MC group and 5 cases in the IC group. A total of 56 cycles of infusion were completed, with one cycle in 24 cases, two cycles in 5 cases, three cycles in 2 cases and four cycles in 4 cases. The median infusion time [M (Q1, Q3)] from the first to the fourth cycle was 14 (14, 28) days, 28 (28, 28) days, 28 (28, 28) days and 28 (26, 28) days, respectively. In terms of adverse reactions, the incidence of grade 1-2 cytokine release syndrome(CRS) was 57.1% (32/56), with grade 1 CRS accounting for 84.4% (27/32). The incidence rate of immune effector cell-associated neurotoxicity syndrome(ICANS) (grade 4) was 1.8% (1/56). In the RR group, 6 cases were treated effectively, and minimal residual disease(MRD) turned negative, before treatment, MRD levels were all less than 20%. Among them, 3 cases had MRD turning positive again 14 to 42 days after discontinuation of Belintoumab. Four cases were treated ineffectively, with MRD >20% before treatment. All MRD positive cases in MC group turned negative and all MRD negative cases in the IC group remained negative after treatment. The median follow-up time of RR group was 5.7 (3.8, 9.4) months, and 1 year median survival rate and event-free survival rate were 40.0%±21.9% and 33.3%±19.2%, respectively. The median follow-up time for MC and IC group patients was 6.7 (5.2, 12.5) months and 7.1 (5.1, 7.6) months, respectively, with an event free survival rate of 100%. The safety and efficacy of using belintoumab in partial RR, MRD clearance, and chemotherapy intolerance are good.

Rare Drivers at Low Prevalence with High Cancer Effects in T-Cell and B-Cell Pediatric Acute Lymphoblastic Leukemia.

The genomic analyses of pediatric acute lymphoblastic leukemia (ALL) subtypes, particularly T-cell and B-cell lineages, have been pivotal in identifying potential therapeutic targets. Typical genomic analyses have directed attention toward the most commonly mutated genes. However, assessing the contribution of mutations to cancer phenotypes is crucial. Therefore, we estimated the cancer effects (scaled selection coefficients) for somatic substitutions in T-cell and B-cell cohorts, revealing key insights into mutation contributions. Cancer effects for well-known, frequently mutated genes like NRAS and KRAS in B-ALL were high, which underscores their importance as therapeutic targets. However, less frequently mutated genes IL7R, XBP1, and TOX also demonstrated high cancer effects, suggesting pivotal roles in the development of leukemia when present. In T-ALL, KRAS and NRAS are less frequently mutated than in B-ALL. However, their cancer effects when present are high in both subtypes. Mutations in PIK3R1 and RPL10 were not at high prevalence, yet exhibited some of the highest cancer effects in individual T-cell ALL patients. Even CDKN2A, with a low prevalence and relatively modest cancer effect, is potentially highly relevant for the epistatic effects that its mutated form exerts on other mutations. Prioritizing investigation into these moderately frequent but potentially high-impact targets not only presents novel personalized therapeutic opportunities but also enhances the understanding of disease mechanisms and advances precision therapeutics for pediatric ALL.

Enhancer engagement sustains oncogenic transformation and progression of B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: Enhancer reprogramming plays a significant role in the heterogeneity of cancer. However, we have limited knowledge about the impact of chromatin remodeling in B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) patients, and how it affects tumorigenesis and drug response. Our research focuses on investigating the role of enhancers in sustaining oncogenic transformation in children with BCP-ALL. METHODS: We used ATAC-seq to study the accessibility of chromatin in pediatric BCP-ALL at three different stages-onset, remission, and relapse. Using a combination of computational and experimental methods, we were able to analyze the accessibility landscape and focus on the most significant cis-regulatory sites. These sites were then functionally validated through the use of Promoter capture Hi-C in a primary cell line model called LAL-B, followed by RNA-seq and genomic deletion of target sites using CRISPR-Cas9 editing. RESULTS: We found that enhancer activity changes during cancer progression and is mediated by the production of enhancer RNAs (eRNAs). CRISPR-Cas9-mediated validation of previously unknown eRNA productive enhancers demonstrated their capability to control the oncogenic activities of the MYB and DCTD genes. CONCLUSIONS: Our findings directly support the notion that productive enhancer engagement is a crucial determinant of the BCP-ALL and highlight the potential of enhancers as therapeutic targets in pediatric BCP-ALL.

Identification of a novel MEF2C::SS18L1 fusion in childhood acute B-lymphoblastic leukemia.

PURPOSE: Leukemia-associated fusion genes are closely related to the occurrence, development, diagnosis, and treatment of leukemia. DNA microarrays and second-generation sequencing have discovered multiple B-ALL fusion genes. We identified a novel MEF2C::SS18L1 fusion gene in a child diagnosed with B-ALL. This study investigates the oncogenicity and prognosis of this fusion gene in B-ALL. METHODS: A child with B-ALL who has a MEF2C::SS18L1 fusion is reported as a newly discovered case. Compared the breakpoints, structural domains, clinical phenotypes, and differential expression genes of MEF2C::SS18L1 and MEF2D::SS18.Using "ONCOFUSE" software, the carcinogenicity of MEF2C::SS18L1 is predicted. Using whole transcriptome sequencing, we analyze the breakpoints and the secondary structure of the fusion protein. Further, we compared the structures, differentially expressed genes, and clinical phenotypes of MEF2D and MEF2C fusion genes by DESeq, GO functional enrichment, and flow cytometry immunophenotyping analysis. RESULTS: Whole transcriptome sequencing identified a MEF2C::SS18L1 fusion transcript in a 3-year-old child with B-ALL. The MADS box, MEF structural domain, HJURP_C structural domain, and TAD I structural domain of MEF2C, and the QPGY structural domain of SS18L1, make up the fusion protein. "Oncofuse" found a 0.99 Bayesian probability that the fusion gene drives cancer. The breakpoint positions, fusion protein secondary structures, differentially expressed genes, and clinical characteristics of this patient were identical to those with MEF2D::SS18 fusion gene. CONCLUSION: We identified a novel MEF2C::SS18L1 fusion gene in childhood ALL, which shares similar structural and clinical characteristics with MEF2D::SS18. Further studies with more samples should be conducted in future.

Bispecific T-cell engagers in childhood B-acute lymphoblastic leukemia.

Immunotherapy has revolutionized treatment for a wide variety of cancers yet its use has been relatively limited in childhood malignancies. With the introduction of bispecific T-cell engagers (BiTE®) and chimeric antigen T-cell receptor technologies, previously refractory patients have attained remission, including molecularly negative states of disease, thus providing the possibility of long-term cure. Blinatumomab is a widely available CD3-CD19 BiTE that has dramatically changed the landscape of therapy for some children with precursor-B acute lymphoblastic leukemias (B-ALL) and lymphoblastic lymphomas. Challenges remain with using BiTE in a broader population although the appeal of now-confirmed reduced toxicity and deeper molecular remissions suggests that this approach will be an essential part of future treatment of childhood B-ALL. Herein, we review some of the pertinent literature covering clinical trials with blinatumomab and address future approaches and combination trials including BiTE.

Allogeneic chimeric antigen receptor T cells for children with relapsed/refractory B-cell precursor acute lymphoblastic leukemia.

Chimeric antigen receptor (CAR) T-cell therapy has emerged as a breakthrough cancer therapy over the past decade. Remarkable outcomes in B-cell lymphoproliferative disorders and multiple myeloma have been reported in both pivotal trials and real-word studies. Traditionally, the use of a patient's own (autologous) T cells to manufacture CAR products has been the standard practice. Nevertheless, this approach has some drawbacks, including manufacturing delays, dependence on the functional fitness of the patient's T cells, which can be compromised by both the disease and prior therapies, and contamination of the product with blasts. A promising alternative is offered by the development of allogeneic CAR-cell products. This approach has the potential to yield more efficient drug products and enables the use of effector cells with negligible alloreactive potential and a significant CAR-independent antitumor activity through their innate receptors (i.e., natural killer cells, γδ T cells and cytokine induced killer cells). In addition, recent advances in genome editing tools offer the potential to overcome the primary challenges associated with allogeneic CAR T-cell products, namely graft-versus-host disease and host allo-rejection, generating universal, off-the-shelf products. In this review, we summarize the current pre-clinical and clinical approaches based on allogeneic CAR T cells, as well as on alternative effector cells, which represent exciting opportunities for multivalent approaches and optimized antitumor activity.

Single-cell transcriptomic analysis of the immune microenvironment in pediatric acute leukemia.

Relapse and treatment resistance pose significant challenges in the management of pediatric B cell acute lymphoblastic leukemia (B-ALL) and acute myeloid leukemia (AML). The efficacy of immunotherapy in leukemia remains limited due to factors such as the immunosuppressive tumor microenvironment (TME) and lack of suitable immunotherapeutic targets. Thus, an in-depth characterization of the TME in pediatric leukemia is warranted to improve the efficacy of immunotherapy. Here, we used single-cell RNA sequencing (scRNA-seq) to characterize the TME of pediatric B-ALL and AML, focusing specifically on bone-marrow-derived T cells. Moreover, we investigated the transcriptome changes during the initiation, remission, and relapse stages of pediatric AML. Our findings revealed that specific functional expression programs correlated with fluctuations in various T cell subsets, which may be associated with AML progression and relapse. Furthermore, our analysis of cellular communication networks led to the identification of VISTA, CD244, and TIM3 as potential immunotherapeutic targets in pediatric AML. Finally, we detected elevated proportions of γδ T cells and associated functional genes in samples from pediatric patients diagnosed with B-ALL and AML, which could inform the development of novel therapeutic approaches, potentially focusing on γδ T cells.

Blinatumomab as postremission therapy replaces consolidation and substantial parts of maintenance chemotherapy and results in stable MRD negativity in children with newly diagnosed B-lineage ALL.

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.

The impact of MCCK1, an inhibitor of IKBKE kinase, on acute B lymphocyte leukemia cells.

B-cell acute lymphoblastic leukemia (B-ALL) is a malignant blood disorder, particularly detrimental to children and adolescents, with recurrent or unresponsive cases contributing significantly to cancer-associated fatalities. IKBKE, associated with innate immunity, tumor promotion, and drug resistance, remains poorly understood in the context of B-ALL. Thus, this research aimed to explore the impact of the IKBKE inhibitor MCCK1 on B-ALL cells. The study encompassed diverse experiments, including clinical samples, in vitro and in vivo investigations. Quantitative real-time fluorescence PCR and protein blotting revealed heightened IKBKE mRNA and protein expression in B-ALL patients. Subsequent in vitro experiments with B-ALL cell lines demonstrated that MCCK1 treatment resulted in reduced cell viability and survival rates, with flow cytometry indicating cell cycle arrest. In vivo experiments using B-ALL mouse tumor models substantiated MCCK1's efficacy in impeding tumor proliferation. These findings collectively suggest that IKBKE, found to be elevated in B-ALL patients, may serve as a promising drug target, with MCCK1 demonstrating potential for inducing apoptosis in B-ALL cells both in vitro and in vivo.

The impact of exosomes derived from B-cell acute lymphoblastic leukemia as a growth factor on bone marrow mesenchymal stromal cells.

Background The incidence of various types of cancers, including leukemia, is on the rise and many challenges in both drug resistance and complications related to chemotherapy appeared. Recently, the development and application of extracellular vesicles (EV) such as exosomes in the management of cancers, especially leukemia, holds great significance. In this article, we extracted exosomes from NALM6 cells and assessed their regulatory effects on proliferation and apoptosis in mesenchymal stem cells (MSCs). Method and result We first verified the exosomes using various techniques, including flow cytometry, transient electron microscopy, dynamic light scattering (DLS), and BCA protein assay. Then MTT analysis and flowcytometry (apoptosis and cell cycle assay) besides gene expressions were employed to determine the state of MSC proliferations. The results indicated that exosome-specific pan markers like CD9, CD63, and CD81 were present. Through DLS, we found out that the mean size of the exosomes was 89.68 nm. The protein content was determined to be 956.292 µg/ml. Analysis of MTT, flow cytometry (cell cycle and apoptosis assay), and RT-qPCR showed that in the dose of 50 µg/ml the proliferation of MSCs was increased significantly (p-value < 0.05). Conclusion All these data showed that exosomes use several signaling pathways to increase the MSCs' proliferation and drug resistance, ultimately leading to high mortalities and morbidities of acute lymphoblastic leukemia.

Application and prognostic relevance of CD34 detection in immunophenotyping of pediatric acute B lymphoblastic leukemia.

OBJECTIVE: We aimed to investigate the application of CD34 detection in immunophenotypic discrimination and its prognostic relevance in children with acute B-lymphoblastic leukemia (B-ALL). PATIENTS AND METHODS: A retrospective analysis was conducted on clinical follow-up data of 105 children with newly diagnosed B-ALL treated at our hospital from January 2022 to December 2023. Based on the expression of CD34 in the bone marrow, patients were divided into a CD34 positive group (positive cells ≥10%) and a CD34 negative group (positive cells <10%). The study compared the positive rates of common leukemia cell antigens, clinical characteristics, initial treatment responses, and long-term follow-up outcomes between the two groups. RESULTS: Among all 105 B-ALL cases, 87 children (82.9%) had bone marrow CD34 positive cells ≥10%, classified into the CD34 positive group, while the remaining 18 children (17.1%) had bone marrow CD34 positive cells <10%, classified into the CD34 negative group. The CD34 positive group exhibited significantly higher positive rates of CD13 expression, standard-risk B-ALL, and risk stratification than the CD34 negative group. In contrast, the proportions of early pre-B-ALL, E2A-PBX1 fusion gene, and MLL-AF4 fusion gene were significantly lower in the CD34 negative group, with statistically significant differences (p<0.05). No significant differences were found in the positive rates of leukemia cell antigens such as CD10, CD19, CD20, CD22, CD79a, CD13, CD33, and CD38 between the two groups (p>0.05). The occurrence rates of minimal residual disease (MRD) and relapse after induction chemotherapy in the CD34 positive group were significantly lower than those in the CD34 negative group (p<0.05). However, the sensitivity to the first prednisone treatment and bone marrow treatment efficacy on the 19th and 33rd days after chemotherapy showed no significant differences between the groups (p>0.05). CONCLUSIONS: A higher positive rate of bone marrow CD34 expression in children with B-ALL is associated with a favorable prognosis. Children with negative CD34 expression are relatively more prone to MRD and tumor relapse after chemotherapy.